Federico Focher

Istituto di Genetica Molecolare, Ticinum, Lombardy, Italy

Are you Federico Focher?

Claim your profile

Publications (101)80.65 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: Because guanine-based herpes simplex virus thymidine kinase inhibitors are not orally available, we synthesized various 6-deoxy prodrugs of these compounds and evaluated them with regard to solubility in water, oral bioavailability, and efficacy to prevent herpes simplex virus-1 reactivation from latency in a mouse model. Methods: Organic synthesis was used to prepare compounds, High Performance Liquid Chromatography (HPLC) to analyze hydrolytic conversion, Mass Spectrometry (MS) to measure oral bioavailability, and mouse latent infection and induced reactivation to evaluate the efficacy of a specific prodrug. Results: Aqueous solubilities of prodrugs were improved, oxidation of prodrugs by animal cytosols occurred in vitro, and oral absorption of the optimal prodrug sacrovir™ (6-deoxy-mCF3PG) in the presence of the aqueous adjuvant Soluplus® and conversion to active compound N(2)-[3-(trifluoromethyl)pheny])guanine (mCF3PG) were accomplished in mice. Treatment of herpes simplex virus-1 latent mice with sacrovir™ in 1% Soluplus in drinking water significantly suppressed herpes simplex virus-1 reactivation and viral genomic replication. Conclusions: Ad libitum oral delivery of sacrovir™ was effective in suppressing herpes simplex virus-1 reactivation in ocularly infected latent mice as measured by the numbers of mice shedding infectious virus at the ocular surface, numbers of trigeminal ganglia positive for infectious virus, number of corneas that had detectable infectious virus, and herpes simplex virus-1 genome copy numbers in trigeminal ganglia following reactivation. These results demonstrate the statistically significant effect of the prodrug on suppressing herpes simplex virus-1 reactivation in vivo.
    Antiviral chemistry & chemotherapy 10/2015; DOI:10.1177/2040206615608722
  • [Show abstract] [Hide abstract]
    ABSTRACT: Most nucleoside kinases, besides the catalytic domain, feature an allosteric domain which modulates their activity. Generally, non-substrate analogs, interacting with allosteric sites, represent a major opportunity for developing more selective and safer therapeutics. We recently developed a series of non-nucleoside non-competitive inhibitors of human Adenosine Kinase (hAK), based on a pyrrolobenzoxa(thia)zepinone scaffold. Based on computational analysis, we hypothesized the existence of a novel allosteric site on hAK, topographically distinct from the catalytic site. In this study, we have adopted a multidisciplinary approach including molecular modeling, biochemical studies and site-directed mutagenesis to validate our hypothesis. Based on a three-dimensional model of interaction between hAK and our molecules, we designed, cloned and expressed specific, single and double point mutants of hAK (Q74A, Q78A, H107A, K341A, F338A and Q74A-F338A). Kinetic characterization of recombinant enzymes indicated that these mutations did not affect enzyme functioning; conversely, mutated enzymes are endowed of reduced susceptibility to our non-nucleoside inhibitors, while maintaining comparable affinity for nucleoside inhibitors to the wild type enzyme. This study represents the first characterization and validation of a novel allosteric site in hAK, and may pave the way to the development of novel selective and potent non-nucleoside inhibitors of hAK endowed with therapeutic potential. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Chemical Biology &amp Drug Design 08/2015; DOI:10.1111/cbdd.12630 · 2.49 Impact Factor
  • Federico Focher ·

