No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Biochemical changes associated with ??-difluoromethylornithine uptake and resistance in Trypanosoma brucei
Abstract
Procyclic Trypanosoma brucei grown in semi-defined media are sensitive to alpha-difluoromethylornithine (DFMO) (EC50 100 microM), an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis. Organisms resistant to 5 mM DFMO (EC50 greater than 20 mM) were obtained by passage in incremental amounts of drug. Resistant and wild-type cells accumulated DFMO by passive diffusion with a consequent decrease in polyamine levels, indicating inhibition of ODC in both cell types. The resistant phenotype was stable in the absence of DFMO, in which state there was no increase in ODC abundance or activity. By kinetic analysis, the ODC of resistant cells appeared normal. In wild-type and resistant cells, [3H]DFMO equally and uniquely affinity-labelled a 50 kDa polypeptide corresponding to the ODC subunit. Levels of ODC and tubulin mRNAs were elevated 4-fold in resistant cells grown in the presence of DFMO, although there was no indication of gene amplification. The intracellular concentration of dihydrotrypanothione (N1,N8-bis(glutathionyl)-spermidine), a redox intermediate unique to kinetoplastids, was unchanged in resistant cells growing in DFMO but was halved in wild-type cells exposed to DFMO for 48 h. The exceptionally elevated levels of ornithine found in DFMO-treated resistant cells most likely play a crucial role in cell survival by maintaining intracellular concentrations of dihydrotrypanothione by competing with DFMO for ODC.
... Difluoromethylornithine (DFMO, Eflornithine), a drug against late-stage T. b. gambiense sleeping sickness, is an irreversible inhibitor of ornithine decarboxylase (ODC), the enzyme generating putrescine, the precursor for spermidine synthesis. Treatment of T. brucei with DFMO decreases the levels of spermidine and T(SH) 2 and slightly increases the level of GSH (Bellofatto et al., 1987;Fairlamb et al., 1987;Xiao et al., 2009). All enzymes involved in T(SH) 2 biosynthesis and also TR, which catalyses the NADPH-dependent reduction of trypanothione disulfide (TS 2 ) as well as glutathionylspermidine disulfide (Gsp 2 ), appear to be restricted to the cytosol. ...
... After both H 2 O 2 -and diamide-stress, rereduction of Tpx-roGFP2 was fastest, followed by roGFP2-hGrx1 and roGFP2, corroborating the kinetic superiority of the Tpx-coupled sensor also under in vivo conditions. Inhibition of trypanothione biosynthesis affects the oxidative stress response of the parasite Treatment of T. brucei with DFMO results in impaired de novo synthesis of T(SH) 2 (Bellofatto et al., 1987;Fairlamb et al., 1987). Wildtype (WT) parasites and Tpx-roGFP2-expressing cells were cultured in the presence or absence of 5 mM DFMO. ...
... In cells cultured for 48 hr in the presence of DFMO, the GSH and T(SH) 2 concentrations were 322 ± 24 mM and 210 ± 17 mM, respectively ( Figure 4B). Thus, in DFMO treated cells, the GSH concentration was doubled, whereas the T(SH) 2 concentration was halved, in agreement with published data (Bellofatto et al., 1987). When the parasites were disintegrated in the absence of the reducing agent TCEP, the values in all samples were 10-20% lower, indicating that the cellular thiols are mainly in the reduced state as previously shown for BS T. brucei (Ulrich et al., 2017). ...
Trypanosomes have a trypanothione redox metabolism that provides the reducing equivalents for numerous essential processes, most being mediated by tryparedoxin (Tpx). While the biosynthesis and reduction of trypanothione are cytosolic, the molecular basis of the thiol redox homeostasis in the single mitochondrion of these parasites has remained largely unknown. Here we expressed Tpx-roGFP2, roGFP2-hGrx1 or roGFP2 in either the cytosol or mitochondrion of Trypanosoma brucei. We show that the novel Tpx-roGFP2 is a superior probe for the trypanothione redox couple and that the mitochondrial matrix harbors a trypanothione system. Inhibition of trypanothione biosynthesis by the anti-trypanosomal drug Eflornithine impairs the ability of the cytosol and mitochondrion to cope with exogenous oxidative stresses, indicating a direct link between both thiol systems. Tpx depletion abolishes the cytosolic, but only partially affects the mitochondrial sensor response to H2O2. This strongly suggests that the mitochondrion harbors some Tpx and, another, as yet unidentified, oxidoreductase.
... Indeed, it would seem extraordinary for the passive diffusion of a charged mole- cule such as an amino acid analogue to equilibrate to intra- cellular concentrations at millimolar levels by passive diffusion alone, even after 1 h. Two groups have independently reported reduced rates of DFMO uptake in resistant procyclic T. b. brucei lines ( Bellofatto et al., 1987;. Uptake in one of these lines appeared to be equilibrative, and the internal DFMO concentration was ultimately identical to the external concentration ( Bellofatto et al., 1987). ...
... Two groups have independently reported reduced rates of DFMO uptake in resistant procyclic T. b. brucei lines ( Bellofatto et al., 1987;. Uptake in one of these lines appeared to be equilibrative, and the internal DFMO concentration was ultimately identical to the external concentration ( Bellofatto et al., 1987). This group concluded that DFMO resistance was not linked to the reduced rate of DFMO uptake, as intracellular concentra- tions of up to 4 mM were measured in the resistant line R5 ± over 30 times the K i for ODC (130 mM) ( Bitonti et al., 1985). ...
... A putative facilitative diffu- sion carrier with the reported K m for DFMO would play a major role in the ef®cient uptake of this drug, whereas the fact that DFMO may diffuse into the trypanosome at 10 mM is of limited therapeutic value. Mean- while, various mechanisms for DFMO resistance in different laboratory strains have been suggested ( Bellofatto et al., 1987;Iten et al., 1997;, but, as far as the author is aware, no T. b. gambiense resistance has so far been reported in the ®eld. Clearly, more research into DFMO uptake, particularly in T. b. gambiense or T. b. brucei blood- stream forms, and into the causes of DFMO resistance is required to settle these questions. ...
Sleeping sickness is an increasing problem in many parts of sub-Saharan Africa. The problems are compounded by the lack of new medication, and the increasing resistance against traditional drugs such as melarsoprol, berenil and isometamidium. Over the last few years, much progress has been made in understanding how drug action, and the development of resistance, is related to the mechanisms by which the parasite ingests the drugs. In some cases novel transporters have been identified. In other cases, transporters do not appear to be involved in drug uptake, and selectivity must lie with other parasite features, such as a specific target or activation of the drug. Lessons learned from studying the uptake of drugs currently in use may assist the design of a much needed new generation of trypanocides.
... However, studies on eflornithine resistant procyclic trypanosomes showed reduced accumulation of eflornithine [11] and uptake of eflornithine was by a saturable process typical of a transporter. Bellofatto et al [12] also found uptake of eflornithine to be temperature dependent and thus likely to be transporter mediated. Indeed as a zwitterionic, charged amino acid, eflornithine would not be expected to diffuse across membranes and transport mediated uptake would be a pre-requisite for uptake. ...
... Furthermore, the evolution of the AAT family [22] makes it impossible, currently, to define specific functionality to any of these transporters based on homology alone. Previous work with bloodstream and procyclic form trypanosomes also revealed a relative simplicity in selecting eflornithine resistance [11,12,29] . In procyclic forms reduced rates of eflornithine uptake were identified [11,12] with possible changes to other transporters for ornithine and putrescine also suggested. ...
... Previous work with bloodstream and procyclic form trypanosomes also revealed a relative simplicity in selecting eflornithine resistance [11,12,29] . In procyclic forms reduced rates of eflornithine uptake were identified [11,12] with possible changes to other transporters for ornithine and putrescine also suggested. In bloodstream forms reduction in eflornithine uptake was noted in two of six eflornithine refractory T. b. rhodesiense lines [29], but in the majority of cases no difference in eflornithine uptake was noted leading the authors to dismiss altered drug uptake as an underlying mechanism for the natural refractoriness of many strains of T. b. rhodesiense in the field [30] . ...
Human African trypanosomiasis, endemic to sub-Saharan Africa, is invariably fatal if untreated. Its causative agent is the protozoan parasite Trypanosoma brucei. Eflornithine is used as a first line treatment for human African trypanosomiasis, but there is a risk that resistance could thwart its use, even when used in combination therapy with nifurtimox. Eflornithine resistant trypanosomes were selected in vitro and subjected to biochemical and genetic analysis. The resistance phenotype was verified in vivo. Here we report the molecular basis of resistance. While the drug's target, ornithine decarboxylase, was unaltered in resistant cells and changes to levels of metabolites in the targeted polyamine pathway were not apparent, the accumulation of eflornithine was shown to be diminished in resistant lines. An amino acid transporter gene, TbAAT6 (Tb927.8.5450), was found to be deleted in two lines independently selected for resistance. Ablating expression of this gene in wildtype cells using RNA interference led to acquisition of resistance while expression of an ectopic copy of the gene introduced into the resistant deletion lines restored sensitivity, confirming the role of TbAAT6 in eflornithine action. Eflornithine resistance is easy to select through loss of a putative amino acid transporter, TbAAT6. The loss of this transporter will be easily identified in the field using a simple PCR test, enabling more appropriate chemotherapy to be administered.
... There was no difference in DFMO uptake between T. b. rhodesiense and T. b. gambiense [143]. However, several early studies showed reduced eflornithine uptake in resistant cells, which were readily produced in the laboratory [144,145]. ...
... One debate [146] with respect to the eflornithine mechanism of action was whether its uptake might be transporter-mediated [145], or by simple diffusion [143,144,147]. This debate has been definitively settled in favour of mediated uptake, as should be expected of a highly soluble, zwitterionic compound with an experimental LogP of −2.7. ...
With the incidence of sleeping sickness in decline and genuine progress being made towards the WHO goal of eliminating sleeping sickness as a major public health concern, this is a good moment to evaluate the drugs that ‘got the job done’: their development, their limitations and the resistance that the parasites developed against them. This retrospective looks back on the remarkable story of chemotherapy against trypanosomiasis, a story that goes back to the very origins and conception of chemotherapy in the first years of the 20 century and is still not finished today.
... Eflornithine acts as an irreversible suicide inhibitor of ornithine decarboxylase; hence, inhibiting the synthesis of polyamines in trypanosomes as well as in mammalian cells (Van, I & Haemers, 1989;Bellofatto et al, 1987). But trypanosomes are more sensitive to eflornithine than mammalian cells (Bellofatto et al, 1987). ...
... Eflornithine acts as an irreversible suicide inhibitor of ornithine decarboxylase; hence, inhibiting the synthesis of polyamines in trypanosomes as well as in mammalian cells (Van, I & Haemers, 1989;Bellofatto et al, 1987). But trypanosomes are more sensitive to eflornithine than mammalian cells (Bellofatto et al, 1987). Inhibition of ornithine decarboxylase leads to a decrease in the levels of putrescine, spermidine and trypanothione, which causes a general decrease in the biosynthesis of DNA, RNA and proteins (including the variant surface glycoprotein) (Fairlamb, 2003). ...
