Hepatitis C virus (HCV) drug development has resulted in treatment regimens composed of interferon‐free, all‐oral combinations of direct‐acting antivirals. While the new regimens are potent and highly efficacious, the full clinical impact of HCV drug resistance, its implications for retreatment, and the potential role of baseline resistance testing remain critical research and clinical questions. In this report, we discuss the viral proteins targeted by HCV direct‐acting antivirals and summarize clinically relevant resistance data for compounds that have been approved or are currently in phase 3 clinical trials. Conclusion: This report provides a comprehensive, systematic review of all resistance information available from sponsors’ trials as a tool to inform the HCV drug development field. (Hepatology 2015;62:1623–1632)

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target–inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations’ health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine.

Oseltamivir (Tamiflu®) is the most widely used drug against influenza infections and is extensively stockpiled worldwide as part of pandemic preparedness plans. However, resistance is a growing problem and in 2008–2009, seasonal human influenza A/H1N1 virus strains in most parts of the world carried the mutation H274Y in the neuraminidase gene which causes resistance to the drug. The active metabolite of oseltamivir, oseltamivir carboxylate (OC), is poorly degraded in sewage treatment plants and surface water and has been detected in aquatic environments where the natural influenza reservoir, dabbling ducks, can be exposed to the substance. To assess if resistance can develop under these circumstances, we infected mallards with influenza A/H1N1 virus and exposed the birds to 80 ng/L, 1 µg/L and 80 µg/L of OC through their sole water source. By sequencing the neuraminidase gene from fecal samples, we found that H274Y occurred at 1 µg/L of OC and rapidly dominated the viral population at 80 µg/L. IC50 for OC was increased from 2–4 nM in wild-type viruses to 400–700 nM in H274Y mutants as measured by a neuraminidase inhibition assay. This is consistent with the decrease in sensitivity to OC that has been noted among human clinical isolates carrying H274Y. Environmental OC levels have been measured to 58–293 ng/L during seasonal outbreaks and are expected to reach µg/L-levels during pandemics. Thus, resistance could be induced in influenza viruses circulating among wild ducks. As influenza viruses can cross species barriers, oseltamivir resistance could spread to human-adapted strains with pandemic potential disabling oseltamivir, a cornerstone in pandemic preparedness planning. We propose surveillance in wild birds as a measure to understand the resistance situation in nature and to monitor it over time. Strategies to lower environmental levels of OC include improved sewage treatment and, more importantly, a prudent use of antivirals.

Finger insertion mutations of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (T69S mutations followed by various dipeptide insertions) have a multinucleoside resistance phenotype that can be explained by decreased sensitivity to deoxynucleoside triphosphate (dNTP) inhibition of the nucleotidedependent unblocking activity of RT. We show that RTs with SG or AG (but not SS) insertions have three-to fourfold-increased unblocking activity and that all three finger insertion mutations have threefold-decreased sensitivity to dNTP inhibition. The additional presence of M41L and T215Y mutations increased unblocking activity for all three insertions, greatly reduced the sensitivity to dNTP inhibition, and resulted in defects in in vitro DNA chain elongation. The DNA chain elongation defects were partially repaired by additional mutations at positions 210, 211, and 214. These results suggest that structural communication between the regions of RT defined by these mutations plays a role in the multinucleoside resistance phenotype.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is responsible for replication of the HIV-1 genome and is therefore an important target for antiretroviral therapy. Many inhibitors of HIV-1 RT are nucleoside analogues that are converted to their active triphosphate forms by host cell kinases and are incorporated into the viral genome by HIV-1 RT. Because nucleoside analogues lack a 3Ј-OH group, their incorporation prevents further extension of the DNA chain. HIV-1 RT becomes resistant to nucleoside analogues primarily through two mechanisms (8, 12): (i) increased discrimination against the compounds (9, 13, 35) leading to decreased incorporation and less chain termination and (ii) increased ability to remove chain terminators from blocked DNA chains, allowing DNA synthesis to resume (1,3,19,20,22,26). The removal occurs through excision of the 3Ј-terminal nucleotide by transfer to one of several potential acceptor substrates, including pyrophosphate (PP i ) or ATP, generating the triphosphate form of the chain terminator or a dinucleoside polyphosphate, respectively. Most zidovudine (AZT) resistance mutations, including M41L, D67N, K70R, T215F/Y, and K219Q (29), confer increased removal, compared to wildtype (WT) RT (3,19,22,25,26). The effect of these mutations is greatest when a nucleoside triphosphate, such as ATP, is used as the acceptor substrate, whereas there is little if any effect of AZT resistance mutations on removal when a nucleoside diphosphate or PP i is used as the substrate (3,16,20,22,25).The efficiency of the removal reaction under physiological conditions will depend on the rate of the reaction and the ability of deoxynucleoside triphosphates (dNTPs) to inhibit it. HIV-1 RT bound to a chain-terminated primer-template will still bind the dNTP complementary to the next nucleotide on the template strand (34) to form a stable complex. Since chemical bond formation is prevented due to lack of a 3Ј-OH, this complex has been named a dead-end...

