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ABSTRACT: Pradimicin S (PRM-S) is a highly water-soluble, negatively charged derivative of the antibiotic pradimicin A (PRM-A) in which the terminal xylose moiety has been replaced by 3-sulfated glucose. PRM-S does not prevent human immunodeficiency virus (HIV) adsorption on CD4(+) T cells, but it blocks virus entry into its target cells. It inhibits a wide variety of HIV-1 laboratory strains and clinical isolates, HIV-2, and simian immunodeficiency virus (SIV) in various cell culture systems (50% and 90% effective concentrations [EC(50)s and EC(90)s] invariably in the lower micromolar range). PRM-S inhibits syncytium formation between persistently HIV-1- and SIV-infected cells and uninfected CD4(+) T lymphocytes, and prevents dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN)-mediated HIV-1 and SIV capture and subsequent virus transmission to CD4(+) T cells. Surface plasmon resonance (SPR) studies revealed that PRM-S strongly binds to gp120 in a Ca(2+)-dependent manner at an affinity constant (K(D)) in the higher nanomolar range. Its anti-HIV activity and HIV-1 gp120-binding properties can be dose-dependently reversed in the presence of an (alpha-1,2)mannose trimer. Dose-escalating exposure of HIV-1-infected cells to PRM-S eventually led to the isolation of mutant virus strains that had various deleted N-glycosylation sites in the envelope gp120 with a strong preference for the deletion of the high-mannose-type glycans. Genotypic resistance development occurred slowly, and significant phenotypic resistance occurred only after the sequential appearance of up to six mutations in gp120, pointing to a high genetic barrier of PRM-S. The antibiotic is nontoxic against a variety of cell lines, is not mitogenic, and does not induce cytokines and chemokines in peripheral blood mononuclear cells as determined by the Bio-Plex human cytokine 27-plex assay. It proved stable at high temperature and low pH. Therefore, PRM-S may qualify as a potential anti-HIV drug candidate for further (pre)clinical studies, including its microbicidal use.
Antimicrobial Agents and Chemotherapy 04/2010; 54(4):1425-35. · 4.84 Impact Factor
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ABSTRACT: Alcian Blue (AB), a phthalocyanine derivative, is able to prevent infection by a wide spectrum of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strains in various cell types [T cells, (co)receptor-transfected cells, and peripheral blood mononuclear cells]. With the exception of herpes simplex virus, AB is inactive against a broad variety of other (DNA and RNA) viruses. Time-of-addition studies show that AB prevents HIV-1 infection at the virus entry stage, exactly at the same time as carbohydrate-binding agents do. AB also efficiently prevents fusion between persistently HIV-1-infected HUT-78 cells and uninfected (CD4(+)) lymphocytes, DC-SIGN-directed HIV-1 capture, and subsequent transmission to uninfected (CD4(+)) T lymphocytes. Prolonged passaging of HIV-1 at dose-escalating concentrations of AB resulted in the selection of mutant virus strains in which several N-glycans of the HIV-1 gp120 envelope were deleted and in which positively charged amino acid mutations in both gp120 and gp41 appeared. A mutant virus strain in which four N-glycans were deleted showed a 10-fold decrease in sensitivity to the inhibitory effect of AB. These data suggest that AB is likely endowed with carbohydrate-binding properties and can be considered an important lead compound in the development of novel synthetic nonpeptidic antiviral drugs targeting the glycans of the envelope of HIV.
Antimicrobial Agents and Chemotherapy 09/2009; 53(11):4852-9. · 4.84 Impact Factor
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ABSTRACT: It was recently shown that capture of HIV-1 by DC-SIGN-expressing cells and the subsequent transmission of HIV to CD4+ T-lymphocytes can be prevented by carbohydrate-binding agents (CBAs), whereas polyanions were unable to block virus capture by DC-SIGN. In this study, we could show that a short pre-exposure of HIV-1 to both mannose- and N-acetylglucosamine (GlcNAc)-specific CBAs or polyanions dose-dependently prevented virus capture by L-SIGN-expressing 293T-REx/L-SIGN cells and subsequent syncytia formation in co-cultures of the drug-exposed HIV-1-captured 293T-REx/L-SIGN cells and uninfected C8166 CD4+ T-lymphocytes. Additionally, the inhibitory potential of the compounds against L-SIGN-mediated HIV-1 capture and transmission was more pronounced than observed for DC-SIGN expressing293T-REx/DC-SIGN cells. The excess value of CBAs and polyanions to prevent HIV-1 capture and transmission by DC-SIGN and L-SIGN-expressing cells to susceptible T-lymphocytes could be of interest for the development of new drug leads targeting HIV entry/fusion.
