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A series of 2′-fluorinated C -nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2′-fluoro-2′- C -methyl adenosine C -nucleoside ( 15 ) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant ( 53 ) with favorable pharmacokinetic properties including efficient liver delivery in animals.

Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2′-C-methyl-1′-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection., Delivery of pharmacologically active nucleoside triphosphate analogs to sites of viral infection is challenging. In prior work we identified a 2′-C-methyl-1′-cyano-7-deaza-adenosine C-nucleotide analog with desirable selectivity and potency for the treatment of hepatitis C virus (HCV) infection. However, the prodrug selected for clinical development, GS-6620, required a high dose for meaningful efficacy and had unacceptable variability due to poor oral absorption as a result of suboptimal solubility, intestinal metabolism, and efflux transport. While obtaining clinical proof of concept for the nucleotide analog, a more effective prodrug strategy would be necessary for clinical utility. Here, we report an alternative prodrug of the same nucleoside analog identified to address liabilities of GS-6620. A phosphoramidate prodrug containing the nonproteinogenic amino acid methylalanine, an isopropyl ester and phenol in the (S) conformation at phosphorous, GS2, was found to have improved solubility, intestinal stability, and hepatic activation. GS2 is a more selective substrate for hepatically expressed carboxyl esterase 1 (CES1) and is resistant to hydrolysis by more widely expressed hydrolases, including cathepsin A (CatA) and CES2. Unlike GS-6620, GS2 was not cleaved by intestinally expressed CES2 and, as a result, was stable in intestinal extracts. Levels of liver triphosphate following oral administration of GS2 in animals were higher than those of GS-6620, even when administered under optimal conditions for GS-6620 absorption. Combined, these properties suggest that GS2 will have better oral absorption in the clinic when administered in a solid dosage form and the potential to extend the clinical proof of concept obtained with GS-6620.

A sulfonamide replacement of the P2–P3 amide bond in the context of macrocyclic HCV NS3 protease inhibitors was investigated. These analogs displayed good inhibitory potency in the absence of any P3 capping group. The synthesis and preliminary SAR are described.

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.

Objectives: Very little is known about the elasticity of the human ventricular fold (false vocal fold). To better understand the potential role of the false fold in the fluid dynamics and aeroacoustics of phonation, we made some measurements on the elastic properties of human ventricular fold tissues in vitro. Methods: Uniaxial tensile stress-strain characteristics of 6 male and 6 female false fold specimens were quantified with sinusoidal stretch-release deformation. Midcoronal sections of 3 specimens were examined to quantify the relative densities of collagen, elastin, seromucous glandular tissue, and adipose tissue by digital image analysis. Results: Nonlinear stress-strain curves with hysteresis (viscous energy loss) were observed, with large interindividual differences. A hybrid linear-exponential model was used to determine the elastic modulus (tangent Young's modulus) of the false fold. On average, the male false fold was twice as stiff as the female at a tensile strain of 20% to 30%. Conclusions: This preliminary gender-related difference in elasticity could be attributed to a higher proportion of glandular tissue in the female false fold, due to the lower elastic modulus of glands. The present data allow one to develop a more comprehensive biomechanical model of phonation, for optimizing postoperative voice production following laryngeal reconstruction procedures.

The exploration of novel inhibitors of the HCV NS4B protein that are based on a 2-oxadiazoloquinoline scaffold is described. Optimization to incorporate activity across genotypes led to a potent new series with broad activity, of which inhibitor 1 displayed the following EC50 values: 1a, 0.08 nM; 1b, 0.10 nM; 2a, 3 nM; 2b, 0.6 nM, 3a, 3.7 nM; 4a, 0.9 nM; 6a, 3.1 nM.

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.

The use of a tri-substituted acylhydrazine as an isostere of a tertiary amide was explored in a series of HCV NS5B thumb site II inhibitors. Direct replacement generated an analog with similar conformational and physicochemical properties. The series was extended to produce compounds with potent binding affinities and encouraging levels of cellular potency.

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.

A novel series of HCV replication inhibitors based on a pyrido[3,2-d]pyrimidine core were optimized for pharmacokinetics (PK) in rats. Several associations between physicochemical properties and PK were identified and exploited to guide the design of compounds. In addition, a simple new metric that may aid in the prediction of bioavailability for compounds with higher polar surface area is described (3*HBD-cLogP).

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.