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As a class, nucleotide inhibitors (NIs) of the hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase offer advantages over other direct-acting antivirals, including properties, such as pangenotype activity, a high barrier to resistance, and reduced potential for drug-drug interactions. We studied the in vitro pharmacology of a novel C -nucleoside adenosine analog monophosphate prodrug, GS-6620. It was found to be a potent and selective HCV inhibitor against HCV replicons of genotypes 1 to 6 and against an infectious genotype 2a virus (50% effective concentration [EC 50 ], 0.048 to 0.68 μM). GS-6620 showed limited activities against other viruses, maintaining only some of its activity against the closely related bovine viral diarrhea virus (EC 50 , 1.5 μM). The active 5′-triphosphate metabolite of GS-6620 is a chain terminator of viral RNA synthesis and a competitive inhibitor of NS5B-catalyzed ATP incorporation, with K i / K m values of 0.23 and 0.18 for HCV NS5B genotypes 1b and 2a, respectively. With its unique dual substitutions of 1′-CN and 2′- C -Me on the ribose ring, the active triphosphate metabolite was found to have enhanced selectivity for the HCV NS5B polymerase over host RNA polymerases. GS-6620 demonstrated a high barrier to resistance in vitro . Prolonged passaging resulted in the selection of the S282T mutation in NS5B that was found to be resistant in both cellular and enzymatic assays (>30-fold). Consistent with its in vitro profile, GS-6620 exhibited the potential for potent anti-HCV activity in a proof-of-concept clinical trial, but its utility was limited by the requirement of high dose levels and pharmacokinetic and pharmacodynamic variability.

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10 8 50% tissue culture infective doses (TCID 50 )/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.

This study examined the contributions of collagen and elastin to the tensile elastic properties of the vocal fold lamina propria. Uniaxial stress-strain responses of vocal fold cover and vocal ligament specimens from 20 human larynges (12 males, 8 females) were quantified with sinusoidal stretch-release deformation in vitro. Mid-coronal sections of 12 specimens were examined histologically with Masson's trichrome and elastin van Gieson stain to quantify the relative densities of collagen and elastin fibers. Results showed that significantly higher levels of collagen were found in the male vocal fold than female, for both the cover and the ligament. For male there was a significantly higher level of elastin in the cover than in the ligament. On average, the elastic modulus of the male cover was about twice that of the female at high-tensile strain (35-40%), whereas the male ligament was 3-5 times stiffer than the female in the same range. The ligament was stiffer than the cover for male, but the opposite was observed for female. These findings suggested that collagen and elastin could contribute differentially to elasticity of the cover and the ligament. The data may provide guidance for surgical reconstruction and tissue engineering of different lamina propria layers.

Viscoelastic response of the human vocal fold under tension has been reported previously, demonstrating nonlinearity and hysteresis of stress–strain curves. However, the importance of various matrix molecules for tensile elasticity of the vocal fold is not well understood. It is important to correlate the biomechanical behavior of the vocal fold with its histological microstructure, so as to examine the relative contributions of the matrix proteins such that cultured tissue constructs for repairing voice disorders may be engineered accordingly. This study attempted to test the hypothesis that collagen and elastin play a major role in determining the tensile elasticity of the vocal fold. Uniaxial tensile elastic properties of the vocal cover and vocal ligament were quantified using a servo‐control lever system. The specimens were also studied histologically with elastin van Gieson stain. Digital image analysis of the vocal fold cover showed that a more profoundly nonlinear stress–strain curve and a higher elastic modulus (tangent Young’s modulus) were associated with higher densities of collagen and elastin in the specimens. These findings suggested that both collagen and elastin fibers likely contribute significantly to elasticity of the vocal fold under tension, thereby regulating vocal fold length changes and fundamental frequency control. [Work supported by NIH.]

The extracellular matrix (ECM) of the human vocal fold is a highly specialized soft connective tissue with a layered microstructure that is optimally tuned for vibration and sound production in response to a unique set of biomechanical stimuli in vivo, including oscillation at amplitudes up to 3–4 mm at magnitudes of acceleration > 200g and at high frequencies (> 100Hz). The vocal fold ECM, commonly called the lamina propria or mucosa, consists of biomacromolecules of two major classes distributed in different densities: (1) fibrous proteins including collagen and elastin fibers that are denser in the deep layers of the ECM, and (2) interstitial proteins like glycosaminoglycans and structural glycoproteins that are scattered throughout the entire ECM [1,2]. Nonlinear viscoelastic response of the vocal fold ECM under different loading conditions has been reported, including strain rate-dependence and hysteresis of tensile stress-strain curves, and nonlinear stress-strain behavior under large-strain shear [3,4].

Hepatitis C virus (HCV) chronically infects close to 3% of the world's population, with 30% of carriers expected to develop serious liver-related diseases, including hepatocellular carcinoma. There is an ongoing effort to develop novel antivirals to improve the therapeutic outcome of anti-HCV treatment.We explore the mechanism of action of nonnucleotide inhibitors (NNIs) of HCV replication that bind to the thumb site II pocket on NS5B polymerase, exemplified by GS-9669 which is in phase II clinical trials. Despite a wealth of enzymatic and structural information regarding NS5B it is not fully understood how binding at this remote pocket inhibits the active site. Stabilization of the closed, inactive enzyme conformation has been suggested, although it has not been described on the mechanistic level.We employed a combination of enzyme activity, direct binding, and calorimetric studies with GS-9669 and other classes of NNIs and several truncation mutants of NS5B that disrupt critical interactions at the interface of the thumb domain, β-loop and C-terminus. Our studies demonstrated unique binding and inhibition profiles for each NNIs. An intact interface between the β-loop and the C-terminus is critical for inhibitory potency of thumb site II NNIs, although both elements are dispensable for binding. In addition, calorimetric studies revealed the pivotal role of both regulatory elements in communication between polymerase domains. We postulate that binding at the thumb site II pocket locks the entire NS5B in an enzymatically inactive, closed conformation. This stabilization of the closed conformation is a consequence of the communication between the binding pocket on the thumb domain and other domains of polymerase that is mediated by the interactions between two key regulatory elements: the β-loop and the C-terminal tail.