Antiviral research 08/2009; 83(1):61-70. · 3.61 Impact Factor
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ABSTRACT: Reduction of intramolecular disulfides in the HIV-1 envelope protein gp120 occurs after its binding to the CD4 receptor. Protein disulfide isomerase (PDI) catalyzes the disulfide reduction in vitro and inhibition of this enzyme blocks viral entry. PDI belongs to the thioredoxin protein superfamily that also includes human glutaredoxin-1 (Grx1). Grx1 is secreted from cells and the protein has also been found within the HIV-1 virion. We show that Grx1 efficiently catalyzes gp120, and CD4 disulfide reduction in vitro, even at low plasma levels of glutathione. Grx1 catalyzes the reduction of two disulfide bridges in gp120 in a similar manner as PDI. Purified anti-Grx1 antibodies were shown to inhibit the Grx1 activity in vitro and block HIV-1 replication in cultured peripheral blood mononuclear cells. Also, the polyanion PRO2000, that was previously shown to prevent HIV entry, inhibits the Grx1- and PDI-dependent reduction of gp120 disulfides. Our findings suggest that Grx1 activity is important for HIV-1 entry and that Grx1 and the gp120 intramolecular disulfides are novel pharmacological targets for rational drug development.
The international journal of biochemistry & cell biology 12/2008; 41(6):1269-75. · 4.89 Impact Factor
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ABSTRACT: Carbohydrate-binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA), but also the nonpeptidic antibiotic pradimicin A (PRM-A), inhibit entry of HIV into its target cells by binding to the glycans of gp120. Given the high sequence identity and similarity between the envelope gp120 glycoproteins of HIV and simian immunodeficiency virus (SIV), the inhibitory activity of a variety of CBAs were evaluated against HIV-1, HIV-2, and SIV. There seemed to be a close correlation for the inhibitory potential of CBAs against HIV-1, HIV-2, and SIV replication in cell culture and syncytia formation in cocultures of persistently SIV-infected HUT-78 cell cultures and uninfected CEM cells. CBAs also inhibit transmission of the SIV to T lymphocytes after capture of the virus by dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN)-expressing cells. A total of 8 different SIV strains were isolated after prolonged HHA, UDA, and PRM-A exposure in virus-infected cell cultures. Each virus isolate consistently contained at least 2 or 3 glycan deletions in its gp120 envelope and showed decreased sensitivity to the CBAs and cross-resistance toward all CBAs. Our data revealed that CBAs afford SIV and HIV-1 inhibition in a similar manner regarding prevention of virus infection, DC-SIGN-directed virus capture-related transmission, and selection of drug-resistant mutant virus strains. Therefore, SIV(mac251)-infected monkeys might represent a relevant animal model to study the efficacy of CBAs in vivo.
Molecular pharmacology 06/2008; 74(2):330-7. · 4.53 Impact Factor
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ABSTRACT: The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.
Molecular Pharmacology 10/2005; 68(3):652-9. · 4.88 Impact Factor
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Jan Balzarini, Joeri Auwerx,
Fátima Rodríguez-Barrios,
Allel Chedad,
Viktor Farkas,
Francesca Ceccherini-Silberstein,
Carlos García-Aparicio,
Sonsoles Velázquez,
Erik De Clercq,
Carlo-Federico Perno,
María-José Camarasa,
Federico Gago
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ABSTRACT: The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.
Molecular Pharmacology 08/2005; 68(1):49-60. · 4.88 Impact Factor
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Joeri Auwerx,
Joke Van Nieuwenhove,
Fátima Rodríguez-Barrios,
Sonia de Castro,
Sonsoles Velázquez,
Francesca Ceccherini-Silberstein,
Erik De Clercq,
María-José Camarasa,
Carlo-Federico Perno,
Federico Gago,
Jan Balzarini
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ABSTRACT: Amino acids N137 and P140 in the p51 subunit of HIV-1 reverse transcriptase (RT) are part of the beta7-beta8-loop that contributes to the formation of the base of the non-nucleoside RT inhibitor (NNRTI)-binding pocket and makes up a substantial part of the dimerization interface. Amino acid P95 in p66 also markedly contributes to the dimerization binding energy. Nine RT mutants at amino acid 137 were constructed bearing the mutations Y, K, T, D, A, Q, S, H or E. The prolines at amino acid positions 95 and 140 were replaced by alanine in separate enzymes. We found that all mutant RT enzymes showed a dramatically decreased RNA-dependent DNA polymerase activity. None of the mutant RT enzymes showed marked resistance against any of the clinically used NNRTIs but they surprisingly lost significant sensitivity for NRTIs such as ddGTP. The denaturation analyses of the mutant RTs by urea are suggestive for a relevant role of N137 in the stability of the RT heterodimer and support the view that the beta7-beta8 loop in p51 is a hot spot for RT dimerization and instrumental for efficient polymerase catalytic activity. Consequently, N137 and P140 in p51 and P95 in p66 should be attractive targets in the design of new structural classes of RT inhibitors aimed at compromising the optimal interaction of the beta7-beta8 loop in p51 at the p66/p51 dimerization interface.