Animal trypanosomiasis is a major hinderance to the growth of livestock farming in sub-Saharan Africa. Chemotherapy using isometamidium, diminazene and ethidium bromide has been the main control method in the absence of a vaccine against this disease. The effectiveness of these few trypanocides is severely threatened by the widespread development of resistance. Therefore, an understanding of the mechanism(s) involved in the development of resistance will assist in the development of screening protocols for easy identification of resistant cases prior to treatment, and also in finding ways to reverse the resistance. We studied the mechanism of resistance to isometamidium in bloodstream forms of Trypanosoma brucei. Resistance to isometamidium in Trypanosoma brucei was found to be composed of a reduced uptake of the drug and the modification of the F1F0 ATPase complex; active drug efflux by ABC transporters was not involved in the resistance mechanism, although efflux of ISM could be observed in both wild-type and resistant lines. Expression of the transporter gene TbAT1, as well as of TbAT-E and TbAT-A, in yeast, each resulted in increased ISM uptake. In addition, the Vmax for the LAPT1 drug transport activity (Low Affinity Pentamidine Transporter) in ISM-resistant trypanosomes (clone ISMR1) was significantly reduced (P<0.05; Student’s t-test) compared to the wild type control. Also, two point mutations, namely G37A and C851A were found in the ATP synthase gamma subunit of the F1F0 ATPase complex of isometamidium-resistant trypanosomes. The resistant clones also lost their mitochondrial DNA and mitochondrial membrane potential and displayed various levels of cross-resistance to ethidium, diminazene, pentamidine and oligomycin. The C851A mutation introduced a stop codon in the open reading frame of the ATP synthase gamma gene. This mutation, when introduced into the wild type Tb427, produced resistance to isometamidium, and cross resistance to diminazene, ethidium, pentamidine and oligomycin. C851A-ATP synthase gamma proves to be a dominant mutation that allows the rapid loss of mitochondrial DNA after just three days exposure of the parasites to 20 nM ISM or ethidium bromide. Finally, following a recent genome-wide loss-of-function RNAi screen that linked TbAQP2 with pentamidine and melarsoprol cross resistance, we were able to demonstrate that TbAQP2 encodes the HAPT1 in T. brucei, thus leaving us with the LAPT1 as the only known T. b. brucei drug transporter of unknown genetic origin. We however identified specific inhibitors for this transporter (LAPT1) that will be of use in its further characterization.
... Eflornithine passive diffusion across the plasma membrane was proposed to account for the uptake of eflornithine in both bloodstream forms of T.b. [99] even if evidence from genomic studies suggests the presence in T.b. of genes encoding amino acid transporters which could carry eflornithine [100,101]. Doses beyond 100 mg/kg of eflornithine given per os fail to increase the drug level in the plasma, suggesting that the drug is accumulated by a saturable transporter [102]. ...
... Eflornithine resistance of T.b. procyclic forms has been shown to be related to a reduction of drug uptake [99,105], suggesting that resistance could be related to loss or changes of eflornithine transport into cells. ...
Human African trypanosomiasis (HAT) is a fatal if untreated fly-borne neuroinflammatory disease caused by protozoa of the species Trypanosoma brucei (T.b.). The increasing trend of HAT cases has been reversed, but according to WHO experts, new epidemics of this disease could appear. In addition, HAT is still a considerable burden for life quality and economy in 36 sub-Saharan Africa countries with 15–20 million persons at risk. Following joined initiatives of WHO and private partners, the fight against HAT was re-engaged, resulting in considerable breakthrough. We present here what is known at this day about HAT etiology and pathogenesis and the new insights in the development of accurate tools and tests for disease staging and severity monitoring in the field. Also, we elaborate herein the promising progresses made in the development of less toxic and more efficient trypanocidal drugs including the potential of medicinal plants and related alternative drug therapies.
... had a considerably higher thiol content than Trypanosoma brucei bloodstream or procyclic trypomastigote forms ( ; Bellofatto et al., 1987). At least a 4-fold excess of monobromobimane reagent was necessary for complete derivatization. ...
... Trypanothione is essential in these organisms for maintenance of intracellular thiol states and in defence against oxidative damage (see review by ). Glutathionylspermidine has been identified also in T. brucei bloodstream () and procyclic trypomastigotes (Bellofatto et al., 1987) and Leishmania (A. H. Fairlamb & J. S. Keithly, unpublished) and is an intermediate in the biosynthetic pathway to T[SHI2 (). Reduction of glutathionylspermidine disulphide does not necessarily require mediation by thiol-disulphide exchange with T[SH],, since this compound serves as an effective substrate for trypanothione reductase from either C. fasciculata (Henderson et al., 19876) or Trypanosoma cruzi (Krauth-Siege1 et al., 1987 ). ...
Levels of the polyamines spermidine and putrescine and the major intracellular thiols glutathione (GSH), glutathionylspermidine (GSH-SPD) and dihydrotrypanothione [bis-(glutathionyl)spermidine); T[SH]2] were measured by high performance liquid chromatography throughout the growth cycle of the insect trypanosomatid Crithidia fasciculata. The amount of total spermidine, putrescine and glutathione (free and conjugated to spermidine) was found to be elevated during growth. Of the total spermidine, 30 to 50% was found conjugated to glutathione during the exponential growth phase, increasing to 60 to 70% at stationary phase. T[SH]2 was the principal intracellular thiol during exponential growth (12.1 to 17.4 nmol per 10(8) cells), whereas GSH-SPD was the major thiol in stationary phase (26.2 nmol per 10(8) cells). GSH levels changed little during the growth cycle and represented a constant proportion (10 to 12%) of the total intracellular glutathione. On dilution of stationary phase cells into fresh medium, a rapid decrease in GSH-SPD levels was observed to be associated with synthesis of T[SH]2. This process reached 90% completion by 15 min, with steady state achieved by 120 min. As the total spermidine and glutathione pools did not increase during this interval, it could be calculated that this rapid redistribution of metabolites resulted in the release of 13 nmol per 10(8) cells unconjugated spermidine without de novo synthesis. This mechanism for rapidly elevating the intracellular concentration of free spermidine may be advantageous to this organism in rapidly adapting to favourable growth conditions.
... Early parasite studies could not demonstrate saturable transport of eflornithine into T. brucei brucei between the concentrations of 5 μM-10 mM. This suggests eflornithine accumulation was by passive diffusion (Bellofatto et al., 1987) and/or possibly involved ...
Human African Trypanosomiasis (HAT) is a neglected parasitic disease that continues to persist in sub-Saharan Africa. It is fatal if untreated. The first stage of the disease is associated with the presence of the parasite in the periphery and the second stage with the presence of the parasites in the CNS. The treatment of CNS stage HAT requires the drugs to cross the blood-brain barrier (BBB). Eflornithine is an amino acid analogue that is used to treat second stage HAT gambiense both alone and in combination with nifurtimox. Recent studies have identified that accumulation of eflornithine into the parasites (trypanosomes) involves the amino acid transporter ( Trypanosoma brucei AAT6). In this study we tested the hypothesis that eflornithine uses a cationic amino acid transport system to cross the BBB. We particularly focused on system y ⁺ and system B 0,+ . To do this we utilized specialist databases to compare the physicochemical characteristics of relevant molecules and an in vitro model of the BBB to explore the mechanisms of eflornithine delivery into the CNS. Our results confirmed that eflornithine is related to the endogenous amino acid, ornithine. At pH 7.4, eflornithine is predominately (92.39%) a zwitterionic (dipolar) amino acid and ornithine is predominately (99.08%) a cationic (tripolar) amino acid. In addition, the gross charge distribution at pH 7.4 of eflornithine is much smaller (+0.073) than that of ornithine (+0.99). Further results indicated that eflornithine utilized a saturable transport mechanism(s) to cross the hCMEC/D3 cell membranes and that transport was inhibited by the presence of other amino acids including ornithine. Eflornithine transport was also sodium-independent and sensitive to a y ⁺ system inhibitor, but not a B 0,+ system inhibitor. Eflornithine transport was also inhibited by pentamidine, suggestive of transport by organic cation transporters (OCT) which are expressed in this cell line. We confirmed expression of the y ⁺ system protein, CAT1, and the B 0,+ system protein, ATB 0,+ , in the hCMEC/D3 cells. We conclude that eflornithine uses the cationic amino acid transporter, system y ⁺ , and OCT to cross the BBB. This research highlights the potential of system y ⁺ to deliver drugs, including eflornithine, across the BBB to treat brain diseases.
... DFMO does not show selective toxicity towards T. brucei ODC versus the human enzyme as it is an effective inhibitor of both; the selective toxicity has been attributed to differences in intracellular turnover rates (88). DFMO resistant parasites have been generated in vitro showing a loss of DFMO uptake (62,89). Characterization of such a mutant led to the identification of the DFMO transporter, which is an amino acid transporter TbAAT6 (90). ...
Polyamines are polycationic organic amines that are required for all eukaryotic life, exemplified by the polyamine spermidine, which plays an essential role in translation. They also play more specialized roles that differ across species, and their chemical versatility has been fully exploited during the evolution of protozoan pathogens. These eukaryotic pathogens, which cause some of the most globally widespread infectious diseases, have acquired species-specific polyamine-derived metabolites with essential cellular functions and have evolved unique mechanisms that regulate their core polyamine biosynthetic pathways. Many of these parasitic species have lost enzymes and or transporters from the polyamine metabolic pathway that are found in the human host. These pathway differences have prompted drug discovery efforts to target the parasite polyamine pathways, and indeed the only clinically approved drug targeting the polyamine biosynthetic pathway is used to manage human African trypanosomiasis. This review will primarily focus on polyamine metabolism and function in Trypanosoma, Leishmania,and Plasmodium species, which are the causative agents of human African trypanosomiasis (HAT) and Chagas disease, Leishmaniasis, and malaria, respectively. Aspects of polyamine metabolism across a diverse group of protozoan pathogens will also be explored.
... 96,97 DFMO is a small water-soluble molecule whose uptake by the parasite, initially thought to occur by passive diffusion, 98 involves active and selective transport by an amino acid permease. 76,99,100 The drug exerts a cytostatic effect in the pathogen that is associated to a rapid and significant depletion of spermidine and trypanothione. [101][102][103] Aberrant methylation of proteins due to the accumulation of S-adenosylmethionine and decarboxylated S-adenosylmethionine (both metabolites of the polyamine biosynthetic pathway) has also been proposed as an additional mode of action of DFMO in T. brucei. ...
Diseases caused by several (sub)species of parasites from the genus Trypanosoma are a major concern for public and animal health. Trypanosomes have a digenetic life cycle that involves an insect host that acts as the vector for transmission to different mammals, some of which are natural reservoirs. The zoonotic nature of the disease together with the capacity of the parasites to evade the host immune response or become dormant hampers pathogen eradication and immunoprophylactic control. A handful of drugs are available for the treatment, although none of them has been specifically designed toward these pathogens. The drugs are efficacious when applied at very early stages of the infections, but display high toxicity, require a long or assisted administration, present a limited efficacy against late stages of the diseases and are prone to develop resistance.
Here we review the state-of-the-art for the drugs used to treat trypanosomiasis that causes African sleeping sickness and Chagas’ disease in humans. The state-of-the-art on drug uptake, mode of action, and resistance is here reviewed. The prospects for developing new molecules that circumvent the current challenges for treating trypanosomiasis are also discussed.
... Several experimental and therapeutic drugs inhibit various steps in the biosynthetic pathway ( Figure 2). DFMO, the first new drug to be licensed for the treatment of human African trypanosomiasis in the past 40 years, inhibits ODC, the first enzyme in polyamine biosynthesis (10,11,142,171,172), resulting in a complete loss of putrescine and in a marked decrease in spennidine and trypanothione (16,65). At the other end of the pathway, trivalent arsenical drugs sequester T[SH]2 as a dithioarsane adduct, MelT (67), which in turn is a competitive inhibitor of TR (62, 67). ...
Trypanosomatids differ from all other organisms in their ability to conjugate the sulfur-containing tripeptide, glutathione, and the polyamine, spermidine, to form trypanothione [N1,N8-bis(glutathionyl)spermidine]. Together with the NADPH-dependent flavoprotein, trypanothione reductase, the dithiol form of trypanothione provides an intracellular reducing environment in these parasites, substituting for glutathione and glutathione reductase found in the mammalian host. Trypanothione and its related enzymes are involved in defense against damage by oxidants, certain heavy metals, and possibly xenobiotics. Trypanothione and its metabolic precursor, glutathionylspermidine, are also implicated in the modulation of spermidine levels during growth. Several existing trypanocidal drugs interact with the trypanothione system, suggesting that trypanothione metabolism may be a good target for the development of new drugs. The purification and properties of three key enzymes (glutathionylspermidine synthetase, trypanothione synthetase, and trypanothione reductase) are discussed, and the catalytic mechanism, substrate-specificity, and the three-dimensional structure of trypanothione reductase are compared to that of glutathione reductase.
