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A transient kinetic procedure was used in examining the catalytic efficiency during single nucleotide incorporation of the M184V mutant of HIV-1 reverse transcriptase (RT). HIV-1 viral resistance is triggered when an amino acid substitution from methionine-184 to valine evokes the 1000-fold 3TC. Results indicate that the fidelity in M184V RT increases by a maximum of 2.4 times over the wild-type HIV-1 RT. Both the wild-type and 3TC-resistant mutant RT have higher fidelity when using an RNA template.

Research was conducted to examine the kinetics for the incorporation of dCMP, ddCMP and (+)SddCMP and its unnatural beta-L-(-) enantiomer, (-)SdCMP, into DNA/DNA and DNA/RNA primer-templates catalyzed by HIV-1 RT. The objective was to gain an insight into the differences between RNA-dependent and DNA-dependent polymerization. Results suggest that perturbations on the ribose ring of cytidine analogues decrease the rate and efficiency of incorporation but improves the binding affinity.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 ( 1 ), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C -nucleoside GS-646089 ( 2 ) with broad antiviral activity against RSV (EC 50 = 3-46 nM), human metapneumovirus (EC 50 = 210 nM), human rhinovirus (EC 50 = 54-61 nM), and enterovirus (EC 50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [( S )-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo . 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

Remdesivir 1 is an phosphoramidate prodrug that releases the monophosphate of nucleoside GS-441524 ( 2 ) into lung cells, thereby forming the bioactive triphosphate 2-NTP . 2-NTP , an analog of ATP, inhibits the SARS-CoV-2 RNA-dependent RNA polymerase replication and transcription of viral RNA. Strong clinical results for 1 have prompted interest in oral approaches to generate 2-NTP . Here, we describe the discovery of a 5'-isobutyryl ester prodrug of 2 (GS-5245, Obeldesivir, 3 ) that has low cellular cytotoxicity and 3-7-fold improved oral delivery of 2 in monkeys. Prodrug 3 is cleaved presystemically to provide high systemic exposures of 2 that overcome its less efficient metabolism to 2-NTP , leading to strong SARS-CoV-2 antiviral efficacy in an African green monkey infection model. Exposure-based SARS-CoV-2 efficacy relationships resulted in an estimated clinical dose of 350-400 mg twice daily. Importantly, all SARS-CoV-2 variants remain susceptible to 2 , which supports development of 3 as a promising COVID-19 treatment.

We describe the discovery of three structurally differentiated potent and selective MTH1 inhibitors and their subsequent use to investigate MTH1 as an oncology target, culminating in target (in)validation. Tetrahydronaphthyridine 5 was rapidly identified as a highly potent MTH1 inhibitor (IC 50 = 0.043 nM). Cocrystallization of 5 with MTH1 revealed the ligand in a Φ- cis - N -(pyridin-2-yl)acetamide conformation enabling a key intramolecular hydrogen bond and polar interactions with residues Gly34 and Asp120. Modification of literature compound TH287 with O - and N -linked aryl and alkyl aryl substituents led to the discovery of potent pyrimidine-2,4,6-triamine 25 (IC 50 = 0.49 nM). Triazolopyridine 32 emerged as a highly selective lead compound with a suitable in vitro profile and desirable pharmacokinetic properties in rat. Elucidation of the DNA damage response, cell viability, and intracellular concentrations of oxo-NTPs (oxidized nucleoside triphosphates) as a function of MTH1 knockdown and/or small molecule inhibition was studied. Based on our findings, we were unable to provide evidence to further pursue MTH1 as an oncology target., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

Hepatitis C virus (HCV) infection presents an unmet medical need requiring more effective treatment options. Nucleoside inhibitors (NI) of HCV polymerase (NS5B) have demonstrated pan-genotypic activity and durable antiviral response in the clinic, and they are likely to become a key component of future treatment regimens. NI candidates that have entered clinical development thus far have all been N-nucleoside derivatives. Herein, we report the discovery of a C-nucleoside class of NS5B inhibitors. Exploration of adenosine analogs in this class identified 1'-cyano-2'-C-methyl 4-aza-7,9-dideaza adenosine as a potent and selective inhibitor of NS5B. A monophosphate prodrug approach afforded a series of compounds showing submicromolar activity in HCV replicon assays. Further pharmacokinetic optimization for sufficient oral absorption and liver triphosphate loading led to identification of a clinical development candidate GS-6620. In a phase I clinical study, the potential for potent activity was demonstrated but with high intra- and interpatient pharmacokinetic and pharmacodynamic variability.