FEBS Letters 05/2005; 579(11):2294-300. · 3.54 Impact Factor
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ABSTRACT: The phenylmethylthiazolylthiourea (PETT) derivative MSK-076 shows, besides high potency against human immunodeficiency virus type 1 (HIV-1), marked activity against HIV-2 (50% effective concentration, 0.63 microM) in cell culture. Time-of-addition experiments pointed to HIV-2 reverse transcriptase (RT) as the target of action of MSK-076. Recombinant HIV-2 RT was inhibited by MSK-076 at 23 microM. As was also found for HIV-1 RT, MSK-076 inhibited HIV-2 RT in a noncompetitive manner with respect to dGTP and poly(rC).oligo(dG) as the substrate and template-primer, respectively. MSK-076 selected for A101P and G112E mutations in HIV-2 RT and for K101E, Y181C, and G190R mutations in HIV-1 RT. The selected mutated strains of HIV-2 were fully resistant to MSK-076, and the mutant HIV-2 RT enzymes into which the A101P and/or G112E mutation was introduced by site-directed mutagenesis showed more than 50-fold resistance to MSK-076. Mapping of the resistance mutations to the HIV-2 RT structure ascertained that A101P is located at a position equivalent to the nonnucleoside RT inhibitor (NNRTI)-binding site of HIV-1 RT. G112E, however, is distal to the putative NNRTI-binding site in HIV-2 RT but close to the active site, implying a novel molecular mode of action and mechanism of resistance. Our findings have important implications for the development of new NNRTIs with pronounced activity against a wider range of lentiviruses.
Journal of Virology 08/2004; 78(14):7427-37. · 5.40 Impact Factor
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ABSTRACT: To map the determinants of the lack of susceptibility of feline immunodeficiency virus (FIV) reverse transcriptase (RT) to anti human immunodeficiency virus type 1 (HIV-1) non-nucleoside RT inhibitors (NNRTIs), a variety of chimeric HIV-1/FIV RTs were constructed. The majority of chimeric RTs had an affinity (Km) for their natural substrates comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy was decreased. Whereas HIV-1 RT could be made entirely insensitive to NNRTIs by exchanging the amino acid sequence 97 through 205 of FIV RT, none of the reverse FIV/HIV-1 RT chimeras gained susceptibility to NNRTIs. The amino acids that are thought to be involved in NNRTI susceptibility and that are different from those in HIV-1 RT have also been introduced in FIV RT. These mutant RTs gained virtually no susceptibility to efavirenz or capravirine. Vice versa, when these HIV-1-specific amino acids were replaced by their FIV RT counterparts in HIV-1 RT, susceptibility to the NNRTIs was lost. Thus, replacing segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive toward NNRTIs and was often accompanied by a decrease or even total loss of polymerase activity. It is postulated that, in contrast to the results found for HIV-1/HIV-2 RT chimeras and supported by the crystal structure of HIV-2 RT, there exist significant differences in the structure and/or flexibility of FIV RTs that may prevent NNRTIs from interacting with the FIV RT.
Molecular Pharmacology 02/2004; 65(1):244-51. · 4.88 Impact Factor
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ABSTRACT: Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are specific for human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and do not inhibit HIV-2. Given that the amino acids lining the NNRTI-specific pocket of HIV-1 RT display higher similarity to the corresponding feline immunodeficiency virus (FIV) RT amino acids than to HIV-2 RT, the susceptibility of FIV RT and chimeric HIV-1/FIV RTs to NNRTIs and the role of the p51 subunit in the inhibitory action of NNRTIs were investigated. We found that the wild-type FIV RT and the FIVp66/HIVp51 chimeric enzyme showed no susceptibility for NNRTIs. On the other hand, the chimeric HIVp66/FIVp51 RT retained a sensitivity spectrum for NNRTIs similar to that of the wild-type HIV-1 RT. The noncompetitive nature of inhibition of HIV-1 RT by nevirapine was also observed with the HIVp66/FIVp51 chimeric enzyme. Inhibition of the chimeric RTs by nucleoside reverse transcriptase inhibitors and foscarnet was in the same range as observed for the corresponding HIVp66/HIVp51 and FIVp66/FIVp51 wild-type enzymes. The chimeric RTs had an affinity (K(m)) for their dNTP substrate and template/primer comparable with that of the wild-type HIV-1 and FIV RTs, but their catalytic efficacy (k(cat)) was markedly decreased. This decreased catalytic efficacy of the RT chimeras may suggest suboptimal interactions between p66 and p51 in the chimeric enzymes. Our results point to a minor role of the p51 subunit in the sensitivity to RT inhibitors.