Using a large panel of human immunodeficiency virus type 1 site-directed mutants, we have observed a higher correlation than has previously been demonstrated between zidovudine (AZT)-triphosphate resistance data at the reverse transcriptase (RT) level and corresponding viral AZT resistance. This enhanced-resistance effect at the RT level was seen with ATP and to a lesser extent with PP i when ATP was added at physiological concentrations. The ATP-dependent mechanism (analogous to pyrophosphorolysis) appears to be dominant in the mutants bearing the D67N and K70R or 69 insertion mutations, whereas the Q151M mutation seems independent of ATP for decreased binding to AZT-triphosphate.

No abstract

We have found a close correlation between viral stavudine (d4T) resistance and resistance to d4T-triphosphate at the human immunodeficiency virus type 1 reverse transcriptase (RT) level. RT from site-directed mutants with 69S-XX codon insertions and/or conventional zidovudine resistance mutations seems to be involved in an ATP-dependent resistance mechanism analogous to pyrophosphorolysis, whereas the mechanism for RT with the Q151M or V75T mutation appears to be independent of added ATP for reducing binding to d4T-triphosphate.Although the use of nucleoside analog reverse transcriptase (RT) inhibitors (NRTIs) (in combination with nonnucleoside RT inhibitors and/or protease inhibitors) has proved highly successful in human immunodeficiency virus type 1 (HIV-1) therapy, this has not eliminated the selection of drug-resistant isolates (13,22,25). The mutational profile of these resistant viruses usually reflects which drugs have been used. For instance, the mutation M184V in RT results in specific and high-level resistance to lamivudine (24), and the changes at codons 41, 67, 70, 210, 215, and 219 contribute to high-level zidovudine (AZT) resistance (4,8,14). However, patient virus samples that are stavudine (d4T) resistant are also cross resistant to other nucleoside analogs rather than having d4T-specific resistance, and the magnitude of d4T resistance is usually low (6, 25). The biochemical mechanism of HIV-1 RT resistance to NRTIs is due in many cases to decreased binding of mutated RT to the respective NRTI-triphosphates (NRTITPs) (9). However, this does not appear to be the situation for AZT resistance, as no significant differences in discrimination between AZT-TP and dTTP in the polymerase reaction are seen when studying RTs with conventional AZT resistance the mutation (9,11,16). Recently, a new biochemical mechanism of AZT resistance has been reported where the RTs containing mutations D67N, K70R, T215F, or K219Q are able to remove AZT-monophosphate (AZT-MP) from blocked primers more efficiently than wild-type RT, thereby enabling resumption of DNA synthesis. This has been demonstrated to occur by either pyrophosphate-dependent pyrophosphorolysis (1) or by an ATP-mediated mechanism similar to pyrophosphorolysis, in which ATP at a physiological concentration (3.2 mM) acts as an acceptor to form, together with AZT-MP, a dinucleoside polyphosphate product (19). We have recently reported a close correlation between resistance to AZT-TP at the enzyme level and viral AZT resistance (16a). When we studied 11 mutants with either conventional AZT resistance mutations or multidrug resistance mutations (T69S-XX insertions or Q151M), we found this biochemical resistance mechanism to be mainly ATP dependent, with the exception of the Q151M mutant. Without added ATP, all the mutants (except the Q151M mutant) behaved similarly to wild-type RT. Mutations that appeared to be dominant in the ATP-dependent reaction were those in the ␤2-␤3 loop in the RT "fingers" domain, i.e., D67N, K70R, and insertions T69S-SG and T69S-AG...