... Intracellular levels of a charged molecule such as eflornithine are unlikely to reach such levels by passive diffusion alone. On the contrary, in an earlier study, it was shown that eflornithine uptake in trypanosomes was indeed temperature dependent, reminiscent of transport activity (Bellofatto, Fairlamb, Henderson, & Cross, 1987). Work by our group showed that eflornithine poorly traversed the murine BBB and perhaps this was an explanation as to why such an intensive treatment regime using this is necessary-to get sufficient quantities of eflornithine into the brain to exhibit trypanocidal effects (Sanderson, Dogruel, Rodgers, Bradley, & Thomas, 2008). ...
Human African trypanosomiasis (HAT or sleeping sickness) is a potentially fatal disease caused by the parasite, Trypanosoma brucei sp. The parasites are transmitted by the bite of insect vectors belonging to the genus Glossina (tsetse flies) and display a life cycle strategy that is equally spread between human and insect hosts. T.b. gambiense is found in western and central Africa whereas, T.b. rhodesiense is found in eastern and southern Africa. The disease has two clinical stages: a blood stage after the bite of an infected tsetse fly, followed by a central nervous system (CNS) stage where the parasite penetrates the brain; causing death if left untreated. The blood-brain barrier (BBB) makes the CNS stage difficult to treat because it prevents 98% of all known compounds from entering the brain, including some anti-HAT drugs. Those that do enter the brain are toxic compounds in their own right and have serious side effects. There are only a few drugs available to treat HAT and those that do are stage specific. This review summarizes the incidence, diagnosis, and treatment of HAT and provides a close examination of the BBB transport of anti-HAT drugs and an overview of the latest drugs in development.
... These are the polyamine biosynthetic pathway for the production of Spd, the glutathione biosynthetic pathway for the production of glutathione and pentose phosphate pathway for the production of NADPH mediating the reduced trypanothione redox cycle from oxidised trypanothione disulfide. Spermine, which is a critical polyamine in mammalian cells, is not taken into account due to its negligible role in T. brucei9101112. Here we study the contribution of the polyamine biosynthetic pathway to regulation of the total trypanothione contents (the summation of both reduced and oxidised trypanothione, TSH tot for short). ...
We present the first computational kinetic model of polyamine metabolism in bloodstream-form Trypanosoma brucei, the causative agent of human African trypanosomiasis. We systematically extracted the polyamine pathway from the complete metabolic network while still maintaining the predictive capability of the pathway. The kinetic model is constructed on the basis of information gleaned from the experimental biology literature and defined as a set of ordinary differential equations. We applied Michaelis-Menten kinetics featuring regulatory factors to describe enzymatic activities that are well defined. Uncharacterised enzyme kinetics were approximated and justified with available physiological properties of the system. Optimisation-based dynamic simulations were performed to train the model with experimental data and inconsistent predictions prompted an iterative procedure of model refinement. Good agreement between simulation results and measured data reported in various experimental conditions shows that the model has good applicability in spite of there being gaps in the required data. With this kinetic model, the relative importance of the individual pathway enzymes was assessed. We observed that, at low-to-moderate levels of inhibition, enzymes catalysing reactions of de novo AdoMet (MAT) and ornithine production (OrnPt) have more efficient inhibitory effect on total trypanothione content in comparison to other enzymes in the pathway. In our model, prozyme and TSHSyn (the production catalyst of total trypanothione) were also found to exhibit potent control on total trypanothione content but only when they were strongly inhibited. Different chemotherapeutic strategies against T. brucei were investigated using this model and interruption of polyamine synthesis via joint inhibition of MAT or OrnPt together with other polyamine enzymes was identified as an optimal therapeutic strategy.
... However, passive diffusion of the polar zwitterionic eflornithine seems unlikely, as its biophysical characteristics would preclude partitioning into the lipid environment of the membrane, and even in that study temperature sensitivity of uptake indicated that passive diffusion was not likely. Two independent studies in procyclic trypanosomes selected for resistance to eflornithine actually pointed to them possessing a transporter for the drug, which was lost in the selection of resistance [41,42]. Recently, two bloodstream form lines were selected for resistance to eflornithine in vitro and both were shown to have decreased their ability to accumulate eflornithine, while changes to ornithine decarboxylase were not apparent and the cellular metabolome was not changed between wild-type and eflornithineresistant derivatives [43]. ...
Human African trypanosomiasis or 'sleeping sickness' is a neglected tropical disease caused by the parasite Trypanosoma brucei. A decade of intense international cooperation has brought the incidence to fewer than 10,000 reported cases per annum with anti-trypanosomal drugs, particularly against stage 2 disease where the CNS is involved, being central to control. Treatment failures with melarsoprol started to appear in the 1990s and their incidence has risen sharply in many foci. Loss of plasma membrane transporters involved in drug uptake, particularly the P2 aminopurine transporter and also a transporter termed the high affinity pentamidine transporter, relate to melarsoprol resistance selected in the laboratory. The same two transporters are also responsible for the uptake of the stage 1 drug pentamidine and, to varying extents, other diamidines. However, reports of treatment failures with pentamidine have been rare from the field. Eflornithine (difluoromethylornithine) has replaced melarsoprol as first-line treatment in many regions. However, a need for protracted and complicated drug dosing regimens slowed widespread implementation of eflornithine monotherapy. A combination of eflornithine with nifurtimox substantially decreases the required dose and duration of eflornithine administration and this nifurtimox-eflornithine combination therapy has enjoyed rapid implementation. Unfortunately, selection of resistance to eflornithine in the laboratory is relatively easy (through loss of an amino acid transporter believed to be involved in its uptake), as is selection of resistance to nifurtimox. The first anecdotal reports of treatment failures with eflornithine monotherapy are emerging from some foci. The possibility that parasites resistant to melarsoprol on the one hand, and eflornithine on the other, are present in the field indicates that genes capable of conferring drug resistance to both drugs are in circulation. If new drugs, that act in ways that will not render them susceptible to resistance mechanisms already in circulation do not appear soon, there is also a risk that the current downward trend in Human African trypanosomiasis prevalence will be reversed and, as has happened in the past, the disease will become resurgent, only this time in a form that resists available drugs.
... There are conflicting reports about whether transport of the drug into the cell is responsible Phillips, M. A. and Wang, C. C., 1987), with others suggesting an increased level of ornithine, which competes with DFMO for the binding site of ODC, gives rise to the resistance (Bellofatto, V. et al, 1987). ...
African trypanosomiasis is a disease caused by the Kinetoplastida parasites Trypanosoma brucei rhodesiense and T. b. gambiense. The distribution of the disease is split geographically with T. b. rhodesiense found in eastern sub-Saharan Africa and T. b. gambiense in the west of the continent. Current treatment for this fatal disease is wholly unsatisfactory with problems such as extreme toxicity, affordability and the emergence of resistance. The case for the generation of new potential chemotherapies is compelling and urgent. Phosphodiesterase (PDE) enzymes degrade the secondary signalling molecule cyclic adenosine monophosphate (cAMP) to AMP by hydrolysis, thereby modulating and regulating the signal transduction to the effector proteins. The phosphodiesterase enzymes in the PDEB family in T. brucei were shown to be essential to the host-infective bloodstream forms and validated as good drug targets using RNA-interference (Zoraghi, R. and Seebeck, T., 2002; Oberholzer, M., 2007). Prompted by these findings, two series of putative trypanosomal PDE inhibitors, from different sources, were thoroughly assessed in this project for their anti-trypanosomal activity and their intracellular effects on the trypanosome. The whole-cell in vitro efficacy for each compound, against T. brucei wildtype and the drug-resistant strain TbAT1 knockout, was established by the standard resazurin reduction assay. 25 compounds from Series 1 had EC50 values below 0.5 µM, with 7 under 100 nM and the most active having an EC50 value of 5.8 ± 3.4 nM. For the much smaller Series 2 (GJS Compounds), the most active compound was GJS-128 with an EC50 value of 79.4 ± 10.3 nM. This demonstrates that a number of compounds from both series have potent in vitro activity against trypanosomes that is better than or equal to the current chemotherapeutic compound diminazene, and some Series 1 compounds are on a par with pentamidine and melarsoprol. No major cross-resistance was displayed by the TbAT1 knockout strain to either Series 1 or the GJS series. Similarly, a panel of Series 1 compounds tested against the B48 strain (resistant to pentamidine and melaminophenyl arsenical drugs), and also against Trypanosoma equiperdum wildtype and diminazene resistant (PBR) strains, showed no major cross-resistance displayed by the other resistant strains. This suggests that there would also be little or no cross-resistance from refractory strains in the field, and also that the compounds are active against multiple Trypanosoma species. A small panel of Series 1 compounds were also tested for efficacy against trypanosomes in infected mice. 4 daily doses of 20 mg/kg bodyweight of Compound 48 significantly reduced parasitaemia by approximately 60% compared to untreated controls, however higher concentrations were not tolerated by the mice so a cure could not be demonstrated. A high-throughput method for monitoring the speed of action of test compounds on trypanosomes in real time was developed, based on the fluorescence of propidium iodide when bound with DNA. Optimisation of the protocol to 96-well plates and low cell densities provided higher resolution and accurate traces of the lysis of trypanosomes in a cell suspension compared to previously used methods, as well as a greatly increased capacity. The propidium iodide assay could also be converted to provide end-point EC50 values that were directly comparable to those established by the standard resazurin reduction assay. The majority of Series 1 compounds did not increase the intracellular concentration of cAMP on incubation with bloodstream form trypanosomes; those that did only induced a minor elevation of the intracellular concentration of the signalling molecule. Since genetic disruption to phosphodiesterase enzymes resulted in large increases in cAMP levels (Oberholzer, M. et al, 2007; Zoraghi, R. and Seebeck, T., 2002), the lack of increase in cAMP by the Series 1 compounds strongly suggest that they do not sufficiently inhibit the PDEs in live trypanosomes and kill the cells via an alternative pathway. In contrast, incubation with the GJS compounds did result in significant increases in intracellular cAMP concentration with the most active being GJS-128 recording an approximately 3-fold increase in cAMP over 3 hours at just 30 nM. The concentrations that begin to increase cAMP level are consistent with the EC50 values for trypanosomes cultured in vitro (this study), and is also in line with inhibition data of recombinant TbrPDEB enzymes (work conducted by Dr. Herrmann Tenor, ALTANA Pharma, and Prof. Thomas Seebeck, University of Bern). This gives a clear and consistent link between the cause of cAMP rise (inhibition of PDEB by GJS compounds) and the effect of that concentration increase on bloodstream form trypanosomes (cell death), demonstrating that the GJS series are inhibitors of trypanosomal PDEs and chemically validate PDEs as drug targets for potential new chemotherapies against African trypanosomiasis. The effect of PDE inhibition on the physiology of the bloodstream form trypanosomes was also investigated. Flow cytometry analysis and the assessment of DNA configuration by fluorescence microscopy after DAPI staining determined that PDE inhibition by GJS-128 resulted in a precise block of the cell cycle in cytokinesis. The replicating trypanosome synthesized and segregated its DNA into two nuclei and kinetoplasts as normal and proceeded to initiate the physical separation of mother and daughter cells. The cleavage furrow between the old and new flagella progressed normally until the point of abscission, at which point division was halted with only a small section of plasma membrane connecting the two almost separated cells. Both cells appeared viable and underwent subsequent rounds of DNA replication, segregation and attempted physical separation that was always blocked near completion. This indicates cAMP signalling plays an important role in the correct physical separation of the replicating bloodstream form trypanosomes. A trypanosome cell line resistant to GJS-128 was developed by chemical mutagenesis and continuous culture with gradually increasing, but sub-lethal concentrations of the PDE inhibitor. This cell line, termed R0.8, was >15-fold less sensitive to GJS-128 and displayed significant cross-resistance to the other GJS compounds, as well as to stable, membrane permeable cAMP analogues. The mode of resistance was investigated by comparing the cAMP profile of the R0.8 and parental wildtype strains on incubation with GJS-128. No major differences were observed suggesting that both the adenylyl cyclase and phosphodiesterase activities remained unchanged in the PDE inhibitor-resistant strain. In support of this, the sequencing of TbrPDEB1 and TbrPDEB2 in both strains, while uncovering the loss of heterozygosity in the R0.8 line, revealed no mutations that would impact on enzyme function or inhibitor binding in the resistant cell line. These data strongly suggest that the adaptation resulting in resistance to PDE inhibitors is located in the effector proteins downstream of the PDEs and adenylyl cyclases in the cAMP signalling pathway. Identifying a compound that inhibits phosphodiesterases in trypanosomes and elevates cAMP concentrations, along with the generation of a PDE inhibitor-resistant cell line will allow more detailed examination of all aspects of the cAMP signalling pathway in T. brucei and across the Kinetoplastida. Phosphodiesterases have also been demonstrated to be chemically inhibitable in trypanosomes and could prove to be the target of a new generation of chemotherapies against African trypanosomiasis.