Molecular Pharmacology 03/2002; 61(2):400-6. · 4.88 Impact Factor
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Joeri Auwerx
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ABSTRACT: Niet-nucleoside reverse transcriptase inhibitoren (NNRTIs) interageren specifiek met het reverse transcriptase (RT) van het humaan immunodeficiëntie virus type 1 (HIV-1). NNRTIs zijn erg actieve inhibitoren van HIV-1 met in het algemeen een lage toxiciteit en ze vertonen geen activiteit tegen andere lentivirussen. NNRTIs binden aan een specifieke plaats in het RT die dicht bij de actieve substraat DNA polymerase site ligt. Uitgebreid gebruik van NNRTIs in anti-HIV chemotherapie veroorzaakteen snelle opkomst van NNRTI-resistente virusstammen. In deze studie concentreerden we ons op de NNRTI-interactie met lentivirale RTs die gecodeerd worden door HIV-1, HIV-2 en het katten (feline) immunodeficiëntie virus (FIV).
Onze eerste doelstelling was de constructie van een FIV RT met hoge gevoeligheid voor NNRTIs. Zulk RT kan later gebruikt worden voor de constructie van hybride RT-FHIVs ter evaluatie van nieuwe NNRTIs in katten. Vervolgens bestudeerden we de moleculaire interacties en resistentiepatronen van MSK-076, een fenylmethylthiazolylthiourea (PETT) NNRTI derivaat welk, naast anti-HIV-1 activiteit, ook een uitzonderlijke activiteit tegenover HIV-2vertoont. Deze studies hebben mogelijk belangrijke gevolgen voor de ontwikkeling van nieuwe NNRTIs met activiteit tegen een ruimere verscheidenheid aan lentivirussen, inclusief HIV-2. Ten derde trachtten we aminozuren in de NNRTI-specifieke bindingsplaats op te sporen die kunnen interageren met NNRTIs en welke de binding van NNRTIs aan het RT en het profiel van NNRTIs ten opzichte van resistentie kunnen verbeteren. De interactiepunten waarop we ons toespitsten zijn gelegen aan de basis van de HIV-1 NNRTI-bindingsplaats en maken ook deel uit van de interfase van het p66/p51 heterodimeer. Op deze manier pogen we bij te dragen tot een beter begrip van de rol en de interacties van welbepaalde aminozuren in de NNRTI-bindingsplaats. Deze inzichten kunnen leiden tot een rationeler design van nieuwe NNRTIs en/of de ontwikkeling van nieuwe types van RT dimerisatie-inhibitoren die een betere remming van resistentie ontwikkeling tegenover NNRTIs tot gevolg hebben.
Als eerste onderzochten we de moleculaire factoren die verantwoordelijk zijn voor de natuurlijke resistentie van FIV RT tegenover NNRTIs. In parallel werd een poging ondernomen om FIV RT gevoelig te maken aan NNRTIs via de rationele constructie van acht verschillende chimere HIV-1/FIV RTs. Gemodificeerde FIV RTs met hoge gevoeligheid aan NNRTIs kunnen bovendien gebruikt worden in een recombinant virus en later in een diermodel waarbij katten geïnfecteerd worden met een moleculaire FIV kloon die een NNRTI-gevoelig RT bevat. Uit onze resultaten bleek echter dat alle geconstrueerde chimere FIV/HIV-1 RTs in de FIV RT genetische achtergrond hun natuurlijke resistentie tegen NNRTIs behielden, terwijl chimere HIV-1/FIV RTs in een HIV-1 genetische achtergrond volledig ongevoelig gemaakt konden worden voor NNRTIs. Bijkomend vonden we dat de meeste van de chimere FIV/HIV-1 RTs een sterk verlaagde katalytische efficiëntie vertoonden terwijl hun affiniteit voor het natuurlijk substraat vergelijkbaar was met dat van wild type HIV-1 en FIV RTs.