    Journal of Genetics 08/2014; 93(2):607-608. DOI:10.1007/s12041-014-0376-x · 1.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100 kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100 kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 raised with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.
    Biochimica et Biophysica Acta 04/2013; 1833(8). DOI:10.1016/j.bbamcr.2013.03.030 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Herpes simplex virus (HSV) types 1 and 2 thymidine kinases (TK) are responsible for phosphorylation of antiherpes acyclonucleosides such as acyclovir (ACV) and 9-(4-hydroxybutyl)guanine (HBG). Related compounds, the N(2)-phenyl-9-(hydroxyalkyl)guanines, are devoid of direct antiviral activity, but potently inhibit the viral TKs and block viral reactivation from latency in vivo. The similarity in structure between the acyclonucleosides and TK inhibitors raised the question of the relevance of phosphorylation of certain of the latter analogs in their mechanisms of action. Using recombinant TKs and HPLC analysis of reaction mixtures, we report that the lead TK inhibitor N(2)-phenyl-9 -(4-hydroxybutyl)guanine (HBPG) and its pentyl homolog (HPnPG) are excellent substrates for the enzymes, approaching the efficiency with which the natural substrate thymidine is phosphorylated, and significantly better than ACV or HBG. Other 9-hydroxyalkyl congeners are substrates for the enzymes, but with much poorer efficiency. HBPG triphosphate was a poor inhibitor of HSV DNA polymerase, the target of acyclonucleoside triphosphates, suggesting that phosphorylation of HBPG is not important in its mechanism of blocking viral reactivation in vivo. The fact that HBPG is an efficient substrate is consistent, however, with its binding mode based both on molecular modeling studies and x-ray structure of the HBPG:TK complex.
    01/2012; 1(1):21-25.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Based on the finding that aerobic Gram-positive antibacterials that inhibit DNA polymerase IIIC (pol IIIC) were potent inhibitors of the growth of anaerobic Clostridium difficile (CD) strains, we chose to clone and express the gene for pol IIIC from this organism. The properties of the recombinant enzyme are similar to those of related pol IIICs from Gram-positive aerobes, e.g. B. subtilis. Inhibitors of the CD enzyme also inhibited B. subtilis pol IIIC, and were competitive with respect to the cognate substrate 2'-deoxyguanosine 5'-triphosphate (dGTP). Significantly, several of these inhibitors of the CD pol IIIC had potent activity against the growth of CD clinical isolates in culture.
    Current Enzyme Inhibition 10/2011; 7(3):147-153. DOI:10.2174/157340811798807597
  • [Show abstract] [Hide abstract]
    ABSTRACT: We investigated some pyrrolobenzoxazepinone (PBOs, 3e-i) analogues of early described effective non-nucleoside inhibitors of HIV-1 reverse transcriptase (RT). Enzymological studies of 3e-i enantiomers, with wild type (wt) RT and some drug-resistant mutants, revealed a stereoselective mode of action and selectivity for RT ternary complex. Unexpectedly (+)-3g was found more potent towards the L100I mutant than towards the wt RT, whereas (+)-3h inhibited the K103N mutant and RT wt with comparable potency.
    Bioorganic & medicinal chemistry letters 07/2011; 21(13):3935-8. DOI:10.1016/j.bmcl.2011.05.020 · 2.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Adenosine kinase (AK) catalyzes the phosphorylation of adenosine (Ado) to AMP by means of a kinetic mechanism in which the two substrates Ado and ATP bind the enzyme in a binary and/or ternary complex, with distinct protein conformations. Most of the described inhibitors have Ado-like structural motifs and are nonselective, and some of them (e.g., the tubercidine-like ligands) are characterized by a toxic profile. We have cloned and expressed human AK (hAK) and searched for novel non-substrate-like inhibitors. Our efforts to widen the structural diversity of AK inhibitors led to the identification of novel non-nucleoside, noncompetitive allosteric modulators characterized by a unique molecular scaffold. Among the pyrrolobenzoxa(thia)zepinones (4a-qq) developed, 4a was identified as a non-nucleoside prototype hAK inhibitor. 4a has proapoptotic efficacy, slight inhibition of short-term RNA synthesis, and cytostatic activity on tumor cell lines while showing low cytotoxicity and no significant adverse effects on short-term DNA synthesis in cells.
    Journal of Medicinal Chemistry 02/2011; 54(5):1401-20. DOI:10.1021/jm101438u · 5.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Molecular modelling studies of complexes of 2-phenylamino-6-oxopurines and HSV1 thymidine kinases (TK) revealed two distinct modes of binding. The “acyclovir mode” was occupied by 9R (9-substituted) compounds, and was identical to that revealed by crystal structures of acyclovir and 2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine (HBPG) bound to HSV1 TK. The “base mode” was occupied by 9H compounds such as 2-[3-(trifluoromethyl)phenylamino]-6- oxopurine (m-CF3PG) , and is characterized by rotation of the inhibitor by 180° around the minor axis of the purine ring. In an attempt to understand the molecular basis for affinity of 2-phenylamino-6-oxopurines for TKs, we cloned and expressed site-directed HSV2 TK mutants to create proteins with inhibitor-interacting domains identical with those of HSV1 TK. The enzyme kinetic properties and inhibitory action of several 2-phenylamino-6-oxopurines showed that the changes were not consistently correlated with differences in affinity of inhibitors to the TKs.
    Letters in Drug Design &amp Discovery 12/2010; 8(1):1-8. DOI:10.2174/157018011793663877 · 0.77 Impact Factor