... Simmaro, WHO, personal communication, Dr. Truc, OCEAC, personal communication, Dr. Matovu, MUK, personal communication). In vitro selection for resistance to eflornithine by passage in increased drug concentration has been demonstrated in T. brucei procyclics which were found to have a 7-fold decrease in drug uptake and increased levels of ornithine (Phillips & Wang, 1987;Bellofatto et al, 1987). In ...
A growing problem with drug resistance in Human African Trypanosomiasis has necessitated the implementation of screening programmes to monitor for its spread. This thesis describes the study of several factors that can influence the selection and propagation of drug resistance in T. brucei. Human African Trypanosomiasis (HAT) is caused by T. brucei gambiense and T. brucei rhodesiense. The few drugs used for the treatment of the disease are either toxic, cause severe side effects or suffer from parasite resistance. The T. brucei P2 transporter, which is encoded by the gene TbAT1, mediates uptake of melaminophenyl arsenicals and diamidines. Reduced P2 uptake is associated with drug resistance. A number of point mutations found in a laboratory derived melarsoprol resistant T. brucei stock (STIB 777R) allowed development of a PCR/RFLP based molecular method to identify resistance alleles. By 1999, 20-30% of patients treated in Omugo, NW Uganda were failing to respond to melarsoprol. PCR/RFLP analysis indicated that mutant alleles accounted for 58.5% of those in circulation. Melarsoprol was withdrawn in 2001 and by 2003 mutant TbAT1 alleles accounted for only 14% of those in circulation in NW Uganda. The current study aimed to determine the incidence of the PCR/Sfa NI TbAT1 mutant alleles in 2006, some five years after melarsoprol had been withdrawn as first-line treatment. Successful molecular analysis of 91 of 132 (68.9%) T. b. gambiense field isolates from Omugo and Moyo in NW Uganda indicated the presence of only TbAT1 wild type alleles. Mutant alleles thus appear to have disappeared. This may be the result of parasite fitness cost following the withdrawal of melarsoprol as a stage II first-line drug from Omugo health centre, Arua, since 2001. This apparent instability of TbAT1 mutants in the field may be exploited for rational or alternating use of melarsoprol and eflornithine (DFMO) to ensure a longer life for eflornithine, delaying the onset of resistance. Insight into the overall population structure of the T. b. gambiense from Omugo, Arua (N=54) and Moyo (N=17) was obtained using mini/microsatellite marker analysis. Genetic diversity was observed to be more intra than inter regional. Multilocus genotype data analysis revealed the Omugo, Arua, population was genetically distinct from the Moyo population (Nei’s genetic distance=0.176). The evidence indicated surprisingly little genetic exchange with an excess in homozygosity (Fis >0) and alleles in linkage disequilibrium (P<0.05) within the Omugo, trypanosome population. This excess in homozygosity may be due to population sub-structuring, trypanosome inbreeding, or migration of patients. The latter is likely occurring from the neighbouring T. b. gambiense endemic disease focus in Southern Sudan. The findings suggested that the T. b. gambiense from Arua is not panmictic, clonal or epidemic but there is some level of genetic exchange. The possibility that T. b. gambiense can infect animals raises the prospect that wild or domestic animals may act as a reservoir and that a veterinary link to gambiense Human African Trypanosomiasis exists. Treatment of animals for babesiosis and trypanosomes with diminazene, uptake of which is mediated through TbAT1/P2 could select for P2-defective drug resistant trypanosomes, thereby threatening control of the human disease as well. Species detection by PCR for animal and human trypanosomes in dog isolates (N=190) from the tsetse fly endemic Jos Plataeu, Nigeria did not reveal T. b. gambiense, but multiple infections with T. brucei (95%), T. vivax (89%), and subspecies T. congolense forest (54%) and savannah (50%) were detected. The dogs were also infected with other parasites, including Babesia canis (22%) and Hepatozoon canis (16%). Multiple infections can make correct diagnosis difficult and the infections are likely to be missed by the less sensitive microscopy method. The trypanocidal action of the diamidine group of trypanocides, diminazene, pentamidine and furamidine (DB75) are principally mediated through the TbAT1/P2. In addition, pentamidine is taken up by two additional T. brucei transporters called High Affinity Pentamidine Transporter (HAPT1) and the Low Affinity Pentamidine Transporter (LAPT1). DB75 also has a secondary unknown route. Loss of TbAT1/P2 leads to significant resistance to DB75 and diminazene but not pentamidine. Identification of other markers of resistance is necessary to determine if other routes of drug entry do exist apart from P2 and whether these can be exploited for the delivery of new trypanocides into the trypanosomes. Adaptation of the T. brucei tbat1 knock-out cell line to higher concentrations of diminazene by in vitro selection for resistance led to loss of HAPT1. The resultant phenotype was similar to the previously characterised pentamidine resistant clone B48, but more resistant to diminazene and DB75. The adapted line was still capable of accumulating 1 µM radiolabelled diminazene suggesting both HAPT1 and LAPT1 as possible routes for diminazene uptake. Adaptation of the T. brucei tbat1 knock-out cell line to a high concentration of DB75 over the same 6 months period did not lead to increased resistance. Overall the project has confirmed an important role for tbat1/P2 in development of resistance to melarsoprol in the field. Importantly, it appears that removal of the selection pressure of melarsoprol leads to a loss of tbat1 alleles associated with resistance in a population of trypanosomes capable of genetic exchange in NW Uganda. Although evidence for a dog reservoir for T. b. gambiense in Nigeria was lacking in this study, a risk of selecting resistance in animals must remain high on any list of consideration. I have further shown that the diamidine drug, diminazene, used in veterinary medicine also appears to enter T. brucei via the HAPT1 transporter, as well as the P2 transporter. Loss of HAPT1 through selection with diminazene leads to high level pentamidine resistance, which could indicate a further risk in selection of human infectious trypanosomes also resistant to drugs like pentamidine.
... Several experimental and therapeutic drugs inhibit various steps in the biosynthetic pathway ( Figure 2). DFMO, the first new drug to be licensed for the treatment of human African trypanosomiasis in the past 40 years, inhibits ODC, the first enzyme in polyamine biosynthesis (10,11,142,171,172), resulting in a complete loss of putrescine and in a marked decrease in spennidine and trypanothione (16,65). At the other end of the pathway, trivalent arsenical drugs sequester T[SH]2 as a dithioarsane adduct, MelT (67), which in turn is a competitive inhibitor of TR (62, 67). ...
Trypanosomatids differ from all other organisms in their ability to conjugate the sulfur-containing tripeptide, glutathione, and the polyamine, spermidine, to form trypanothione [N1,N8-bis(glutathionyl)spermidine]. Together with the NADPH-dependent flavoprotein, trypanothione reductase, the dithiol form of trypanothione provides an intracellular reducing environment in these parasites, substituting for glutathione and glutathione reductase found in the mammalian host. Trypanothione and its related enzymes are involved in defense against damage by oxidants, certain heavy metals, and possibly xenobiotics. Trypanothione and its metabolic precursor, glutathionylspermidine, are also implicated in the modulation of spermidine levels during growth. Several existing trypanocidal drugs interact with the trypanothione system, suggesting that trypanothione metabolism may be a good target for the development of new drugs. The purification and properties of three key enzymes (glutathionylspermidine synthetase, trypanothione synthetase, and trypanothione reductase) are discussed, and the catalytic mechanism, substrate-specificity, and the three-dimensional structure of trypanothione reductase are compared to that of glutathione reductase.
Onchocerciasis, also called river blindness, is a neglected tropical disease which is in desperate need of a therapeutic revolution. This debilitating disease, endemic in 31 countries in sub-Saharan Africa, Yemen, and Latin America, is a leading cause of blindness in the developing world. The infection is caused by the filarial parasitic nematode Onchocerca volvulus that is transmitted to humans by the black fly Simulium spp. Its pathology, whose symptoms are onchodermatitis, musculoskeletal pain, and various stages of blindness, is a result of the death of the microfilariae in the skin and eyes. This review covers the drug design and early detection fields of this pathology and pays particular attention to the period after the introduction of ivermectin, which is the only drug available for mass treatment. The emergence of ivermectin resistance justifies the crucial need to identify new drug targets and agents that can effectively treat onchocerciasis.
Leishmaniasis is causing a major health problem worldwide and only five to six drugs are available for its control. Among them, antimonials, amphotericin, and miltefosine have been introduced starting in the 1940s and have proved to be effective for many years. However, the appearance of resistance threatens the success of anti-Leishmania treatment and spontaneous resistance to classical first- and second-line anti-Leishmania therapy reveals the major need to develop novel and less toxic drugs. A deep understanding of the mechanisms of drug resistance may help in the design of novel anti-Leishmania agents to be exploited in combination or as second-line therapy. Moreover, the discovery and validation of novel and druggable targets represent a useful tool to tackle drug resistance in leishmaniasis. Some biological processes such as redox metabolism, folate biosynthesis, protein synthesis, and turnover are potentially suitable to develop novel effective agents since they involve proteins showing a unique biological profile with respect to the human metabolism. Phenotypic screening and target-based drug discovery are promising tools to be exploited in drug discovery and development for leishmaniasis. Herein, we outline the most recent efforts in the identification of novel chemical scaffolds using both drug discovery strategies. We finally discuss the breakthroughs in the identification and validation of new biological targets, including Leishmania infantum initiation factor 4A, Leishmania casein kinase 1.2 (LmCK1.2), and methionyl-tRNA synthetase in Leishmania parasites. Different medicinal chemistry teams are currently exploring these targets aiming to pick out compounds displaying an improved profile to reinforce the drug pipeline.
Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.
Over a century has passed since the causative organism of cutaneous leishmaniasis was discovered. The history of trypanothione is considerably shorter than that, and, following the initial observations in 1982, our knowledge of trypanothione has increased rapidly. Thus, reviews of this unusual spermidine-containing peptide need to be frequently updated. The purpose of this article is two-fold: to revise an earlier review in the NATO-ASI series (Fairlamb and Henderson, 1987); and to highlight specific areas of the biochemistry of trypanothione that may be exploited for chemotherapeutic intervention against leishmaniasis. Most of our present knowledge has been obtained from studies on other trypanosomatids, but preliminary studies on leishmania suggests that these findings are also applicable to this group of organisms.
