Summary The most recent Ebola virus outbreak in West Africa – unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected – highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae1. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we describe the discovery of a novel anti-EBOV small molecule antiviral, GS-5734, a monophosphoramidate prodrug of an adenosine analog. GS-5734 exhibits antiviral activity against multiple variants of EBOV in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternate substrate and RNA-chain terminator in primer-extension assays utilizing a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life = 14 h) and distribution to sanctuary sites for viral replication including testes, eye, and brain. In a rhesus monkey model of EVD, once daily intravenous administration of 10 mg/kg GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two of six treated animals. These results provide the first substantive, post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses – including filoviruses, arenaviruses, and coronaviruses – suggests the potential for expanded indications. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.

The recent Ebola virus (EBOV) outbreak in West Africa was the largest recorded in history with over 28,000 cases, resulting in >11,000 deaths including >500 healthcare workers. A focused screening and lead optimization effort identified 4b (GS-5734) with anti-EBOV EC 50 = 86 nM in macrophages as the clinical candidate. Structure activity relationships established that the 1′-CN group and C-linked nucleobase were critical for optimal anti-EBOV potency and selectivity against host polymerases. A robust diastereoselective synthesis provided sufficient quantities of 4b to enable preclinical efficacy in a non-human-primate EBOV challenge model. Once-daily 10 mg/kg iv treatment on days 3−14 postinfection had a significant effect on viremia and mortality, resulting in 100% survival of infected treated animals [Nature 2016, 531, 381−385]. A phase 2 study (PREVAIL IV) is currently enrolling and will evaluate the effect of 4b on viral shedding from sanctuary sites in EBOV survivors.

Background:Competitors of LEDGF binding to HIV-1 integrase could prevent targeted integration to chromatin. Results: LEDGF competitors like tBPQAs were also found to inhibit integrase enzyme activity by preventing proper integraseviral DNA assembly. Conclusion: tBPQAs are allosteric inhibitors of integrase with a dual mode of action. Significance: Interference with two distinct steps of integration through the same binding site represents a new antiviral paradigm.

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Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders, presumably secondary to the activation and production of transforming growth factor-␤ (TGF-␤), a key inducer of fibroblast proliferation and matrix synthesis. The CTGF gene promoter has a TGF-␤ response element that regulates its expression in fibroblasts but not epithelial cells or lymphocytes. Recent studies have shown that the macrophage-produced cytokine tumor necrosis factor ␣ (TNF␣) is necessary to promote inflammation and to induce genes, such as matrix metalloproteinases, involved with the early stages of wound healing. In this study, we examined the ability of TNF␣ to modulate CTGF gene expression. TNF␣ was found to suppress the TGF-␤-induced expression of CTGF protein in cultured normal fibroblasts. The activity of TNF␣ was blocked by NF-B inhibitors. We showed that sequences between ؊244 and ؊166 of the CTGF promoter were necessary for both TGF-␤ and TNF␣ to modulate CTGF expression. There was a constitutive expression of CTGF by scleroderma fibroblasts that was increased by TGF-␤ treatment. Although TNF␣ was able to repress TGF-␤-induced CTGF and collagen synthesis both in normal and scleroderma skin fibroblasts, fibroblasts cultured from scleroderma patients were more resistant to TNF␣ as TNF␣ was unable to suppress the basal level of CTGF expression in scleroderma fibroblasts. Thus, we suspect that the high level of constitutive CTGF expression in scleroderma fibroblasts and its inability to respond to negative regulatory cytokines may contribute to the excessive scarring of skin and internal organs in patients with scleroderma.

Ribonucleoside analogues have potential utility as anti-viral, -parasitic, -bacterial and -cancer agents. However, their clinical applications have been limited by off target effects. Development of antiviral ribonucleosides for treatment of hepatitis C virus (HCV) infection has been hampered by appearance of toxicity during clinical trials that evaded detection during preclinical studies. It is well established that the human mitochondrial DNA polymerase is an off target for deoxyribonucleoside reverse transcriptase inhibitors. Here we test the hypothesis that triphosphorylated metabolites of therapeutic ribonucleoside analogues are substrates for cellular RNA polymerases. We have used ribonucleoside analogues with activity against HCV as model compounds for therapeutic ribonucleosides. We have included ribonucleoside analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents that are non-obligate chain terminators of the HCV RNA polymerase. We show that all of the anti-HCV ribonucleoside analogues are substrates for human mitochondrial RNA polymerase (POLRMT) and eukaryotic core RNA polymerase II (Pol II) in vitro. Unexpectedly, analogues containing 2′-C-methyl, 4′-methyl and 4′-azido substituents were inhibitors of POLRMT and Pol II. Importantly, the proofreading activity of TFIIS was capable of excising these analogues from Pol II transcripts. Evaluation of transcription in cells confirmed sensitivity of POLRMT to antiviral ribonucleosides, while Pol II remained predominantly refractory. We introduce a parameter termed the mitovir (mitochondrial dysfunction caused by antiviral ribonucleoside) score that can be readily obtained during preclinical studies that quantifies the mitochondrial toxicity potential of compounds. We suggest the possibility that patients exhibiting adverse effects during clinical trials may be more susceptible to damage by nucleoside analogs because of defects in mitochondrial or nuclear transcription. The paradigm reported here should facilitate development of ribonucleosides with a lower potential for toxicity.

A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18 x 10¹¹ particles/ml; 95% confidence interval [CI], 7.89 x 10¹¹ to 1.05 x 10¹² particles/ml), vector genomes ({X}, 3.28 x 10¹⁰ vector genomes/ml; 95% CI, 2.70 x 10¹⁰ to 4.75 x 10¹⁰ vector genomes/ml), transducing units ({X}, 5.09 x 10⁸ transducing units/ml; 95% CI, 2.00 x 10⁸ to 9.60 x 10⁸ transducing units/ml), and infectious units ({X}, 4.37 x 10⁹ TCID₅₀ IU/ml; 95% CI, 2.06 x 10⁹ to 9.26 x 10⁹ TCID₅₀ IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.

Toxicity has emerged during the clinical development of many but not all nucleotide inhibitors (NI) of hepatitis C virus (HCV). To better understand the mechanism for adverse events, clinically relevant HCV NI were characterized in biochemical and cellular assays, including assays of decreased viability in multiple cell lines and primary cells, interaction with human DNA and RNA polymerases, and inhibition of mitochondrial protein synthesis and respiration. NI that were incorporated by the mitochondrial RNA polymerase (PolRMT) inhibited mitochondrial protein synthesis and showed a corresponding decrease in mitochondrial oxygen consumption in cells. The nucleoside released by the prodrug balapiravir (R1626), 4′-azido cytidine, was a highly selective inhibitor of mitochondrial RNA transcription. The nucleotide prodrug of 2′-C-methyl guanosine, BMS-986094, showed a primary effect on mitochondrial function at submicromolar concentrations, followed by general cytotoxicity. In contrast, NI containing multiple ribose modifications, including the active forms of mericitabine and sofosbuvir, were poor substrates for PolRMT and did not show mitochondrial toxicity in cells. In general, these studies identified the prostate cell line PC-3 as more than an order of magnitude more sensitive to mitochondrial toxicity than the commonly used HepG2 cells. In conclusion, analogous to the role of mitochondrial DNA polymerase gamma in toxicity caused by some 2′-deoxynucleotide analogs, there is an association between HCV NI that interact with PolRMT and the observation of adverse events. More broadly applied, the sensitive methods for detecting mitochondrial toxicity described here may help in the identification of mitochondrial toxicity prior to clinical testing.