  • ChemInform 12/2010; 32(49). DOI:10.1002/chin.200149212
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 07/2010; 29(27). DOI:10.1002/chin.199827280

  • Evolving DNA Polymerases: Chemistry meets Biology, Monte Verita, Switzerland; 05/2010
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: 2-Phenylamino-6-oxo-9-(4-hydroxybutyl)purine (HBPG) is a thymidine kinase inhibitor that prevents encephalitic death in mice caused by herpes simplex virus (HSV) types 1 and 2, although its potency is somewhat less than that of acyclovir (ACV). The present study was undertaken to determine the effect of combinations of HBPG and either ACV, phosphonoformate (PFA), or cidofovir (CDF) against HSV encephalitis. BALB/c mice were given ocular infections with HSV-1 or HSV-2, and treated twice daily intraperitoneally for five days with HBPG, alone or in combination with ACV, PFA, or CDF. Animals were observed daily for up to 30 days, and the day of death of each was recorded. All of the combinations showed additivity, and the combination of HBPG + ACV appeared to be synergistic, ie, protected more mice against HSV-1 encephalitis compared with each drug given alone. Delay of treatment with HBPG for up to two days was still effective in preventing HSV-2 encephalitis. The combination of the thymidine kinase inhibitor HBPG and the antiherpes drug ACV may have synergistic activity against HSV encephalitis. The development of a potent and safe combination therapy for the prevention and/or treatment of HSV infection of the central nervous system can improve the outcome of this infection in humans.
    Drug Design, Development and Therapy 12/2009; 3:289-94. · 3.03 Impact Factor
  • A Lossani · A Torti · S Gatti · A Bruno · R Maserati · G E Wright · F Focher ·
    [Show abstract] [Hide abstract]
    ABSTRACT: Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0.27 microm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0.74 and 0.22 mM, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.
    Parasitology 05/2009; 136(6):595-602. DOI:10.1017/S0031182009005964 · 2.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.
    Antimicrobial Agents and Chemotherapy 07/2007; 51(6):2028-34. DOI:10.1128/AAC.01284-06 · 4.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: A human T-lymphoblastoid cell line that is resistant to the antiviral activity of zidovudine (ZDV) and moderately resistant to lamivudine (3TC) has been obtained as a result of prolonged treatment with a combination of three nucleoside analogues (NA), ZDV, 3TC, and abacavir (ABV). These cells, called CEM(ZLA), are fully sensitive to ABV. The cellular resistance of the CEM(ZLA) cells to ZDV correlates with significant reductions in thymidine kinase (TK) activity and in the amount of intracellular TK protein. Interestingly, the reduction in TK activity led to impairment of the ability of CEM(ZLA) to accumulate the triphosphate metabolite of ZDV. However, the moderately 3TC-resistant phenotype of CEM(ZLA) cannot be ascribed to a similar reduction in deoxycytidine kinase activity. Compared to the parental CEM cells, CEM(ZLA) cells express a high level of multidrug resistance protein 4 (MRP4), which could reduce the intracellular concentration of 3TC. This study shows that the exposure of cells to a combination of NAs is capable of simultaneously affecting more than one target site to confer resistance and that NAs display differing abilities to select cellular resistance mechanisms.
    AIDS Research and Human Retroviruses 11/2006; 22(10):960-7. DOI:10.1089/aid.2006.22.960 · 2.33 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A set of deazaguanine derivatives 1-3 targeting human purine nucleoside phosphorylase (hPNP) have been designed and synthesized. The new compounds are characterized by the presence of a structurally simplified "azasugar" motif to be more easily accessible by chemical synthesis than previous inhibitors. In the enzymatic assays, some of the new derivatives proved to be able to inhibit hPNP at low nanomolar concentration, thereby showing the same inhibitory potency in vitro as immucillin-H (IMH). Molecular docking experiments revealed a binding mode to hPNP essentially identical to that of IMH. As a result, the lower in vivo activity exhibited by 1d, compared with that exhibited by IMH, might be ascribed to differences in the pharmacokinetic, rather than pharmacodynamic, profile between these compounds. Derivatives 1a, 1d, and 2c emerged as the most active compounds within this new set and may represent interesting leads in the search for novel hPNP inhibitors.
    Journal of Medicinal Chemistry 11/2006; 49(20):6037-45. DOI:10.1021/jm060547+ · 5.45 Impact Factor
  • Source
    Annalisa Verri · Federico Focher · Guido Tettamanti · Vittorio Grazioli ·