Development of eggs after a blood meal in the yellow fever mosquito Aedes aegypti involves hormonal changes, synthesis of nucleic acids, activation of the digestive enzyme trypsin, and production of the yolk protein vitellogenin. Polyamines have been implicated in growth processes and were here examined for possible involvement during egg development. The data suggest that polyamines are important for normal vitellogenesis in the mosquito. Polyamine levels and activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, key enzymes in the polyamine pathway, were determined in the fat body for two days after a blood meal. During the time that the macromolecules required for vitellogenesis were being synthesized, polyamine levels increased as did the activities of their rate-limiting enzymes. Administration of suicide inhibitors of ornithine decarboxylase, α-difluoromethylornithine (DFMO) and α-monofluoromethyldehydroornithine methylester (MDME), limited increased polyamine levels and disrupted macromolecular syntheses, particularly during the first twenty-four hours after blood feeding. Specific metabolic processes reduced by DFMO included trypsin activity, and production of RNA, DNA and vitellogenin. MDME had differential effects on transcription of some mRNA species made after an oogenic meal. The level of actin mRNA was not affected by inhibiting polyamine synthesis, but the mRNA levels of vitellogenin, trypsin, and the vitelline membrane protein were decreased. Adding polyamines to a meal containing DFMO or MDME partially reversed the effects of these inhibitors. Increases in spermidine and spermine were associated with these reversals.
In the present article, we summarize our studies of antrycide-resistance of Trypanosoma brucei evansi in four aspects in the last recent several years, the analysis of quinapyramine-sensitive situation of T. b. evansi in China, biological characteristics of T. b. evansi population in quinapyramine-resistance and biological materials of quinapyramine-resistance in T. b. evansi population. Firstly, the correlative assays of effective dosage of quinapyramine on T. b. evansi disease between in vivo and in vitro methods showed that their relationship was parabolic with positive correlation. On the other hand, the IC(50) and CD(100) values of 12 T. b. evansi isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB, GDH, GXM, HBM and XJCA, collected from buffaloes, horses, mules and camels across nine provinces of China were examined using the two methods, respectively. Among them, the nine isolates, AHB, GDB1, GDB2, HNB, JSB1, JSB2, YNB, ZJB and GDH, became quinapyramine-sensitive T. b. evansi. Secondly, T. evansi populations could rapidly obtain antrycide-resistance when they were passed through immunosuppressed mice treated with low doses of the drug. But, the replication rate of trypanosomes with antrycide-resistance decreases as the level of drug-resistance increases. Thirdly, the analysis of the HK, G6PDH, ALAT and ASAT isoenzymes showed that they were not involved in the quinapyramine-resistance of T. b. evansi. But the protein bands of 15.79kDa and 19.76kDa might be involved in the antrycide-resistance of T. b. evansi population. At genetic level, the gene, TbTA1, could be amplified from the T. b. evansi isolate sensitive to quinapyramine-sensitivity but the T. b. evansi isolate with quinapyramine-resistance using not only the RT-PCR technique, but also PCR technique. We used the SSH (Suppression Subtractive Hybridization) to clone highly or low expressed cDNA fragments caused by production of antrycide-resistance in T. b. evansi. The 5 low and 9 high expressed new cDNA fragments were amplified. Among them, the 3 low expressed cDNA fragments had the same sequence of 65 amino acids and the 3 high expressed cDNA fragments were located in chromosome VI, like T. brucei. Lastly, more work needs to be done in order to elucidate the mechanism of quinapyramine-resistance of T. b. evansi.
The protozoan parasites Trypanosoma brucei and Trypanosoma cruzi are the causative agents of African trypanosomiasis and Chagas disease, respectively. These are debilitating infections that exert a considerable health burden on some of the poorest people on the planet. Treatment of trypanosome infections is dependent on a small number of drugs that have limited efficacy and can cause severe side effects. Here, we review the properties of these drugs and describe new findings on their modes of action and the mechanisms by which resistance can arise. We further outline how a greater understanding of parasite biology is being exploited in the search for novel chemotherapeutic agents. This effort is being facilitated by new research networks that involve academic and biotechnology/pharmaceutical organisations, supported by public-private partnerships, and are bringing a new dynamism and purpose to the search for trypanocidal agents.
The development of new drugs against Human African Trypanosomiasis is much needed due to toxicity, efficacy and availability problems with current drug treatments for this resurgent parasitic disease. Delivery of drugs into cells is an important determinant of therapeutic efficacy of drugs. An effective means of selective drug delivery is to use plasma membrane transport systems to mediate the entry of drugs into the cell. Some amino acid transporters fulfil the criteria needed for successful exploitation of nutrient transport systems for drug delivery. The Trypanosoma brucei genomic database was screened to identify the full gene repertoire of amino acid transporters. From this, candidate genes were selected and functional genetic approaches were employed to characterise candidate amino acid transporter genes. Further characterisation of TbAATP1, a RNAi cell line shown to be a transporter of small neutral amino acids (serine, glycine, cysteine, asparagine and alanine), showed a role in threonine uptake. Amino acid analogues were tested for trypanocidal activity. Of the 96 tested, two (Azaserine and Levodopa) were investigated in more detail, paying special attention to the nature of their trypanocidal action and possible route of entry through an amino acid transporter. Azaserine showed a trypanostatic action as well multiple routes of entry into the protozoan interior (as shown by inhibition of glutamine, phenylalanine and tyrosine uptake). The trypanocidal Levodopa showed entry through a tyrosine specific transporter. However, it is possible that Levodopa’s trypanocidal activity may not be as a result of the analogue itself, but secondary products of the analogue. Amino acids are important for protozoa as energy sources as well as forming pools of soluble osmolites. Amino acid usage in trypanosomes was investigated. Upregulation of proline transport and catabolism in response to reduced glucose availability was exhibited by the genome strain of T. brucei. Moreover, this metabolic shift could be mimicked by addition of GlcNAc to the medium, which blocks the hexose transporter limiting glucose entry to the cell. Systems biology approaches were initiated to investigate the undergoing metabolic changes. More specifically, mass spectrometry methodologies were employed to investigate underlying metabolite changes in procyclic form trypanosomes grown in differing medium.
A strain of Leishmania donovani has been described that is resistant to DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (OD-Case) activity, and contains 15-fold greater amounts of ODCase activity and protein than the wild type strain from which it was derived (Coons, T., Hanson, S., Bitonti, A.J., McCann, P.P., and Ullman, B. (1990) Mol. Biochem. Pharmacol. 39, 77-90). From this mutant strain, another ODCase overproducing L. donovani strain, DFMO16, was generated by virtue of its ability to proliferate under even higher concentrations of DFMO. To investigate the mechanism by which DFMO-resistant cells overexpress ODCase, the leishmanial ODCase gene was isolated by hybridization to a fragment of the L. donovani ODCase gene that was generated by the polymerase chain reaction. The nucleotide sequence of a 4.5-kilobase DNA fragment encompassed an open reading frame encoding 707 amino acids (Mr = 77,350). The leishmanial protein contained an extra approximately 200 amino acid NH2-terminal extension and lacked the COOH terminus of the mammalian ODCase. Northern blot analysis revealed two leishmanial OD-Case transcripts of 4.8 and 6.5 kilobases, both of which were amplified 10-20-fold in the DFMO16 cells. Genomic Southern blot analysis established that the augmented amount of ODCase activity and ODCase mRNA in the DFMO16 strain could be attributed to a approximately 10-20-fold amplification of the ODCase gene copy number. DFMO16 cells exhibited an unstable phenotype in that the amplification of the ODCase gene, the increased amount of ODCase transcript, the overproduction of ODCase activity, and the DFMO-resistance growth phenotype all reverted synchronously in the absence of selective pressure.
Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.
A modular method for pursuing structure-based inhibitor design in the framework of a design cycle is presented. The approach entails four stages: (1) a design pathway is defined in the three-dimensional structure of a target protein; (2) this pathway is divided into subregions; (3) complementary building blocks, also called fragments, are designed in each subregion; complementarity is defined in terms of shape, hydrophobicity, hydrogen bond properties and electrostatics; and (4) fragments from different subregions are linked into potential lead compounds. Stages (3) and (4) are qualitatively guided by force-field calculations. In addition, the designed fragments serve as entries for retrieving existing compounds from chemical databases. This linked-fragment approach has been applied in the design of potentially selective inhibitors of triosephosphate isomerase from Trypanosoma brucei, the causative agent of sleeping sickness.
A modified cystine-free minimum essential medium has been used to address the question whether cysteine is an essential growth factor for bloodstream form trypanosomes or if its reducing power is sufficient to support parasite growth in axenic culture. Bloodstream-form trypanosomes, taken either from freshly isolated infected mouse blood or from logarithmically growing axenic cultures were transferred to a medium containing 20% dialysed foetal calf serum, 10 microM bathocuproine sulphonate and 250 microM cysteine. Growth curves of these cultures have been compared to those obtained in identical cultures containing no cysteine but cystine and reducing agents (beta-mercaptoethanol, monothioglycerol), or reducing agents alone. The results clearly show that cell growth was only obtained if cysteine was either directly added to the medium or was reduced from cystine by the action of reducing agents. However, neither reducing agents alone, nor D-cysteine, supported cell growth. Since cystine is not taken up by bloodstream form trypanosomes, and methionine is a regular constituent of the medium, we conclude from our results that cysteine is an essential growth factor for Trypanosoma brucei.
Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of D,L-alpha-difluoromethylornithine (DFMO) and D,L-alpha-difluoromethylarginine (DFMA) on growth and polyamine synthesis. In a defined, polyamine-free medium growth was markedly inhibited by DFMO (94% at 50 mM; IC50 = 37 mM) and to a lesser extent by DFMA (65% at 50 mM). Addition of putrescine, but not agmatine, reverses inhibition of growth, suggesting that the site of inhibition is ornithine decarboxylase (ODC). Consistent with this conclusion, DFMO or DFMA results in a complete loss of putrescine and significant reductions in intracellular spermidine, glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione). In addition, significant concentrations of DFMO (0.8 mM) were present in DFMA-treated cells. However, in contrast to other organisms, conversion of DFMA to DFMO is probably not catalysed by arginase. Substantial ornithine decarboxylase activity (63.1 pmol min-1 mg-1; ODC) was observed in control cells, sufficient to account for polyamine synthesis during growth. In addition, a trace arginine decarboxylase (ADC) activity (1.19 pmol min-1 mg-1) was found. Evidence is presented showing that the apparent ADC activity is actually due to the concerted action of arginase (1.5 nmol min-1 mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC.
DL-alpha-Difluoromethylornithine is an enzyme-activated inhibitor of ornithine decarboxylase and an antagonist of polyamine
metabolism that has been successful in clinical trials against West African sleeping sickness caused by Trypanosoma brucei
gambiense. Its potential for use against the more virulent East African form of the disease, caused by T. brucei rhodesiense,
is not certain. We examined 14 East African clinical isolates from the Kenya Trypanosomiasis Research Institute strain bank
plus 2 established isolates for susceptibility to DL-alpha-difluoromethylornithine and to standard trypanocides. Seven of
16 strains were partially or totally refractory to DL-alpha-difluoromethylornithine in our test system. Four strains were
also refractory to arsenical drugs, and five were refractory to diamidines. The results indicate that other novel agents or
combinations of established agents may be needed for chemotherapy of East African disease.
The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.
Chemotherapy of trypanosomiasis and leishmaniasis is far from satisfactory and the discovery of new drugs has been slow. Emphasis
on the empirical approach to drug development is now shifting towards a more rational, directed approach, which employs the
modern tools of science. This is illustrated with respect to the newly discovered drug target, trypanothione, a unique metabolite
which is critical for survival of Trypanosoma and Leishmania and absent from the host.
