The Substitutions L50F, E166A, and L167F in SARS-CoV-2 3CLpro Are Selected by a Protease Inhibitor In Vitro and Confer Resistance To Nirmatrelvir.

The SARS-CoV-2 main protease (3CLpro) has an indispensable role in the viral life cycle and is a therapeutic target for the treatment of COVID-19. The potential of 3CLpro-inhibitors to select for drug-resistant variants needs to be established. Therefore, SARS-CoV-2 was passaged in vitro in the presence of increasing concentrations of ALG-097161, a probe compound designed in the context of a 3CLpro drug discovery program. We identified a combination of amino acid substitutions in 3CLpro (L50F E166A L167F) that is associated with a >20× increase in 50% effective concentration (EC50) values for ALG-097161, nirmatrelvir (PF-07321332), PF-00835231, and ensitrelvir. While two of the single substitutions (E166A and L167F) provide low-level resistance to the inhibitors in a biochemical assay, the triple mutant results in the highest levels of resistance (6× to 72×). All substitutions are associated with a significant loss of enzymatic 3CLpro activity, suggesting a reduction in viral fitness. Structural biology analysis indicates that the different substitutions reduce the number of inhibitor/enzyme interactions while the binding of the substrate is maintained. These observations will be important for the interpretation of resistance development to 3CLpro inhibitors in the clinical setting. IMPORTANCE Paxlovid is the first oral antiviral approved for treatment of SARS-CoV-2 infection. Antiviral treatments are often associated with the development of drug-resistant viruses. In order to guide the use of novel antivirals, it is essential to understand the risk of resistance development and to characterize the associated changes in the viral genes and proteins. In this work, we describe for the first time a pathway that allows SARS-CoV-2 to develop resistance against Paxlovid in vitro. The characteristics of in vitro antiviral resistance development may be predictive for the clinical situation. Therefore, our work will be important for the management of COVID-19 with Paxlovid and next-generation SARS-CoV-2 3CLpro inhibitors

Activation Pathway of a Nucleoside Analog Inhibiting Respiratory Syncytial Virus Polymerase

Human respiratory syncytial virus (RSV) is a negative-sense RNA virus and a significant cause of respiratory infection in infants and the elderly. No effective vaccines or antiviral therapies are available for the treatment of RSV. ALS-8176 is a first-in-class nucleoside prodrug inhibitor of RSV replication currently under clinical evaluation. ALS-8112, the parent molecule of ALS-8176, undergoes intracellular phosphorylation, yielding the active 5′-triphosphate metabolite. The host kinases responsible for this conversion are not known. Therefore, elucidation of the ALS-8112 activation pathway is key to further understanding its conversion mechanism, particularly given its potent antiviral effects. Here, we have identified the activation pathway of ALS-8112 and show it is unlike other antiviral cytidine analogs. The first step, driven by deoxycytidine kinase (dCK), is highly efficient, while the second step limits the formation of the active 5′-triphosphate species. ALS-8112 is a 2′- and 4′-modified nucleoside analog, prompting us to investigate dCK recognition of other 2′- and 4′-modified nucleosides. Our biochemical approach along with computational modeling contributes to an enhanced structure–activity profile for dCK. These results highlight an exciting potential to optimize nucleoside analogs based on the second activation step and increased attention toward nucleoside diphosphate and triphosphate prodrugs in drug discovery

Preclinical Characteristics of the Hepatitis C Virus NS3/4A Protease Inhibitor ITMN-191 (R7227) ▿ †

Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C

Molecular Basis for the Selective Inhibition of Respiratory Syncytial Virus RNA Polymerase by 2'-Fluoro-4'-Chloromethyl-Cytidine Triphosphate

<div><p>Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections, yet no vaccines or effective therapeutics are available. ALS-8176 is a first-in-class nucleoside analog prodrug effective in RSV-infected adult volunteers, and currently under evaluation in hospitalized infants. Here, we report the mechanism of inhibition and selectivity of ALS-8176 and its parent ALS-8112. ALS-8176 inhibited RSV replication in non-human primates, while ALS-8112 inhibited all strains of RSV in vitro and was specific for paramyxoviruses and rhabdoviruses. The antiviral effect of ALS-8112 was mediated by the intracellular formation of its 5'-triphosphate metabolite (ALS-8112-TP) inhibiting the viral RNA polymerase. ALS-8112 selected for resistance-associated mutations within the region of the L gene of RSV encoding the RNA polymerase. In biochemical assays, ALS-8112-TP was efficiently recognized by the recombinant RSV polymerase complex, causing chain termination of RNA synthesis. ALS-8112-TP did not inhibit polymerases from host or viruses unrelated to RSV such as hepatitis C virus (HCV), whereas structurally related molecules displayed dual RSV/HCV inhibition. The combination of molecular modeling and enzymatic analysis showed that both the 2'F and the 4'ClCH₂ groups contributed to the selectivity of ALS-8112-TP. The lack of antiviral effect of ALS-8112-TP against HCV polymerase was caused by Asn291 that is well-conserved within positive-strand RNA viruses. This represents the first comparative study employing recombinant RSV and HCV polymerases to define the selectivity of clinically relevant nucleotide analogs. Understanding nucleotide selectivity towards distant viral RNA polymerases could not only be used to repurpose existing drugs against new viral infections, but also to design novel molecules.</p></div

Contribution of the 4'ClCh₂ group to the QUAD-mutant resistance to ALS-8112-TP.

<p><b>(A)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of either CTP or ALS-8112-TP. Product formation was quantified and expressed as % primer extension from the +3 position (see calculation in Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.s001" target="_blank">S1 Text</a>). QUAD K_{m CTP} = 0.056±0.010 μM (<i>n</i> = 2), and QUAD K_{m ALS-8112-TP} = 1.74±0.34 μM (<i>n</i> = 2). K_m values for the WT enzyme were reported in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.g004" target="_blank">Fig 4</a>. <b>(B)</b> Fold discrimination for each enzyme was calculated as K_{m CTP analog} / K_{m CTP}. <b>(C and D)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 2'F-CTP. WT K_{m 2'F-CTP} = 0.12±0.014 μM (<i>n</i> = 2), and QUAD K_{m 2'F-CTP} = 0.07±0.017 μM (<i>n</i> = 2). <b>(E and F)</b> The RSV L-P proteins (WT and QUAD) were incubated in the presence of GTP* + ATP and increasing concentrations of 4'ClCH₂-CTP. WT K_{m 4'ClCH2-CTP} = 16±2.7 μM (<i>n</i> = 2), and QUAD K_{m 4'ClCH2-CTP} = 198±18 μM (<i>n</i> = 2).</p

Rational design of ALS-8112 as a selective RSV inhibitor.

<p><b>(A)</b> The nucleotide analog 2'-F-CTP is a substrate for both RSV and HCV polymerase, but it does not cause any inhibition by immediate chain termination. The addition of a 4'ClCH₂ group (ALS-8112-TP) makes the molecule a selective inhibitor of RSV polymerase. The addition of a 2'Me group favors recognition by HCV polymerase, and the addition of a 4'N₃ group causes dual RSV/HCV polymerase inhibition. <b>(B)</b> X-ray structure of natural CDP in the active site of HCV polymerase (PDB 4WTC, [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004995#ppat.1004995.ref028" target="_blank">28</a>]). (<b>C, D</b>, and <b>E</b>) Docked binding modes of 2'F-2'Me-, 2'F-4'ClCH₂-, and 2'F-4'N₃-CDP, respectively.</p