Vermits de overeenkomstige aminozuren van de analoge ‘NNRTI-specifieke bindingsplaats’ in FIV RT slechts in enkele residuen verschillen, werden de mutaties Q101K, D179V en Y227F in de FIV RT genetische achtergrond geïntroduceerd. Er werd bij deze mutante RTs echter geen stijgende gevoeligheid tegenover NNRTIs waargenomen. We kunnen dus stellen dat uitwisseling van aminozuursegmenten of het vervangen van relevante aminozuren in FIV RT door hun HIV-1 RT equivalenten in FIV RT onvoldoende is om FIV RT gevoelig te maken voor NNRTIs. Zulke uitwisselingen bleken zelfs vaak samen te gaan met een daling of zelfs het totale verlies van DNA polymerase activiteit. Hierbij aansluitend hebben we ook onderzocht wat de invloed van de p51 subeenheid van FIV RT was op NNRTI-gevoeligheid van HIV-1 RT. De constructie van enzymatische hybriden met uitgewisselde subeenheden tussen HIV-1 en FIV RT onthulden dat het vervangen van de p51 subeenheid van HIV-1 RT door de p51 subeenheid van FIV RT geen enkele invloed had op de gevoeligheid van zulk hybride HIV-1/FIV RT voor de geanalyseerde NNRTIs. Vice versa , wanneer de p51 subeenheid in FIV RT vervangen werd door de p51 subeenheid van HIV-1 RT werd er geen verandering in het resistentieprofiel van FIV RT voor NNRTIs waargenomen. De geconstrueerde hybride RTs hadden een opmerkelijk lagere katalytische activiteit welke suggereert dat er in de hybride RTs suboptimale interacties plaatsvinden tussen de subeenheden van de verschillende RTs.
Sommige derivaten van de NNRTI klasse van de PETT analogen (nl. MSK-076 en PETT-2) bezitten de ongewone eigenschap om HIV-2 te inhiberen in celcultuur. Om het mechanisme te begrijpen dat hierachter schuil gaat en het doelwit van deze anti-HIV-2 activiteit te bepalen, selecteerden we resistente virusstammen onder druk van MSK-076 en voerden we studies uit om de moleculaire interacties van deze verbinding met HIV RT te onthullen. Er werden resistentiemutaties gevonden in de overeenkomstige NNRTI bindingsplaats van HIV-2 RT (nl. A101P), maar ook verwijderd van deze bindingsplaats en in nabijheid van de actieve substraatsbindingsplaats (nl. G112E). Onze studies toonden aan dat sommige verbindingen met een NNRTI-structuur, ondanks de intrinsieke ongevoeligheid van HIV-2 voor NNRTIs, kunnen interageren met HIV-2 RT via eenbindingsplaats die verschillend is van (maar erg gelijkend op) deze in HIV-1 RT waardoor de virus replicatie en de katalytische RT activiteit onderdrukt wordt.
Om bij te dragen tot de rationele ontwikkeling van nieuwe of gewijzigde NNRTIs met een beter resistentieprofiel zochten we naar aminozuren in de NNRTI bindingsplaats die na mutatie de katalytische activiteit van HIV-1 RT gevoelig verlagen of zelfs vernietigen. Meer bepaald zochten we naar interactiepunten binnen RT die zich bevinden in de beta7-beta8 lus van de p51 subeenheid van HIV-1 RT welke de basis van de NNRTI-bindingsplaats vormt en cruciaal is voor dimerisatie-interacties tussen de p66 en p51 subeenheid. Deze benadering maakt het mogelijk om een nieuwe klasse NNRTIs te ontwikkelen die eveneens kan interageren met de interfase van het heterodimeer RT, wat een nieuw doelwit kan worden voor anti-HIV therapie. We concentreerden ons voornamelijk op twee aminozuren, N136 en N137, die (i) deel van de interfase uitmaken tussen p66 en p51, (ii) zich bevinden aan de basis van de NNRTI-bindingsplaats, (iii) sterk geconserveerd zijn in lentivirale RTs en (iv) nog niet beschreven zijn als resistentie-geassocieerde aminozuren in celcultuur of in behandelde patiënten. Met behulp van plaatsgerichte mutagenese werden zeven mutante N136 (nl. A, Q, Y, K, T, S, L en D) en negen mutante N137 (nl. A, Q, Y, K, T, E, D, H en S) RTs geconstrueerd. Analyse van de katalytische activiteiten van deze mutante RTs onthulde sterk gereduceerde DNA polymerase (RT) activiteit voor alle N136 en N137 mutante RTs behalve voor N137H en N137S (welke beiden gevonden worden in behandelde patiënten), en N137A en N137Q.We toonden aan dat de sterk gereduceerde DNA polymerase activiteit van de meeste N136 en N137 mutante RTs wijst op een belangrijke functionele of structurele rol van deze aminozuren in het behoud van de heterodimere vorm van het RT. De verstoring van de interfase door mutaties ter hoogte van deze aminozuren werd bevestigd door denaturatie studies van verschillende N136 en N137 mutante RTs met behulp van urea of acetonitrile, gelchromatografie en circulair dichroïsme spectra. Wanneer echterde mutante RTs geëvalueerd werden voor hun gevoeligheid/resistentie tegenover NNRTIs behielden ze over het algemeen een sterke gevoeligheid voor deze producten. Enkel wanneer NRTIs bestudeerd werden, werd een resistentiepatroon waargenomen dat sterk correleerde met het gereduceerde niveau van katalytische RT activiteit. Onze resultaten identificeerden zowel N136 als N137 in HIV-1 RT als aminozuren met een cruciale functionele rol in het behouden van de heterodimere vorm van het RT en met een structurele rol in de vorming van de basis van de NNRTI-bindingsplaats. Wanneer deze aminozuren dan als doelwit gebruikt worden door nieuwe of geoptimaliseerde NNRTIs wordt verwacht dat de affiniteit van de NNRTIs voor het RT zal vergroten en/of de drugresistentie ontwikkeling zal worden uitgesteld door het onvermogen van deze aminozuren om te muteren zonder een serieuze daling van de katalytische activiteit van HIV-1 RT te veroorzaken. Belangrijk is ook het feit dat zulke NNRTI derivaten kunnen interageren met een nieuw potentieel doelwit (nl. dimerisatie) van het RT dat mogelijk resulteert in een ander resistentie- en kruisgevoeligheidsprofiel dan de ‘klassieke’ NNRTIs.
Een gekende NNRTI die mogelijk interacties maakt met de interfase van het HIV-1 heterodimeer is (+)-calanolide A, de meest actieve en bestudeerde verbinding onder de anti-HIV coumarines. Dit product selecteert voor de T139I mutatie die zich bevindt in de p51 subeenheid van RT, meer bepaald aan de interfase van de p66/p51 heterodimeer. (+)-Calanolide A heeft een nog onduidelijk werkingsmechanisme en maakt waarschijnlijk gebruik van de pyrofosfaat-bindingsplaats. Door het unieke karakter van (+)-calanolide A en het feit dat dit product selecteert voor mutaties van een aminozuur dat in de p66/p51 interfase gelokaliseerd is, besloten we dit ongewoon resistentie fenomeen te onderzoeken en trachtten we te verklaren waarom de T139I mutatie geselecteerd wordt onder (+)-calanolide A druk. Hiervoor construeerden we zeven HIV-1 RTs gemuteerd op aminozuurpositie 139 (i.e. A, K, Y, D, I, S en Q). We evalueerden de T139 mutante RTs voor hun DNA polymerase activiteit en onderzochten hun resistentieprofiel tegenover (+)-calanolide A en andere NNRTIs. Het mutante T139I RT bewaarde zijn katalytische activiteit goed en vertoonde daarbij ook een uitgesproken resistentie voor (+)-calanolide A. Bovendien is er maar één puntmutatie nodig om T te muteren tot I, terwijl er minimum twee puntmutaties nodig zijn om T te muteren naar Q, S of A. Laatst genoemde aminozuren vertonen eveneens een goede katalytische activiteit. Onze resultaten verklaren dus waarom T139I de meest waarschijnlijke mutatie is die verschijnt onder (+)-calanolide A druk. Deze mutatie ligt op de interfase van de p66 en p51 subeenheden maar we vonden echter dat dit aminozuur waarschijnlijk geen geschikt doelwit is voor optimalisatie van NNRTIs.
