Mingjun Huang

Selection of Replicon Variants Resistant to ACH-806, a Novel Hepatitis C Virus Inhibitor with No Cross-Resistance to NS3 Protease and NS5B Polymerase Inhibitors

ABSTRACT We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.

Small-molecule Factor D inhibitors selectively block the alternative pathway of complement in paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome

Paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome are diseases of excess activation of the alternative pathway of complement that are treated with eculizumab, a humanized monoclonal antibody against the terminal complement component C5. Eculizumab must be administered intravenously, and moreover some patients with paroxysmal nocturnal hemoglobinuria on eculizumab have symptomatic extravascular hemolysis, indicating an unmet need for additional therapeutic approaches. We report the activity of two novel small-molecule inhibitors of the alternative pathway component Factor D using in vitro correlates of both paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome. Both compounds bind human Factor D with high affinity and effectively inhibit its proteolytic activity against purified Factor B in complex with C3b. When tested using the traditional Ham test with cells from paroxysmal nocturnal hemoglobinuria patients, the Factor D inhibitors significantly reduced complement-mediated hemolysis at concentrations as low as 0.01 μM. Additionally the compound ACH-4471 significantly decreased C3 fragment deposition on paroxysmal nocturnal hemoglobinuria erythrocytes, indicating a reduced potential relative to eculizumab for extravascular hemolysis. Using the recently described modified Ham test with serum from patients with atypical hemolytic uremic syndrome, the compounds reduced the alternative pathway-mediated killing of PIGA-null reagent cells, thus establishing their potential utility for this disease of alternative pathway of complement dysregulation and validating the modified Ham test as a system for pre-clinical drug development for atypical hemolytic uremic syndrome. Finally, ACH-4471 blocked alternative pathway activity when administered orally to cynomolgus monkeys. In conclusion, the small-molecule Factor D inhibitors show potential as oral therapeutics for human diseases driven by the alternative pathway of complement, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome.

ACH-806, an NS4A Antagonist, Inhibits Hepatitis C Virus Replication by Altering the Composition of Viral Replication Complexes

ABSTRACT Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro

ABSTRACT Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

Hepatitis C drug discovery: in vitro and in vivo systems and drugs in the pipeline

The combination therapy of ribavirin and pegylated interferon-α for hepatitis C has significant side effects, is often poorly tolerated and is ineffective in many patients, despite causing impressive improvement in the sustained virological response. Discovery and development of more effective and well-tolerated antihepatitis C virus drugs are clearly in great demand. During the past few years, remarkable advances have been made in the establishment of in vitro and in vivo systems. Armed with these systems, a wave of specific antihepatitis C virus compounds have been discovered and are moving into the clinical phase. More effective combination therapies with specific antivirals are predicted to emerge in the near future for the treatment of hepatitis C.

Internal cleavages of hepatitis C virus NS3 induced by P1 mutations at the NS3/4A cleavage site.

Despite being the focus of intensive investigation for its enzymatic activities and its roles in HCV virus replication, little is known about the internal processing of NS3. Here we show that single mutations at P1 position of the NS3/4A junction lead to alternative cleavages. Among the multiple novel cleavage products observed, there were two predominant species of about 12 kDa (p12) and 67 kDa (p67). This p12 species consists of the NS4A and about a 6 kDa long C-terminal region of NS3 and forms a complex with NS3. The remaining NS3 corresponds to the p67 species. This alternative cleavage is an NS3 protease-mediated intra-molecular event and more interestingly can also be induced with low concentrations of one NS3 protease inhibitor examined. Our results led us to propose a model explaining the alternative cleavage observed and its functional role.

A highly sensitive and reproducible HCV RNA hybridization method valuable for antiviral drug discovery

Real-time RT-PCR and Northern blot are employed for the measurement of HCV RNA but suffer from multiple purification steps, high cost, and relatively large variability. In this study, a hybridization method for HCV RNA detection is described. This method does not need RNA purification, and is sensitive enough to detect HCV RNA present in replicon cellular lysates harvested from a single well of a 96-well plate. Fixation of RNA by UV cross-linking is crucial for this sensitivity. A linear relationship exists between hybridization signal and cell density ranging from 10(5) to as few as 300 cells per well. The signal-to-background ratio is greater than 40 and the Z factor is above 0.7. Using several known anti-HCV agents, dose-response curves and EC(50) values generated from hybridization were similar to those obtained from a luciferase assay. This method has been successfully applied to replicons of different HCV subtypes and hepatitis B virus in our laboratory. In summary, this hybridization assay is sensitive, highly reproducible, easy to handle, and a valuable tool for antiviral drug discovery.

Hepatitis C Viral Proteases And Inhibitors

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Chapter 12 – Factor D

Factor D is a serine protease (SP) consisting of a single polypeptide of 228 amino acids. Unlike other SPs in the complement system, factor D circulates in the plasma as a mature but ‘resting-state’ form at a very low concentration and is produced mainly in adipocytes. The function of factor D is to cleave its unique substrate, factor B in Mg++-dependent complex with C3(H2O) or C3b, to generate the alternative pathway C3 convertases C3(H2O)Bb and C3bBb. Therefore, factor D is a key and rate-limiting component in the alternative pathway. Factor D participates in the amplification loop which contributes significantly to responses elicited by the complement classical and lectin pathways. Humans with factor D deficiency are rare and are phenotypically normal but encounter increased infection risk, particularly with Neisseria meningitidis.

Assessment of complement-mediated bacterial killing and the effect of a small molecule factor D inhibitor in vitro

number: 62 XXVI International Complement Workshop, Kanazawa, Ishikawa, Japan, September 4-8, 2016 a. Percentages were calculated relative to NHS incubated at 37oC b. Percentages were calculated relative to the depleted serum fully reconstituted with the missing component(s) at 37oC ACH-4471, fD inhibitor NHS, normal human serum Dpl, depleted serum Neg, negative Pos, positive Heated, NHS heated at 56oC for 30 min INTRODUCTION ACH-4471, fD inhibitor NHS, Normal Human Serum Dpl, Depleted PI, Propidium Iodide FL, Fluorescence Serum/ Treatment % Phagocytic Activity Monocyte Granulocyte 0◦C Neg control 0 a 0a 37◦C Pos control 100 a 100a C1q-Dpl 98b 82b fD-Dpl 99b 144b C1q&fDDpl 31 b 30b C2-Dpl 68b 73b C4-Dpl 55b 80b C5-Dpl 67b 82b Heated 2b 3b 0.1 μM ACH-4471 106 a 90a 1 μM ACH-4471 82 a 82a 10 μM ACH-4471 102 a 91a Monocytes Granulocytes METHODS