Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

A Replication-Defective Human Cytomegalovirus Vaccine Elicits Humoral Immune Responses Analogous to Those with Natural Infection.

Human cytomegalovirus (HCMV) can cause congenital infections, which are a leading cause of childhood disabilities. Since the rate of maternal-fetal transmission is much lower in naturally infected (HCMV-seropositive) women, we hypothesize that a vaccine candidate capable of eliciting immune responses analogous to those of HCMV-seropositive subjects may confer protection against congenital HCMV. We have previously described a replication-defective virus vaccine based on strain AD169 (D. Wang, D. C. Freed, X. He, F. Li, et al., Sci Transl Med 8:362ra145, 2016, https://doi.org/10.1126/scitranslmed.aaf9387). The vaccine, named V160, has been shown to be safe and immunogenic in HCMV-seronegative human subjects, eliciting both humoral and cellular immune responses (S. P. Adler, S. E. Starr, S. A. Plotkin, S. H. Hempfling, et al., J Infect Dis 220:411-419, 2019, https://doi.org/10.1093/infdis/171.1.26). Here, we further showed that sera from V160-immunized HCMV-seronegative subjects have attributes similar in quality to those from seropositive subjects, including high-avidity antibodies to viral antigens, coverage against a panel of genetically distinct clinical isolates, and protection against viral infection in diverse types of human cells in culture. More importantly, vaccination appeared efficient in priming the human immune system, inducing memory B cells in six V160 recipients at frequencies comparable to those of three HCMV-seropositive subjects. Our results demonstrate the ability of V160 to induce robust and durable humoral memory responses to HCMV, justifying further clinical evaluation of the vaccine against congenital HCMV.IMPORTANCEIn utero HCMV infection can lead to miscarriage or childhood disabilities, and an effective vaccine is urgently needed. Since children born to women who are seropositive prior to pregnancy are less likely to be affected by congenital HCMV infection, it has been hypothesized that a vaccine capable of inducing an immune response resembling the responses in HCMV-seropositive women may be effective. We previously described a replication-defective virus vaccine that has been demonstrated safe and immunogenic in HCMV-seronegative subjects. Here, we conducted additional analyses to show that the vaccine can induce antibodies with functional attributes similar to those from HCMV-seropositive subjects. Importantly, vaccination can induce long-lived memory B cells at frequencies comparable to those seen in HCMV-seropositive subjects. We conclude that this vaccine is a promising candidate that warrants further clinical evaluation for prevention of congenital HCMV.

Increased muscle force production and bone mineral density in ActRIIB-Fc-treated mature rodents.

Myostatin is a highly conserved member of the transforming growth factor-ß ligand family known to regulate muscle growth via activation of activin receptors. A fusion protein consisting of the extracellular ligand-binding domain of activin type IIB receptor with the Fc portion of human immunoglobulin G (ActRIIB-Fc) was used to inhibit signaling through this pathway. Here, we study the effects of this fusion protein in adult, 18-month-old, and orchidectomized mice. Significant muscle growth and enhanced muscle function were observed in adult mice treated for 3 days with ActRIIB-Fc. The ActRIIB-Fc-treated mice had enhanced fast fatigable muscle function, with only minor enhancement of fatigue-resistant fiber function. The ActRIIB-Fc-treated 18-month-old mice and orchidectomized mice showed significantly improved muscle function. Treatment with ActRIIB-Fc also increased bone mineral density and serum levels of a marker of bone formation. These observations highlight the potential of targeting ActRIIB receptor to treat age-related and hypogonadism-associated musculoskeletal degeneration.

Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay.

The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.

A cell-based beta-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication.

This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses beta-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4-AM as a beta-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of beta-lactamase, to eliminate residual beta-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.

Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase.

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.

Persistent replication of hepatitis C virus replicons expressing the beta-lactamase reporter in subpopulations of highly permissive Huh7 cells.

Progress toward development of better therapies for the treatment of hepatitis C virus (HCV) infection has been hampered by poor understanding of HCV biology and the lack of biological assays suitable for drug screening. Here we describe a powerful HCV replication system that employs HCV replicons expressing the beta-lactamase reporter (bla replicons) and subpopulations of Huh7 cells that are more permissive (or "enhanced") to HCV replication than naïve Huh7 cells. Enhanced cells represent a small fraction of permissive cells present among naïve Huh7 cells that is enriched during selection with replicons expressing the neomycin phosphotransferase gene (neo replicons). The level of permissiveness of cell lines harboring neo replicons can vary greatly, and the enhanced phenotype is usually revealed upon removal of the neo replicon with inhibitors of HCV replication. Replicon removal is responsible for increased permissiveness, since this effect could be reproduced either with alpha interferon or with an HCV NS5B inhibitor. Moreover, adaptive mutations present in the replicon genome used during selection do not influence the permissiveness of the resulting enhanced-cell population, suggesting that the mechanisms governing the permissiveness of enhanced cells are independent from viral adaptation. Because the beta-lactamase reporter allows simultaneous quantitation of replicon-harboring cells and reporter activity, it was possible to investigate the relationship between genome replication activity and the frequency with which transfected genomes can establish persistent replication. Our study demonstrates that differences in the replication potential of the viral genome are manifested primarily in the frequency with which persistent replication is established but modestly affect the number of replicons observed per replicon-harboring cell. Replicon copy number was found to vary over a narrow range that may be defined by a minimal number required for persistent maintenance and a maximum that is limited by the availability of essential host factors.