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) specifically interact with the reverse transcriptase (RT) of human immunodeficiency type 1 (HIV-1). NNRTIs are highly active inhibitors of HIV-1 with a generally low toxicity, which are as a rule completely inactive against other lentiviruses. NNRTIs bind to a specific site of the RT that is close to, but distinct from the polymerase active site. Widespread use of NNRTIs in anti-HIV chemotherapy has been hampered by the rapid emergence ofNNRTI-resistant virus strains. In this study we focused on the NNRTI-interaction with the lentiviral RTs, encoded by HIV-1, HIV-2 and feline immunodeficiency virus (FIV). Our first goal was to investigate whether an FIV RT could be constructed that is susceptible to NNRTIs and that could be therefore ultimately used as a model for evaluation of new NNRTIs in the in vivo background of a relevant cat model. Second, we studied the molecular interactions and resistance pattern of MSK-076, a member of the PETT NNRTI series that is, besides targeting HIV-1, exceptionally also active against HIV-2 RT. These studies may have important implications for the development of novel NNRTIs with activity against a wider range of lentiviruses, including HIV-2. Third, we wanted to identify new interaction points in the NNRTI-specific binding pocket that can be reached by NNRTIs and may improve the binding and resilience of NNRTIs to resistance development by HIV-1. The interaction points that we focused on are located at the bottom of the HIV-1 NNRTI-pocket and are also part of the p66/p51 heterodimer interface. In this way, we wanted to contribute to a better understanding of the NNRTI-binding pocket and interface interactions. These insights may lead to a more rational design of novel NNRTIs and/or a new type of RT dimerisation inhibitors that can lead to a better suppression of NNRTI resistance development of HIV-1.
We first investigated the molecular determinants that are responsible for the intrinsic resistance of FIV RT towards NNRTIs. In parallel, an attempt was made to sensitize FIV RT to NNRTIs by construction of eight different HIV-1/FIV RT chimeras. Modified FIV RTs with high sensitivity towards NNRTIs could eventually be used in a recombinant virus model and later in animal model studies where cats are infected with a molecular FIV clone containing this NNRTI-sensitive RT. However, all constructed HIV-1/FIV chimeric RTs in the FIV RT background retained resistance towards NNRTIs, whereas chimeric HIV-1/FIV RTs in the HIV-1 background could be made entirely insensitive to NNRTIs. In addition, we found that the majority of the chimeric FIV/HIV-1 RTs had a markedly decreased catalytic efficacy while their affinity for the natural substrates was comparable with that of the wild-type HIV-1 and FIV RTs. Since the corresponding amino acids of the putative NNRTI-specific pocket in FIV RT only differ in a few amino acids, we introduced the mutations Q101K, D179V and Y227F in FIV RT. Surprisingly no susceptibility towards NNRTIs was noticed. Thus, exchanging amino acid segments or substituting relevant amino acids in FIV RT by their HIV-1 RT counterparts did not suffice to make FIV RT sensitive towards NNRTIs. Moreover, such exchanges were often accompanied by a decrease or even total loss of DNA polymerase activity. In addition we studied the influence of the p51 subunit of FIV RT on the sensitivity of HIV-1 RT towards NNRTIs. Construction of enzymatic hybrids with exchanged subunits between HIV-1 and FIV RT revealed that replacing the p51 subunit of HIV-1 RT by that of FIV RT has no influence on the sensitivity of the hybrid HIV-1 RT to any of the analyzed NNRTIs. Vice versa , replacing the p51 subunit in FIV RT by the p51 subunit of HIV-1 RT did not alter the FIV RT resistance to NNRTIs. As a rule, the constructed hybrid RTs had a poor catalytic efficacy that may suggestive for a sub optimal interaction between the subunits of the different RTs.
Some derivatives of the NNRTI class of phenylmethylthiazolylthiourea (PETT) compounds (i.e. MSK-076 and PETT-2) possess the unusual property to inhibit HIV-2 RT. In order to understand the mechanism of inhibition and the target of anti-HIV-2 RT action we selected resistant virus strains under drug (MSK-076) pressure and we performed mechanistic studies to unravel the molecular interaction of the compound with RT. We detected a mode/site of action in HIV-2 RT that was similar to the NNRTI interaction in HIV-1 RT. Resistance mutations in the equivalent presumable NNRTI-binding region of HIV-2 RT (i.e. A101P) but also distal to the NNRTI-pocket and in close proximity of the active site of HIV-2 RT (i.e. G112E) were found. Our studies revealed that, although HIV-2 RT is, as a rule, insensitive to NNRTIs, some compounds still could interact with HIV-2 RT and inhibit the catalytic activity and virus replication at a site in HIV-2 RT that is distinct from, but closely related to the NNRTI-pocket.