Over the last few years a remarkable progress has been made in the understanding of parasites biochemistry, molecular biology, and immunology. This progress is especially encouraging in that emphasis on drug development is shifting from random screening towards a more rational approach. A number of peculiar aspects characteristic of parasites which are not present in other organisms and that might be exploitable for the design of specific agents have been described recently. One of these aspects is their deficiency in defense mechanisms against oxygen toxicity. Catalase is absent in many parasites. Distinct superoxide dismutases have been detected and specific inhibitors of these enzymes have been investigated. Glutathione is absent in some anaerobic protozoa. Peroxidase and reductase activities dependent on a glutathione-spermidine cofactor termed trypanothione have been detected in several trypanosomatids and apparently replace the glutathione peroxidase-glutathione reductase system of other eukaryotic cells. Free radical intermediates have been shown to be involved in the reaction of enzymes present in anaerobic protozoa. In addition, a number of antiparasitic agents have been shown to exert their actions through a free radical metabolism: nitro compounds used against trypanosomatids, anaerobic protozoa and helminths; crystal violet used in blood banks to prevent blood transmission of Chagas' disease; the antimalarial primaquine, chloroquinine, and quinhasou; and quinones active in vitro and in vivo against different parasites.
A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been
developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked
by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation
of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the
orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control
experiments. This system provides a method for defining transcriptional control elements in trypanosomes.
Throughout evolution, enzymes and their metabolites have been highly conserved. Parasites are no exception to this and differ most markedly by the absence of metabolic pathways that are present in the mammalian host. In general, parasites are metabolically lazy and rely on the metabolism of the host both for a supply of prefabricated components such as purines, fatty acids, sterols and amino acids and for the removal of end-products. Nonetheless, parasites are metabolically highly sophisticated in that (1) they retain the genetic capacity to induce many pathways, when needed, and (2) they have developed complex mechanisms for their survival in the host. Certain unique features of the metabolism of trypanosomes, leishmania, malaria and anaerobic protozoa will be discussed. This will include (1) glycolysis and electron transport with reference to the unique organelles: the glycosome and the hydrogenosome, (2) purine salvage, pyrimidine biosynthesis and folic acid metabolism and (3) polyamine and thiol metabolism with special reference to the role of the unique metabolite of trypanosomes and leishmanias, trypanothione.
Ornithine decarboxylase and trypanothione reductase are the key enzymes in polyamine and trypanothione metabolism in kinetoplastids. Using a heterologous Trypanosoma brucei brucei probe for ornithine decarboxylase and a mixed synthetic probe of 29 oligonucleotides for trypanothione reductase, we have detected the putative genes for these enzymes by Southern blot hybridization using genomic DNA of Leishmania braziliensis guyanensis MHOM/SR/80/CUMC 1. The trypanothione reductase probe was constructed both from the conserved codon usage of the redox active site for other flavin oxidoreductases over a wide evolutionary scale, and the preferred codon usage for other genes in species of Leishmania.
The past decade has seen the development of an exciting, new strategy for the chemotherapeutic treatment of infectious disease. All prokaryotic and eukaryotic cells contain polyamines and it is now recognized that these aliphatic cations are essential for cell growth and multiplication. This vital requirement has been demonstrated most effectively by the cytostatic effects of specific and potent inhibitors of polyamine metabolism. The availability of such compounds, particularly DL α-difluoromethylornithine (DFMO; eflornithine), has opened new avenues of research into the effect of polyamine inhibitors on the growth of pathogenic microorganisms. In rapid succession from studies in vitro, through experimental animals to clinical trials it has become apparent that polyamine inhibitors do indeed have considerable chemotherapeutic potential. In particular, the effect of DFMO against parasitic protozoa has provided new therapies both for long established diseases of man and animals such as those caused by various African trydanosomes and for new problems of human medicine such as AIDS-related pneumonia due to opportunist infection by Pneumocystis carinii. It is the purpose of this review to give an account of these major advances and to discuss the other effects of polyamine inhibitors in bacterial, viral and protozoal infections.
Trypanosomatids are unusual in that a considerable proportion of their intracellular spermidine and glutathione (GSH) is found as the conjugate Ni-glutathionylspermidine (GSH-SPD) and N1-, N8-bis-(glutathionyl)spermidine (T[SH]2)1. The latter compound is unique to the parasitic and free-living Trypanosomatidae and has been assigned the trivial name trypano-thione2.
The structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the trypanosomatid parasite Leishmania mexicana has been determined by X-ray crystallography. The protein crystallizes in space group P2(1)2(1)2(1) with unit cell parameters a = 99.0 A, b = 126.5 A, and c = 138.9 A. There is one 156,000 Da protein tetramer per asymmetric unit. The model of the protein with bound NAD+s and phosphates has been refined against 86% complete data from 10.0 to 2.8 A to a crystallographic Rfactor of 0.198. Density modification by noncrystallographic symmetry averaging was used during model building. The final model of the L. mexicana GAPDH tetramer shows small deviations of less than 0.5 degrees from ideal 222 molecular symmetry. The structure of L. mexicana GAPDH is very similar to that of glycosomal GAPDH from the related trypanosomatid Trypanosoma brucei. A significant structural difference between L. mexicana GAPDH and most previously determined GAPDH structures occurs in a loop region located at the active site. This unusual loop conformation in L. mexicana GAPDH occludes the inorganic phosphate binding site which has been seen in previous GAPDH structures. A new inorganic phosphate position is observed in the L. mexicana GAPDH structure. Model building studies indicate that this new anion binding site is well situated for nucleophilic attack of the inorganic phosphate on the thioester intermediate in the GAPDH-catalyzed reaction. Since crystals of L. mexicana GAPDH can be grown reproducibly and diffract much better than those of T. brucei GAPDH, L. mexicana GAPDH will be used as a basis for structure-based drug design targeted against trypanosomatid GAPDHs.
The current rapid growth in the number of known 3‐dimensional protein structures is producing a database of structures that is increasingly useful as a starting point for the development of new medically relevant molecules such as drugs, therapeutic proteins, and vaccines. This development is beautifully illustrated in the recent book, Protein structure: New approaches to disease and therapy (Perutz, 1992). There is a great and growing promise for the design of molecules for the treatment or prevention of a wide variety of diseases, an endeavor made possible by the insights derived from the structure and function of crucial proteins from pathogenic organisms and from man.
We present here 2 illustrations of structure‐based drug design. The first is the prospect of developing antitrypanosomal drugs based on crystallographic, ligand‐binding, and molecular modeling studies of glycolytic glycosomal enzymes from Trypanosomatidae. These unicellular organisms are responsible for several tropical diseases, including African and American trypanosomiases, as well as various forms of leishmaniasis. Because the target enzymes are also present in the human host, this project is a pioneering study in selective design. The second illustrative case is the prospect of designing anti‐cholera drugs based on detailed analysis of the structure of cholera toxin and the closely related Escherichia coli heat‐labile enterotoxin. Such potential drugs can be targeted either at inhibiting the toxin's receptor binding site or at blocking the toxin's intracellular catalytic activity.
Study of the Vibrio cholerae and E. coli toxins serves at the same time as an example of a general approach to structure‐based vaccine design. These toxins exhibit a remarkable ability to stimulate the mucosal immune system, and early results have suggested that this property can be maintained by engineered fusion proteins based on the native toxin structure. The challenge is thus to incorporate selected epitopes from foreign pathogens into the native framework of the toxin such that crucial features of both the epitope and the toxin are maintained. That is, the modified toxin must continue to evoke a strong mucosal immune response, and this response must be directed against an epitope conformation characteristic of the original pathogen.
Within the framework of a project aimed at rational design of drugs against diseases caused by trypanosomes and related hemoflagellate parasites, selective inhibitors of trypanosomal glycolysis were designed, synthesized, and tested. The design was based upon the crystallographically determined structures of the NAD:glyceraldehyde-3-phosphate dehydrogenase complexes of humans and Trypanosoma brucei, the causative agent of sleeping sickness. After one design cycle, using the adenosine part of the NAD cofactor as a lead, the following encouraging results were obtained: (1) a 2-methyl substitution, targeted at a small pocket near Val 36, improves inhibition of the parasite enzyme 12.5-fold; (2) an 8-(thien-2-yl) substitution, aimed at Leu 112 of the parasite enzyme, where the equivalent residue in the mammalian enzyme is Val 100, results in a 167-fold better inhibition of the trypanosomal enzyme, while the inhibition of the human enzyme is improved only 13-fold; (3) exploitation of a "selectivity cleft" created by a unique backbone conformation in the trypanosomal enzyme near the adenosine ribose yields a considerable improvement in selectivity: 2'-deoxy-2'-(3-methoxybenzamido)adenosine inhibits the human enzyme only marginally but enhances inhibition of the parasite enzyme 45-fold when compared with adenosine. The designed inhibitors are not only better inhibitors of T. brucei GAPDH but also of the enzyme from Leishmania mexicana.
Drug resistance is a significant impediment to the therapy of African sleeping sickness in humans. To evaluate molecular mechanisms that contribute to drug resistance in African trypanosomes, a clonal strain of Trypanosoma brucei gambiense, MPA10, was selected in medium containing mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (IMPDH) activity. IMPDH activity in MPA10 cells was approximately 6-fold higher than that of wild type parental cells, although the enzymes in both strains were equally sensitive to inhibition by MPA. To evaluate the mechanism of IMPDH overexpression in MPA10 cells, the gene encoding IMPDH (impdh) was isolated from a T.b. brucei library by cross-hybridization to the Leishmania donovani impdh. Sequence analysis indicated that the T. brucei IMPDH was 76% identical with the L. donovani IMPDH. The T. brucei impdh hybridized to a 2.7-kb transcript that was expressed at approximately 10-fold greater levels in the MPA10 cells, and this impdh overexpression could be ascribed to an approximately 10-fold amplification of the impdh copy number. Pulsed field gel electrophoresis revealed that the extra impdh copies in MPA10 cells were localized to an approximately 6.0-Mb chromosome that comigrated with the wild type chromosome encompassing impdh. The amplification of impdh could be ascribed to an increased copy number of this 6.0-Mb chromosome, and a predicted augmented DNA content in MPA10 cells was confirmed by flow cytometry. This is the first demonstration that DNA amplification can serve as a molecular mechanism by which T. brucei become resistant to cytotoxic drugs, and the amplification of the 6.0-Mb chromosome represents a novel mechanism of drug resistance in parasitic protozoa.
Trypanosoma brucei glycosomes (microbodies containing nine enzymes involved in glycolysis) have been purified to near homogeneity from bloodstream-form trypomastigotes for the purpose of morphologic and biochemical analysis. Differential centrifugation followed by two isopycnic centrifugations in an isotonic Percoll and in a sucrose gradient, respectively, resulted in 12- to 13-fold purified glycosomes with an overall yield of 31%. These glycosomes appeared to be highly pure and contained less than 1% mitochondrial contamination as judged by morphometric and biochemical analyses. In intact cells, glycosomes displayed a remarkably homogeneous size distribution centered on an average diameter of 0.27 micron with a standard deviation of 0.03 micron. The size distribution of isolated glycosomes differed only slightly from that measured in intact cells. One T. brucei cell contained on average 230 glycosomes, representing 4.3% of the total cell volume. The glycosomes were surrounded by a single membrane and contained as phospholipids only phosphatidyl choline and phosphatidyl ethanolamine in a ratio of 2:1. The purified glycosomal fraction had a very low DNA content of 0.18 microgram/mg protein. No DNA molecules were observed that could not have been derived from contaminating mitochondrial or nuclear debris.
Contrary to previous reports in the literature, bloodstream forms of the haemoflagellate protozoan Trypanosoma brucei brucei are not deficient in their ability to metabolize hydrogen peroxide, although they either lack or only possess the normal enzymes for H2O2 detoxification, catalase (EC 1.11.1.6) and glutathione peroxidase (EC 1.11.1.9), at extremely low levels. The hydrogen peroxide which is consumed appears to be reduced by NADPH derived from glucose via the pentose phosphate pathway. This process requires the newly discovered cofactor trypanothione.
