CHARMM (Chemistry at HARvard Macromolecular Mechanics) is a highly flexible computer program which uses empirical energy functions to model macromolecular systems. The program can read or model build structures, energy minimize them by first-or second-derivative techniques, perform a normal mode or molecular dynamics simulation, and analyze the structural, equilibrium, and dynamic properties determined in these calculations. The operations that CHARMM can perform are described, and some implementation details are given. A set of parameters for the empirical energy function and a sample run are included.

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 design, synthesis, and in vitro evaluation of the novel carbocycles as transition-state-based inhibitors of influenza neuraminidase (NA) are described. The double bond position in the carbocyclic analogues plays an important role in NA inhibition as demonstrated by the antiviral activity of 8 (IC50 = 6.3 microM) vs 9 (IC50 > 200 microM). Structure-activity studies of a series of carbocyclic analogues 6a-i identified the 3-pentyloxy moiety as an apparent optimal group at the C3 position with an IC50 value of 1 nM for NA inhibition. The X-ray crystallographic structure of 6h bound to NA revealed the presence of a large hydrophobic pocket in the region corresponding to the glycerol subsite of sialic acid. The high antiviral potency observed for 6h appears to be attributed to a highly favorable hydrophobic interaction in this pocket. The practical synthesis of 6 starting from (-)-quinic acid is also described.

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.

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.