To contribute to the rational design of improved NNRTIs that can cope with, or circumvent the problem of, resistance development, we searched for ‘unmutatable’ amino acids in the NNRTI binding pocket. In particular,we searched for interaction sites located in the beta7-beta8 loop of the p51 subunit of HIV-1 RT, which forms the bottom of the NNRTI binding pocket and is crucial for dimerisation interactions between p66 and p51. This approach makes it possible todevelop novel classes of NNRTIs that can also interfere with the interface of the RT heterodimer, which might become a new target for anti-HIV therapy. We focused on two amino acids, N136 and N137, which are (i) part of the interface, (ii) at the bottom of the NNRTI binding pocket, (iii) highly conserved among lentiviruses and (iv) as yet unidentified as sites for resistance mutations. Using site-directed mutagenesis, seven N136 (i.e. A, Q, Y, K, T, S, L and D) and nine N137 (i.e. A, Q, Y, K, T, E, D, H and S) mutant RTs were constructed. Kinetic analysis of the catalytic activities of these mutant RTs revealed severely impaired RNA- and DNA-polymerase activities for all N136 and N137 mutant RTs with the exception of N137H and N137S (both are found in patients) and N137A and N137Q. We provided evidence that the severely compromised polymerase activities of most of the N136 and N137 mutant RTs suggest an important functional or structural role of these amino acids in maintaining the heterodimeric form of the RT. The disturbance of the interface by mutating these amino acids was confirmed by denaturation analysis by urea and/or acetonitrile of several mutant N136 and N137 RTs, size exclusion chromatography and circular dichroism spectra. When the mutant RTs were evaluated for their sensitivity/resistance towards NNRTIs, they retained in general a marked sensitivity to the NNRTIs. Only when NRTIs were evaluated, a marked resistance was observed that correlated well with the level of decreased catalytic activity. Our data identified both N136 and N137 in the HIV-1 RT as amino acids with a crucial functional role in maintaining the heterodimeric form of RT and a structural function in the formation of the bottom of the NNRTI-binding pocket. These amino acids should be optimally targeted by novel or optimized NNRTIs. By achieving this goal, the affinity of the NNRTIs to HIV-1 RT would be strengthened and it would be expected that drug resistance development could be delayed due to the inability of these amino acids to mutate without seriously compromising the catalytic activity of HIV-1 RT. More importantly, such NNRTI derivatives may interact with a new potential target (i.e. dimerisation) on the RT, resulting in adifferent resistance and cross-sensitivity profile.
An existing drug that may potentially interact with the interface of the HIV-1 p66/p51 heterodimer is (+)-calanolide A, the most active and most studied compound among the anti-HIV-1 coumarines.The compound selects for a T139I mutation located in the p51 subunit at the interface of the p66/p51 heterodimer and has an unclear mode of action that includes at least the pyrophosphate binding site. Because of the unique character of (+)-calanolide A, we choose to investigate this peculiar resistance phenomenon and tried to resolve why a T139I mutation is consistently selected under (+)-calanolide A drug pressure. We therefore constructed seven RTs mutated at amino acid position 139 (i.e. A,K, Y, D, I, S and Q). We evaluated the mutant T139 RTs for their DNA polymerase activities and determined their resistance profile against (+)-calanolide A and several other NNRTIs. Since mutant T139I RT was amongst the RTs that retained most of their catalytic activity and (+)-calanolide A lost part of its activity against mutant T139I RT and against a few other mutant T139 RTs, we could explain why the T139I RT mutation is the most likely mutation to emerge under (+)-calanolide A drug pressure. This mutation in HIV-1 RT is at the interface of p66 and p51, but we found that it is probably not a good target for the design of optimised NNRTIs. Doctor in de Wetenschappen: Biologie Afdeling Virologie en chemotherapie Departement Microbiologie en immunologie Faculteit Wetenschappen Doctoral thesis Doctoraatsthesis
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ABSTRACT: Carbohydrate-binding agents (CBAs), such as the mannose-specific Hippeastrum hybrid agglutinin (HHA) and the GlcNAc-specific Urtica dioica agglutinin (UDA), frequently select for glycan deletions in all different domains of HIV-1 gp120, except in the V1/V2 domain. To reveal the underlying mechanisms, a broad variety of 31 different virus strains containing one or several N-glycan deletions in V1/V2 of the gp120 of the X4-tropic HIV-1NL4.3 were constructed by chimeric virus technology. No co-receptor switch to CCR5 was observed for any of the replication-competent mutant virus strains. With a few exceptions, the more glycans were deleted in the gp120 V1/V2 domain, the more the replication capacity of the mutant viruses became compromised. None of the mutant virus strains showed a markedly decreased sensitivity to the inhibitory activity of HHA and UDA. Instead, an up to 2- to 10-fold higher sensitivity to the inhibitory activity of these CBAs was observed. Our data may provide an explanation why glycan deletions in the gp120 V1/V2 domain rarely occur under CBA pressure and confirm the important functional role of the glycans in the HIV-1 gp120 V1/V2 domain. The gp120 V1/V2 loop glycans of HIV-1 should therefore be considered as a hot spot and novel target for specific therapeutic drug intervention.
Virology.
