Publications

We have developed a new sensitive enzyme immunoassay (EIA) and MTT (tetrazolium salt) assay for screening compounds against two variants of human herpesvirus 6 (HHV-6A, HHV-6B) and human herpesvirus 7 (HHV-7) and evaluated the anti-HHV-6 and HHV-7 activity of a series of anti-herpesvirus compounds and acyclic nucleoside phosphonate analogues. The results indicate that the pattern of activity of these compounds against these betaherpesviruses is similar to that for human cytomegalovirus (HCMV). The highest potency and selectivity against the two variants of HHV-6 and HHV-7 was demonstrated by S2242 (N7-isomer of 6-deoxy-ganciclovir). Also, ganciclovir (GCV), foscarnet, (phosphonoformic acid; PFA) and the acyclic nucleoside phosphonate analogues such as cidofovir (HPMPC) exhibited selective inhibitory activity against these viruses. Thymidine kinase (TK)-dependent drugs (acyclovir, ACV; brivudin, BVDU; and sorivudine, BVaraU) showed little, if any, activity. These results suggest a structural homology of the DNA polymerase and a lack of TK gene among these three betaherpesviruses (HHV-6, HHV-7 and HCMV). The finding that HHV-7 was highly sensitive to GCV also suggests that HHV-7 may have an HCMV-UL97-homologue gene for the phosphorylation of GCV. The present EIA method is more rapid and sensitive than the previously reported procedures and could be useful for the large-scale screening of compounds against HHV-6 and HHV-7.

Abstract The preparation of the title compounds (XII) and (XVIII) is reported.

We recently described the synthesis and antiviral activity of the compounds 5-phenyl-3-(4-cyano-5-phenylisothiazol-3-yl) disulphanyl-4-isothiazolecarbonitrile and S-(4-cyano-5-phenylisothiazol -3-yl)-O-ethyl thiocarbonate, which were found to be effective against both HIV-1 (IIIB) and HIV-2 (ROD). We have now evaluated these compounds against both RNA and DNA viruses, obtaining high selectivity indexes for poliovirus 1 (SI: 223 and 828, respectively) and Echovirus 9 (SI: 334 and 200, respectively). In our previous studies, 3-methylthio-5-(4-OBn-phenyl)-4-isothiazolecarbonitrile was found to exhibit a broad spectrum of action against picornaviruses, we therefore selected this compound and S-(4-cyano-5-phenylisothiazol-3-yl)-O-ethyl thiocarbonate as the model for the synthesis of a new isothiazole derivative, S-[4-cyano-5-(4-OBn-phenyl)isothiazol-3-yl]-O-ethyl thiocarbonate. This compound was evaluated against picornaviruses, measles virus, HIV-1 (IIIB) and HIV-2 (ROD), and some DNA viruses (adenovirus type 2 and herpes simplex virus type 1). The compound was shown to be active against rhinoviruses 2, 39, 86 and 89, Coxsackie B1 and measles virus.

Current standard therapy for the treatment of chronic infections with hepatitis C virus consists of combination therapy with (pegylated) interferon-alpha and ribavirin. 1,5-Anhydrohexitol nucleoside analogues are constrained congeners known to mimic the ribonucleoside conformation. Within this series some analogues are endowed with strong antiviral properties, particularly against herpesviruses. The six-membered anhydrohexitol ring was, therefore, combined with the triazolyl carboxamide moiety of ribavirin, thus providing a new series of ribavirin analogues. None of the newly synthesized compounds elicited any substantial antiviral activity, neither against herpes simplex virus type 1 and 2, nor against bovine viral diarrhoea virus (a surrogate for hepatitis C virus) or the yellow fever virus.

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.

