Emerging viral infections are difficult to control as heterogeneous members periodically cycle in and out of humans and zoonotic hosts, complicating the development of specific antiviral therapies and vaccines. Coronaviruses (CoVs) have a proclivity to spread rapidly into new host species causing severe disease. SARS-CoV and MERS-CoV successively emerged causing severe epidemic respiratory disease in immunologically naïve human populations throughout the globe. Broad-spectrum therapies capable of inhibiting CoV infections would address an immediate unmet medical need and could be invaluable in the treatment of emerging and endemic CoV infections. Here we show that a nucleotide prodrug GS-5734, currently in clinical development for treatment of Ebola virus disease, can inhibit SARS-CoV and MERS-CoV replication in multiple in vitro systems including primary human airway epithelial cell cultures with submicromolar IC50 values. GS-5734 was also effective against bat-CoVs, prepandemic bat-CoVs and circulating contemporary human CoV in primary human lung cells, thus demonstrating broad-spectrum anti-CoV activity. In a mouse model of SARS-CoV pathogenesis, prophylactic and early therapeutic administration of GS-5734 significantly reduced lung viral load and improved clinical signs of disease as well as respiratory functions. These data provide substantive evidence that GS-5734 may prove effective against endemic MERS-CoV in the Middle East, circulating human CoV, and possibly most importantly, emerging CoV of the future.

GS-5734 is a monophosphate prodrug of an adenosine nucleoside analog that showed therapeutic efficacy in a non-human primate model of Ebola virus infection. It has been administered under compassionate use to two Ebola patients, both of whom survived, and is currently in Phase 2 clinical development for treatment of Ebola virus disease. Here we report the antiviral activities of GS-5734 and the parent nucleoside analog across multiple virus families, providing evidence to support new indications for this compound against human viruses of significant public health concern.

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.

To examine the role of the mitochondrial polymerase (Pol ␥) in clinically observed toxicity of nucleoside analogs used to treat AIDS, we examined the kinetics of incorporation catalyzed by Pol ␥ for each Food and Drug Administration-approved analog plus 1-(2-deoxy-2-fluoro-␤-D-arabinofuranosyl)-5-iodouracil (FIAU), ␤-L-(؊)-2,3-dideoxy-3-thiacytidine (؊)3TC, and (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA). We used recombinant exonuclease-deficient (E200A), reconstituted human Pol ␥ holoenzyme in single turnover kinetic studies to measure K d (K m ) and k pol (k cat ) to estimate the specificity constant (k cat /K m ) for each nucleoside analog triphosphate. The specificity constants vary more than 500,000-fold for the series ddC > ddA (ddI) > 2,3-didehydro-2,3-dideoxythymidine (d4T) > > (؉)3TC > > (؊)3TC > PMPA > azidothymidine (AZT) > > Carbovir (CBV). Abacavir (prodrug of CBV) and PMPA are two new drugs that are expected to be least toxic. Notably, the higher toxicities of d4T, ddC, and ddA arose from their 13-36-fold tighter binding relative to the normal dNTP even though their rates of incorporation were comparable with PMPA and AZT. We also examined the rate of exonuclease removal of each analog after incorporation. The rates varied from 0.06 to 0.0004 s ؊1 for the series FIAU > (؉)3TC ϳ (؊)3TC > CBV > AZT > PMPA ϳ d4T > > ddA (ddI) > > ddC. Removal of ddC was too slow to measure (<0.00002 s ؊1 ). The high toxicity of dideoxy compounds, ddC and ddI (metabolized to ddA), may be a combination of high rates of incorporation and ineffective exonuclease removal. Conversely, the more effective excision of (؊)3TC, CBV, and AZT may contribute to lower toxicity. FIAU is readily extended by the next correct base pair (0.13 s ؊1 ) faster than it is removed (0.06 s ؊1 ) and, therefore, is stably incorporated and highly mutagenic. We define a toxicity index for chain terminators to account for relative rates of incorporation versus removal. These results provide a method to rapidly screen new analogs for potential toxicity.Current treatment of HIV 1 includes a mixture that generally consists of a combination of nucleoside and nonnucleoside analogs directed against HIV RT, plus an inhibitor of HIV protease. Treatment with this mixture allows patients to coexist with a low level of virus for years, but treatments are limited by the development of resistance of HIV to the drugs on the one hand and toxicity of nucleoside analogs on the other. Toxicity of nucleoside analogs is particularly troublesome for the long term management of the viral infection. Nucleoside analogs function as chain terminators to suppress viral replication by HIV-1 RT, and because HIV RT lacks a proofreading exonuclease, the specificity of nucleoside analogs toward HIV RT results from selective discrimination during incorporation and/or from removal by the proofreading exonuclease of the host DNA polymerase.Six nucleoside analogs have received Food and Drug Administration approval for treatment of HIV, and these analogs are illustrated with others...

Despite substantial progress in the development of antiretroviral regimens that durably suppress Human Immunodeficiency Virus (HIV) infection, new agents that maintain high efficacy while further optimizing the safety of lifelong, chronic therapy are needed. Tenofovir alafenamide (TAF; formerly known as GS-7340) is a novel prodrug of the antiviral acyclic nucleoside phosphonate tenofovir (TFV) with improved properties relative to tenofovir disoproxil fumarate (TDF). Although potent and generally well tolerated, TDF therapy has been associated with changes in markers of renal function, decreases in bone mineral density and a rare occurrence of serious renal adverse events, including Fanconi's Syndrome. The renal and bone toxicity observed with TDF is associated with high circulating plasma levels of TFV. TAF was discovered to be a more efficient prodrug able to further refine HIV therapy and better address life-long therapy in an older and increasingly comorbid HIV infected population. By enhancing stability in biological matrices while being rapidly activated in cells, TAF produces higher levels of intracellular TFV diphosphate, the pharmacologically active metabolite, in HIV-target cells at substantially reduced oral doses of TFV equivalents. All TFV released in the body is eventually eliminated renally; therefore, lowering the TFV equivalents administered reduces off-target kidney exposure. Effective therapy is thus achieved at approximately 90% lower systemic exposure to TFV, translating to statistically and clinically significant improvement in safety parameters associated with bone mineral density and markers of renal function.