We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
We selected and characterized a series of mouse S49 cell variants that overproduce ornithine decarboxylase (ODC). Previously, we described variants that have an amplified ODC gene and produce about 500-fold more ODC than the wild-type cells of origin (L. McConlogue and P. Coffino, J. Biol. Chem. 258:12083-12086, 1983). We examined a series of independent variants that overproduce ODC to a lesser degree and found that a number of mechanisms other than gene amplification are responsible for the increased ODC activity. Variants were selected for resistance to 0.1 mM difluoromethylornithine, an inhibitor of ODC, by either a single or a multistep process. All showed increased ODC activity and increased ODC mRNA steady-state levels. The half-life of the enzyme was not increased in any of the variants. In one class of variant the increase of ODC mRNA was sufficient to account for ODC overproduction. In a second class, the rate of synthesis of ODC polypeptide per ODC mRNA was at least four- to eightfold higher than that in wild-type cells. Therefore, these variants were altered in the translatability of ODC mRNA. Southern analysis showed that gene amplification does not account for the increased ODC mRNA levels in any of the variants. In both variant and wild-type cells, ODC activity was responsive to changes in polyamine pools; activity was reduced following augmentation of pool size. This change in activity was associated with modification of the rate of synthesis and degradation of ODC but no change in the level of ODC mRNA.
Glutathione reductase from trypanosomes and leishmanias, unlike glutathione reductase from other organisms, requires an unusual
low molecular weight cofactor for activity. The cofactor was purified from the insect trypanosomatid Crithidia fasciculata
and identified as a novel glutathione-spermidine conjugate, N1,N8-bis(L-gamma-glutamyl-L-hemicystinyl-glycyl)spermidine, for
which the trivial name trypanothione is proposed. This discovery may open a new chemotherapeutic approach to trypanosomiasis
and leishmaniasis.
The selection of Chinese hamster ovary cells resistant to ornithine analogues has been extended to very high levels of the suicide inhibitor alpha-difluoromethylornithine. Stepwise selections resulted in stepwise increases in the inducible levels of ornithine decarboxylase activity. The enzyme is shown by several criteria to be normal in every respect: regulation in the cell cycle, inhibition by antizyme, absolute specific activity as determined with the help of alpha-[3H]difluoromethylornithine. The enzyme has been highly purified from such overproducing cells. The subunit molecular weight of the mammalian enzyme has been confirmed to be approximately 54,000. The location of the enzyme in two-dimensional polyacrylamide gel electrophoresis was determined, and the protein could be detected on two-dimensional gels with extracts from our best overproducers.
Leishmania tropica promastigotes that are highly resistant to methotrexate, a dihydrofolate reductase inhibitor, have been developed. Organisms resistant to 1 mM methotrexate have a 40-fold increase in dihydrofolate reductase which is associated with thymidylate synthetase, and they contain amplified regions of DNA that may be directly visualized on stained gels of restriction digests. The amplified DNA in these organisms is about 56 kilobases in length, has a copy number about 80-fold higher than that of wild-type organisms, and constitutes about 10% of the nuclear DNA. When the methotrexate-resistant L. tropica are propagated in drug-free medium, the dihydrofolate reductase-thymidylate synthetase protein and the amplified DNA decrease in a parallel fashion until they are indistinguishable from the levels in wild-type organisms. However, when these apparent revertants are again challenged with 1 mM methotrexate, enzyme overproduction and DNA amplification occur rapidly. As in mammalian cells, it appears that drug resistance in parasitic protozoa may be mediated by gene amplification.
We used mRNA from a mutant S49 mouse lymphoma cell line that produces ornithine decarboxylase (OrnDCase) as its major protein product to synthesize and clone cDNA. Plasmids containing OrnDCase cDNA were identified by hybrid selection of OrnDCase mRNA and in vitro translation. The two of these with the largest inserts together span 2.05 kilobases of cDNA. Southern blot analysis of DNA from wild-type or mutant S49 cells, cleaved with EcoRI or with BamHI, revealed multiple bands homologous to OrnD-Case cDNA, only one of which was amplified in the mutant cells. RNA transfer blot analysis showed that the major OrnD-Case mRNA in the mouse lymphoma cells is 2.0 kilobases long. A similar size mRNA was found in mouse kidney and was more abundant in the kidneys of mice treated with testosterone, an inducer of OrnDCase activity in that tissue.
The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania
enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric
unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem
repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.
A 1986 bp-long histidine decarboxylase cDNA from the human basophilic leukemia cell line, KU-812-F, was inserted into a shuttle vector carrying the isocitrate lyase gene promoter and terminator originated from Candida tropicalis. The plasmid was transfected into Saccharomyces cerevisiae host strain, which was cultivated in a medium containing sodium acetate. Histidine decarboxylase activity in the cell lysate was measured throughout the inoculation period where the maximum histidine decarboxylase activity was obtained at around 20 h, near the end of the log-phase growth. A typical preparation gave the specific activity of 210 pmol/min/mg in the crude cell lysate. This value was two and three times higher than that in the crude extract of mast cells and in mastocytoma P-815 culture cells, respectively. The enriched activity of histidine decarboxylase in the soluble fraction suggests that the yeast expression system provides the properly folded and catalytically active enzyme. Therefore, the present expression system is suitable for structural studies including X-ray crystallography, which require a large amount of enzyme.
The naturally-occurring polyamines exist in the free form, as N-acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.
SYNOPSIS. The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [14C]methionine. Putrescine and spermidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.
Intact ribonucleic acid (RNA) has been prepared from tissues rich in ribonuclease such as the rat pancreas by efficient homogenization in a 4 M solution of the potent protein denaturant guanidinium thiocyanate plus 0.1 M 2-mercaptoethanol to break protein disulfide bonds. The RNA was isolated free of protein by ethanol precipitation or by sedimentation through cesium chloride. Rat pancreas RNA obtained by these means has been used as a source for the purification of alpha-amylase messenger ribonucleic acid.
The sensitive dansyl procedure was used to detect putrescine and spermidine, but not spermine and cadaverine, in pleomorphic Trypanosoma brucei. The polyamines were synthesized in vitro from [3H]ornithine, [14C]arginine and [14C]methionine. Proline, agmatine, and citrulline, but not glutamine, glutamic or pyroglutamic acids, stimulated spermidine formation from [4C]methionine. Putrescine and sperimidine synthesis occurred rapidly from ornithine: putrescine synthesis peaked in 0.5 h, spermidine in 1 h. Trypanosoma brucei assimilated exogenous 14C-labeled putrescine, spermidine, and spermine; spermidine and spermine were taken up 5 times as rapidly as putrescine. Polyamine syntheses may therefore be a practical target for novel trypanocies.
Kinetic and chemical analysis show that the haploid genome of Leishmania donovani has between 4.6 and 6.5 × 107 Kb pairs of DNA. Cot analysis shows that the genome contains 12% rapidly reassociating DNA, 13% middle repetitive DNA with
an average reiteration frequency of 77 and 62% single copy DNA. Saturation hybridization experiments show that 0.82% of the
nuclear DNA is occupied by rRNA coding sequences. The average repetition frequency of these sequences is determined to be
166. Sedimentation velocity studies indicate the two major rRNA species have sedimentation values of 26S and 16S, respectively.
The arrangement of the rRNA genes and their spacer sequences on long strands of purified rDNA has been determined by the examination
of the structure of rRNA:DNA hybrids prepared for electron microscopy by the gene 32-ethidium bromide technique. Long DNA
strands are observed to contain several gene sets (16S + 26S). One repeat unit contains the following sequences in the order
given: (a) A l6S gene of length 2.12 Kb, (b) An internal transcribed spacer (Spl) of length 1.23 Kb, which contains a short
sequence that may code for a 5.8S rRNA (c) The 26S gene with a length of 4.31 Kb which contains an internal gap region of
length 0.581 Kb, (d) An external spacer of average length 5.85 Kb.
Both DL-α-Methyl orinithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (L-orinithine carboxy-lyase EC 4.1.1.17) and DL-α-difluoromethyl orinithine (α-DF MeOrn), a catalytic irreversible inhibitor of this enzyme, decrease the concentrations of putrescine and spermidine but not of spermine in rat hepatoma (HTC) cells and in mouse leukemia cells cultured . The depletion of the two amines is followed by a striking decrease in the rate of cell replication in both cell lines. Growth of human prostate adenoma cells is inhibited by α-DF MeOrn but not by α-MeOrn, illustrating the greater effectiveness of the irreversible inhibitor. These findings again support an essential function for putrescine and spermidine in cell division processes.
RNA molecular weight measurements were carried out by gel electrophoresis under four different denaturing conditions including 99% formamide, 10 mM methyl mercury, 2.2 M formaldehyde, and 6 M urea at pH 3.8. Electrophoresis at a series of gel concentrations and at least two different voltage gradients resulted in some RNA species exhibiting apparent molecular weights that vary with both gel concentration and voltage gradient. Three different deviations from the requirement for hydrodynamically equivalent conformations were observed: (1) deformation of the random coil structure of very large RNAs at moderately high gel concentrations and voltage gradients resulting, in extreme cases, in a molecular weight independent migration of RNA molecules; (2) incomplete denaturation of RNA molecules with very GC rich helical regions; and (3) varying charge/mass ratio due to differential protonation at pH 3.8. Reliable molecular weight measurements of RNA molecules as large as 4.0 × 106 containing GC rich helical regions could only be made on dilute (0.5-1.0%) agarose gels after reaction with either 2.2 M formaldehyde or 10 mM methyl mercury hydroxide. A theoretical justification for the use of the empirical log molecular weight-mobility relation is presented. It is also demonstrated that the gel electrophoretic behavior of a homologous series of random coils can be approximated by that of a series of spheres with radii proportional to the square root of radius of gyration of a random coil. Consequently, molecular weight determinations of denatured RNAs, especially those obtained by extrapolation, are more reliable if the square root of the molecular weight is plotted vs. log mobility.
This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
γ-Aminobutyric acid-α-ketoglutarate transaminase from is irreversibly inhibited by 4-aminohex-5-yhoic acid, a new structural analog of GABA. The fact that this inhibition requires the pyridoxal form of the holoenzyme, and the formation of a Michaelis complex is in support of a catalytic mechanism. The compound is also active and on the same enzyme from mammalian brain.
Stepwise increments of the concentration of 2-difluoromethylornithine, a mechanism-based irreversible inhibitor of mammalian ornithine decarboxylase (EC 4.1.1.17), resulted in a selection of cultured Ehrlich ascites carcinoma cells capable of growing in the presence of up to 50 mM difluoromethylornithine. Dialyzed extracts of drug-resistant tumor cells exhibited a very high ornithine decarboxylase activity and contained large excess of immunoreactive ornithine decarboxylase protein. Hybridization analyses with cloned complementary DNA revealed that the difluoromethylornithine-resistant tumor cells also expressed mRNA of the enzyme at greatly enhanced rate. The overproduction of ornithine decarboxylase by the tumor cells grown under the pressure of difluoromethylornithine was at least partly attributable to a 10 to 20-fold increase in the total gene dosage of ornithine decarboxylase involving an amplification of several genes of the gene family. The gene amplification developed appeared to be stable, as the gene dosage only slowly (during a period of several months) returned towards the normal level upon the removal of difluoromethylornithine. The overproduction of ornithine decarboxylase was accompanied by an enhanced resistance of the enzyme towards difluoromethylornithine in vitro.
Treatment of the chemically transformed fibroblasts BP-A31 and other cell lines with low concentrations of cycloheximide (CHM) for 72 h followed by the removal of the protein synthesis inhibitor leads to the proliferation of alpha-difluoromethylornithine (DFMO)-resistant phenotypes. These drug-resistant cells contain almost no ornithine decarboxylase (ODC) activity and concomitantly very low levels of putrescine and spermidine. Southern blot analysis and measurements of ODC activity and intracellular polyamine levels showed that the described mechanism of inducing resistance to DFMO triggered by CHM does not involve ODC gene amplification, altered transport of the drug or reduced affinity of the enzyme for DFMO.