    Clinical Chemistry 08/2005; 51(7):1282-4. DOI:10.1373/clinchem.2005.048124 · 7.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.
    Journal of Medicinal Chemistry 07/2005; 48(11):3919-29. DOI:10.1021/jm049059x · 5.45 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: New 5-chloro-6-substituted-uracil derivatives have been prepared by microwave assisted-synthesis and tested in vitro as thymidine phosphorylase inhibitors. One of these compounds showed potent inhibitory activity, with an IC50 value in the submicromolar range. The biological activity of the new compounds is discussed in terms of structure-activity relationship.
    Il Farmaco 04/2005; 59(12):987-92. DOI:10.1016/j.farmac.2004.08.007

Publication Stats

2k Citations
80.65 Total Impact Points


  • 2004-2015
    • Istituto di Genetica Molecolare
      Ticinum, Lombardy, Italy
  • 2002-2013
    • National Institute of Molecular Genetics (INGM)
      Milano, Lombardy, Italy
  • 2005-2011
    • Università degli Studi di Siena
      • Department of Medicine, Surgery and Neuroscience
      Siena, Tuscany, Italy
  • 2010
    • University of Birmingham
      • School of Chemistry
      Birmingham, England, United Kingdom
  • 1995-2010
    • INO - Istituto Nazionale di Ottica
      Florens, Tuscany, Italy
  • 2007
    • Oklahoma State University - Stillwater
      • Department of Veterinary Pathobiology
      SWO, Oklahoma, United States
  • 1981-2004
    • National Research Council
      • Institute of Molecular Genetics IGM
      Roma, Latium, Italy
    • University Hospital Brussels
      Bruxelles, Brussels Capital, Belgium
  • 2003
    • University of Rome Tor Vergata
      Roma, Latium, Italy
  • 1993
    • University of Milan
      Milano, Lombardy, Italy
  • 1987-1991
    • University of Zurich
      • Institute of Veterinary Biochemistry and Molecular Biology
      Zürich, Zurich, Switzerland