The conformationally locked nucleoside, (north)-methanocarbathymine (1a), is a potent and selective anti-herpes agent effective against herpes simplex type 1 (HSV1) and type 2 (HSV2) viruses. Hereby, we report on the synthesis and biological evaluation of a small set of 5-substituted pyrimidine nucleosides belonging to the same class of bicyclo[3.1.0]hexane nucleosides. Both the 5-bromovinyl (4) and the 5-bromo analogue (3) appeared to be exclusive substrates of HSV1 thymidine kinase (TK), contrasting with the 5-iodo analogue (2), which was significantly phosphorylated by the human cytosolic TK. The binding affinity constant and catalytic turnover for HSV1 TK were measured to assess the influence of the substitution on these parameters. In the plaque reduction and cytotoxicity assays, the 5-bromo analogue (3) showed good activity against HSV1 and HSV2 with less general toxicity than 1a. Against varicella-zoster virus (VZV), the north-locked 5-bromovinyl analogue (4) proved to be as potent as its conformationally unlocked 2'-deoxyriboside equivalent BVDU. The three compounds were also tested in vitro as prodrugs used in a gene therapy context on three osteosarcoma cell lines, either deficient in TK (TK(-)), nontransduced, or stably transduced with HSV1 TK. The 5-iodo compound (2, CC(50) 25 +/- 7 microM) was more efficient than ganciclovir (GCV, CC(50) 75 +/- 35 microM) in inhibiting growth of HSV1-TK transfected cells and less inhibitory than GCV toward TK(-) cells, whereas compound 3 inhibited transfected and nontransfected cell lines in a relatively similar dose-dependent manner.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are very potent and most promising anti-AIDS drugs that specifically inhibit HIV-1 reverse transcriptase (RT). However, to a great extent, the efficacy of NNRTI drugs is impaired by rapid emergence of drug-resistance mutations. Fortunately, detailed analysis of a wide range of crystal structures of HIV-1 RT/NNRTI complexes together with data on drug resistance mutations has identified factors important for design of inhibitors and resilience to mutations, such as, exhibiting conformational flexibility and positional adaptability of NNRTIs, forming extensive main chain hydrogen bonding, targeting highly conserved residues in HIV-1 RT and possessing unconventional mechanisms for NNRTI-mediated inhibition of RT. Besides, the plasticity of NNRTIs binding pocket (NNIBP) also provides a broad space for the discovery of new generations of NNRTIs. For instance, the composite binding pocket, integrated all available crystal structure information about the NNRTI binding site of HIV RT, was demonstrated to be an effective tool to better understand the flexible nature of the binding pocket and to identify specific inhibitors. The RT/solvent interface proved to be an attractive site for incorporating a moiety to improve water solubility and pharmacokinetics or introducing a second pharmacophore to construct multifunctional ligand. Totally, the characterization of NNRTIs and NNIBP may help in the design of more effective drugs that are potent toward wild type and drug-resistant strains of RT. In this paper we attempt to translate the general knowledge gained from a large number of related literature into a set of medicinal chemistry strategies to improve the drug resistance profile of NNRTIs.

Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impairs the adaptive immune system during acute infection. Still, it remains largely unclear whether the frequency and functions of T and B cells return to normal after the recovery of Coronavirus Disease 2019 (COVID-19). Here, we analyzed immune repertoires and SARS-CoV-2-specific neutralization antibodies in a prospective cohort of 40 COVID-19 survivors with a 6-month follow-up after hospital discharge. Immune repertoire sequencing revealed abnormal T- and B-cell expression and function with large T cell receptor/B cell receptor clones, decreased diversity, abnormal class-switch recombination, and somatic hypermutation. A decreased number of B cells but an increased proportion of CD19⁺ CD138⁺ B cells were found in COVID-19 survivors. The proportion of CD4⁺ T cells, especially circulating follicular helper T (cTfh) cells, was increased, whereas the frequency of CD3⁺ CD4^- T cells was decreased. SARS-CoV-2-specific neutralization IgG and IgM antibodies were identified in all survivors, especially those recorded with severe COVID-19 who showed a higher inhibition rate of neutralization antibodies. All severe cases complained of more than one COVID-19 sequelae after 6 months of recovery. Overall, our findings indicate that SARS-CoV-2-specific antibodies remain detectable even after 6 months of recovery. Because of their abnormal adaptive immune system with a low number of CD3⁺ CD4^- T cells and high susceptibility to infections, COVID-19 patients might need more time and medical care to fully recover from immune abnormalities and tissue damage.