Procyclic Trypanosoma brucei brucei strain 366D is susceptible to DL-alpha-difluoromethylornithine (DFMO) with an in vitro ED50 value of 225 microM. A mutant of the procyclic strain resistant to 20 mM of DFMO was isolated by serial in vitro passages of the organisms in increasing concentrations of the drug. Drug resistance remains unchanged after at least ten serial passages in the absence of DFMO. The mutant contains the same level of ornithine decarboxylase activity as the wild-type procyclic, and the mutant enzyme exhibits a similar susceptibility toward DFMO as the wild type. Neither the rate of decarboxylation of ornithine, nor the membrane potential in the mutant cell is changed. The only observed change in the mutant is its significantly decreased uptake of DFMO which reaches a saturating level of 18 microM inside the cells; a concentration seven times below the Ki value of DFMO on T. brucei ornithine decarboxylase (130 microM). Apparently, the failure of DFMO uptake in the mutant strain has provided the basis of drug resistance. The results also raise the question on whether the uptake of DFMO by T. brucei is by passive diffusion or by transporter(s) mediation. DFMO does not compete with the uptake of ornithine, arginine or putrescine, and the reverse holds also true. However, the mutant strain cultivated under DFMO for several generations has a greatly enhanced uptake of ornithine and a moderately heightened uptake of putrescine. Both are reduced to the normal level upon further propagations of the mutant strain in the absence of DFMO.
Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)
The relative stability of amplified DNA in drug-resistant Leishmania major was previously reported to be dependent on location,
that is, unstable amplified DNA was extrachromosomal and stable amplified DNA was chromosomal. Leishmanial chromosomes have
now been directly examined by means of orthogonal-field-alternation gel electrophoresis (OFAGE). The amplified DNA's in three
resistant cell lines displayed unusual migration and were clearly extrachromosomal, regardless of whether the amplified DNA's
were stable or unstable. Thus, contrary to conclusions from earlier studies of drug resistance in cultured animal cells, stable
amplified DNA in Leishmania can be extrachromosomal. In addition, these amplified DNA's were shown to be circular on the basis
of their resistance to exonuclease III digestion and their behavior on OFAGE. Their mobility was also greatly changed after
treatment with topoisomerase II, suggesting that the amplified DNA's were either supercoiled or concatenated circles.
Studies on the biosynthesis of trypanothione [N1,N8-bis(glutathionyl)-spermidine] in the insect trypanosomatid Crithidia fasciculata have led to the discovery of an additional sulfur-containing peptide conjugated to spermidine. Labelling studies with [3H]spermidine show that 50% of the total intracellular spermidine is incorporated into peptide conjugates, the major component being N1-glutathionylspermidine. This compound has previously been identified in Escherichia coli, as the principal low molecular weight thiol in stationary phase, but not the logarithmic phase of growth. In contrast, in C. fasciculata, this compound is present in all phases of growth. In the presence of glutathione and ATP, extracts of C. fasciculata can catalyse conversion of spermidine to N1-glutathionylspermidine and trypanothione. Both N1- and N8-regioisomers of glutathionylspermidine will replace spermidine in the reaction, suggesting they may be intermediates in the biosynthetic pathway to trypanothione. The antiprotozoal drugs berenil, pentamidine, ethidium bromide, imidocarb, methylglyoxal-bis(guanylhydrazone) and 1,3-diacetylbenzene-bis(guanylhydrazone) had no effect on the synthesis of N1-glutathionylspermidine or trypanothione in vitro.
Ornithine decarboxylase from Trypanosoma brucei brucei was inhibited by several substrate (ornithine) and product (putrescine) analogs both in vitro and in vivo. Since alpha-difluoromethylornithine is effective for the treatment of experimental and clinical African trypanosomiasis, it was possible that the more potent ornithine and putrescine analogs might be more active in treating the disease. However, only alpha-monofluoromethyldehydroornithine methyl ester was more potent than alpha-difluromethylornithine against mouse trypanosomiasis and warrants further study in model infections.
Human KB cells with increasing resistance to colchicine and other chemotherapeutic agents have been isolated in four sequential steps. This report describes the characterization of drug uptake in the parent and four mutant cell lines. Drug uptake in these cell lines occurred via a nonsaturable process. In general, drug accumulation decreased with increasing drug resistance; this relationship was seen best with colchicine, vincristine, vinblastine, and daunomycin and, to a lesser extent, with actinomycin D. The accumulation of dexamethasone, an agent to which all lines were equally sensitive, was similar for the parent and the four mutants. Drug efflux occurred rapidly, and differences among the various cell lines could be detected within the first minute. In the more resistant lines, a greater percentage of the drug was released more rapidly, although the absolute amount of drug released was less. Verapamil partially reversed the multiple drug-resistance phenotype by increasing the initial rate of uptake and accumulation of drugs in the resistant cell lines without an apparent effect on drug efflux. The results suggest that, in this human epithelial cell, the development of resistance to multiple drugs is complex, with changes in drug uptake, accumulation, and efflux.
Salivarian trypanosomes were separated from infected blood by adsorbing the particulate blood components on to DEAE-cellulose columns and eluting the trypanosomes. The optimal ionic strength of buffer was determined for a number of combinations of host-blood and parasite, and sufficient data were obtained to indicate the conditions for successful separation from untried combinations. By the technique, trypanosomes were recovered from lightly parasitemic and subpatent, as well as heavily parasitemic, infections; Trypanosoma brucei brucei alone was separated from T. vivax in a doubly infected blood.The specific adsorption-elution characteristics of the organisms with respect to DEAE-cellulose confirmed previous observations, cyclically transmitted isolations behaving similarly to those which were mechanically transmitted. The characteristics of T. simiae were unlike those of its fellow member of the subgenus Nannomonas, T. congolense, but like those of species in the subgenus Trypanozoon. Drug-resistant T. vivax tended to be more readily adsorbed than drug-sensitive T. vivax. The specific adsorption-elution characteristics of both trypanosomes and erythrocytes are interpreted in terms of their relative surface negative charge.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.
A technique for conveniently radiolabeling DNA restriction endonuclease fragments to high specific activity is described. DNA fragments are purified from agarose gels directly by ethanol precipitation and are then denatured and labeled with the large fragment of DNA polymerase I, using random oligonucleotides as primers. Over 70% of the precursor triphosphate is routinely incorporated into complementary DNA, and specific activities of over 10(9) dpm/microgram of DNA can be obtained using relatively small amounts of precursor. These "oligolabeled" DNA fragments serve as efficient probes in filter hybridization experiments.
Clinical Pharmacology and Therapeutics (1984) 35, 287–300; doi:10.1038/clpt.1984.33
Ornithine decarboxylase, the initial enzyme of polyamine biosynthesis, was induced in vitro in Leptomonas seymouri, a parasite of Diptera, by resuspending stationary phase cells with fresh medium. Induction was biphasic with peaks at 2 and 8 h. Activity increased about 20-fold over 22 h under control conditions. Induction was completely blocked by cycloheximide and was suppressed by actinomycin D, alpha-amanitin, putrescine, spermidine and spermine. The enzyme half-life was 45 min in cells treated with cycloheximide 24 h post induction. These observations suggest the presence of a highly sensitive mechanism for regulation of ornithine decarboxylase as found in mammalian and other eukaryotic cells.
During the growth of mammalian cells the level of ornithine decarboxylase ( OrnDCase ; L-ornithine carboxy-lyase, EC 4.1.1.17), the first enzyme in polyamine biosynthesis, undergoes rapid changes. As an initial step in the study of possible genetic mechanisms involved in these changes, we have isolated cDNA clones encoding OrnDCase . To obtain RNA enriched for OrnDCase messenger, mouse myeloma cells that overproduce OrnDCase were selected in the presence of the OrnDCase inhibitor, difluoromethylornithine. A pBR322 cDNA library was prepared from poly(A)+ RNA isolated from difluoromethylornithine-resistant cells, and the library was probed with [32P]cDNA representing mRNA sequences from resistant or parental (sensitive) cells. All clones hybridizing preferentially to the resistant cell probe shared nucleotide sequences. A representative clone containing 1.1 kilobases of cDNA was shown to encode OrnDCase sequences by in vitro translation of hybrid-selected mRNA followed by precipitation of the translation products with anti- OrnDCase antiserum. Using this cDNA clone as a probe, we found that mouse DNA yielded several restriction fragments that react with the OrnDCase cDNA. In the difluoromethylornithine-resistant myeloma cells, one of these DNA segments is amplified and the level of OrnDCase mRNA is greatly increased compared with that in parental plasmacytoma cells. The level of OrnDCase mRNA is also increased in cultured 3T3 cells stimulated with serum and in mouse kidneys after administration of androgen, indicating that OrnDCase gene transcription and/or mRNA stability are regulated during cell growth.
alpha-Difluoromethylornithine (RMI 71,782), a specific irreversible inhibitor of the first step in polyamine biosynthesis, that is, the formation of putrescine from ornithine by ornithine decarboxylase, cures mice infected with a virulent, rodent-passaged strain of Trypanosoma brucei brucei. This parasite is closely related to the trypanosomes that cause human sleeping sickness. The drug, which is remarkably nontoxic, was effective when administered in drinking water or by intubation. The ability of the compound to inhibit ornithine decarboxylase in vitro was demonstrated by the reduced amounts of putrescine synthesized from tritiated ornithine in Trypanosoma brucei suspensions. These observations direct attention to polyamine metabolism as a target for chemotherapy of parasitic diseases.
The role of polyamines in myoblast proliferation was studied by treating cells of Yaffe's L6 line of rat myoblasts with inhibitors of polyamine synthesis. Both an irreversible inhibitor of ornithine decarboxylase--difluoromethyl-ornithine (DFMO)--and a competitive inhibitor of S-adenosyl-methionine decarboxylase--methylglyoxal-bis(guanylhydrazone) (MGBG)--depressed spermidine levels and inhibited myoblast proliferation. Spermine levels were not significantly depressed by either inhibitor and putrescine levels were decreased only by DFMO. Putrescine and spermidine, but not magnesium, prevented inhibition of myoblast proliferation by DFMO and MGBG; determination of 14C-DFMO uptake in the presence and absence of these compounds demonstrated that they did not reduce the rate or extent of inhibitor uptake and thus prevent its inhibition of ornithine decarboxylase. Thus it seems likely that these inhibitors reduce cell proliferation by inhibiting polyamine formation. Addition of spermidine to the cells led to a substantial reduction in the activity of S-adenosyl-methionine-decarboxylase, suggesting that the enzyme is subject to negative regulation by the products of the polyamine biosynthetic pathway. Unexpectedly, addition of spermidine also increased intracellular putrescine levels; this apparently resulted from conversion of spermidine to putrescine. Addition of putrescine or spermidine in the absence of serum did not increase the rate of myoblast proliferation although it did elevate intracellular polyamine levels as expected. We conclude that some threshold level of one or more polyamines (probably spermidine) is necessary but not sufficient for initiation and maintenance of myoblast proliferation in culture.
Activity of ornithine decarboxylase, the major rate limiting enzyme of polyamine biosynthesis, was determined in bloodstream trypomastigotes of Trypanosoma brucei brucei. The enzyme required pyridoxal-5'-phosphate, dithiothreitol and EDTA for optimal activity. Several properties of the enzyme were investigated and compared to the mammalian enzyme. Most notably, the parasite enzyme was greater than 60-fold more sensitive to the inhibitor DL-alpha-difluoromethylornithine than its mammalian counterpart, thus making it an attractive target for chemotherapy.
We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture from were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1,221-246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.
Transformation ofTrypanosoma brucei bloodstream forms to procyclic forms can be stimulated in vitro by adding 2–10 mM citrate and/or cis-aconitate to the culture medium for at least 3 h. Optimal transformation was obtained in the presence of 3 mM cis-aconitate for 48 h. Citrate and cis-aconitate may activate isocitrate dehydrogenase and/or other Krebs' cycle enzymes and thus act as a metabolic trigger for the morphological changes involved in transformation.
Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.