Molnupiravir maintains antiviral activity against SARS-CoV-2 variants and exhibits a high barrier to the development of resistance.

Molnupiravir, an oral prodrug of N-hydroxycytidine (NHC), previously demonstrated broad in vitro antiviral activity against multiple RNA viruses and has shown a high barrier to the development of resistance. Here, we present the antiviral activity of NHC against recent SARS-CoV-2 variants and the results of resistance selection studies to better understand the potential for viral resistance to NHC. NHC activity against SARS-CoV-2 variants omicron (BA.1, BA.1.1, BA.2, BA.4, BA.4.6, BA.5, BQ.1.1, XBB.1, and XBB.1.5), alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), lambda (C.37), and mu (B.1.621) was evaluated in Vero E6 cells using cytopathic effect assays. Resistance selection studies were performed by passaging SARS-CoV-2 (WA1) in the presence of NHC or a 3C-like protease inhibitor (MRK-A) in Vero E6 cells. Supernatants from cultures exhibiting a cytopathic effect score of ≥2 were re-passaged, and IC50 values were estimated. Whole-genome deep sequencing was performed on viral RNA isolated at each passage. NHC demonstrated similar potency against all SARS-CoV-2 variants evaluated. No evidence of SARS-CoV-2 phenotypic or genotypic resistance to NHC was observed following 30 passages. A random pattern of nucleotide changes was observed in NHC cultures, consistent with the drug's mechanism of action. In contrast, resistance was readily selected in all three MRK-A control cultures with the selection of a T21I substitution in the 3C-like protease. In conclusion, molnupiravir maintains antiviral activity across all major SARS-CoV-2 variants. Furthermore, no evidence of viral resistance to NHC was observed, supporting previous reports that NHC has a high barrier to developing resistance.

SARS-CoV-2 tropism, entry, replication, and propagation: Considerations for drug discovery and development.

Since the initial report of the novel Coronavirus Disease 2019 (COVID-19) emanating from Wuhan, China, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally. While the effects of SARS-CoV-2 infection are not completely understood, there appears to be a wide spectrum of disease ranging from mild symptoms to severe respiratory distress, hospitalization, and mortality. There are no Food and Drug Administration (FDA)-approved treatments for COVID-19 aside from remdesivir; early efforts to identify efficacious therapeutics for COVID-19 have mainly focused on drug repurposing screens to identify compounds with antiviral activity against SARS-CoV-2 in cellular infection systems. These screens have yielded intriguing hits, but the use of nonhuman immortalized cell lines derived from non-pulmonary or gastrointestinal origins poses any number of questions in predicting the physiological and pathological relevance of these potential interventions. While our knowledge of this novel virus continues to evolve, our current understanding of the key molecular and cellular interactions involved in SARS-CoV-2 infection is discussed in order to provide a framework for developing the most appropriate in vitro toolbox to support current and future drug discovery efforts.

Activity of Tricyclic Pyrrolopyrimidine Gyrase B Inhibitor against Mycobacterium abscessus.

Tricyclic pyrrolopyrimidines (TPPs) are a new class of antibacterials inhibiting the ATPase of DNA gyrase. TPP8, a representative of this class, is active against Mycobacterium abscessus in vitro. Spontaneous TPP8 resistance mutations mapped to the ATPase domain of M. abscessus DNA gyrase, and the compound inhibited DNA supercoiling activity of recombinant M. abscessus enzyme. Further profiling of TPP8 in macrophage and mouse infection studies demonstrated proof-of-concept activity against M. abscessus ex vivo and in vivo.

Selective small-molecule inhibition of an RNA structural element.

Riboswitches are non-coding RNA structures located in messenger RNAs that bind endogenous ligands, such as a specific metabolite or ion, to regulate gene expression. As such, riboswitches serve as a novel, yet largely unexploited, class of emerging drug targets. Demonstrating this potential, however, has proven difficult and is restricted to structurally similar antimetabolites and semi-synthetic analogues of their cognate ligand, thus greatly restricting the chemical space and selectivity sought for such inhibitors. Here we report the discovery and characterization of ribocil, a highly selective chemical modulator of bacterial riboflavin riboswitches, which was identified in a phenotypic screen and acts as a structurally distinct synthetic mimic of the natural ligand, flavin mononucleotide, to repress riboswitch-mediated ribB gene expression and inhibit bacterial cell growth. Our findings indicate that non-coding RNA structural elements may be more broadly targeted by synthetic small molecules than previously expected.

In vitro studies evaluating the activity of imipenem in combination with relebactam against Pseudomonas aeruginosa.

BACKGROUND: The prevalence of antibiotic resistance is increasing, and multidrug-resistant Pseudomonas aeruginosa has been identified as a serious threat to human health. The production of ß-lactamase is a key mechanism contributing to imipenem resistance in P. aeruginosa. Relebactam is a novel ß-lactamase inhibitor, active against class A and C ß-lactamases, that has been shown to restore imipenem susceptibility. In a series of studies, we assessed the interaction of relebactam with key mechanisms involved in carbapenem resistance in P. aeruginosa and to what extent relebactam might overcome imipenem non-susceptibility. RESULTS: Relebactam demonstrated no intrinsic antibacterial activity against P. aeruginosa, had no inoculum effect, and was not subject to efflux. Enzymology studies showed relebactam is a potent (overall inhibition constant: 27 nM), practically irreversible inhibitor of P. aeruginosa AmpC. Among P. aeruginosa clinical isolates from the SMART global surveillance program (2009, n = 993; 2011, n = 1702; 2015, n = 5953; 2016, n = 6165), imipenem susceptibility rates were 68.4% in 2009, 67.4% in 2011, 70.4% in 2015, and 67.3% in 2016. With the addition of 4 µg/mL relebactam, imipenem susceptibility rates increased to 87.6, 86.0, 91.7, and 89.8%, respectively. When all imipenem-non-susceptible isolates were pooled, the addition of 4 µg/mL relebactam reduced the mode imipenem minimum inhibitory concentration (MIC) 8-fold (from 16 µg/mL to 2 µg/mL) among all imipenem-non-susceptible isolates. Of 3747 imipenem-non-susceptible isolates that underwent molecular profiling, 1200 (32%) remained non-susceptible to the combination imipenem/relebactam (IMI/REL); 42% of these encoded class B metallo-ß-lactamases, 11% encoded a class A GES enzyme, and no class D enzymes were detected. No relationship was observed between alleles of the chromosomally-encoded P. aeruginosa AmpC and IMI/REL MIC. CONCLUSIONS: IMI/REL exhibited potential in the treatment of carbapenem-resistant P. aeruginosa infections, with the exception of isolates encoding class B, some GES alleles, and class D carbapenemases.

Functional defects in Clostridium difficile TcdB toxin uptake identify CSPG4 receptor-binding determinants.

Clostridium difficile is a major nosocomial pathogen that produces two exotoxins, TcdA and TcdB, with TcdB thought to be the primary determinant in human disease. TcdA and TcdB are large, multidomain proteins, each harboring a cytotoxic glucosyltransferase domain that is delivered into the cytosol from endosomes via a translocation domain after receptor-mediated endocytosis of toxins from the cell surface. Although there are currently no known host cell receptors for TcdA, three cell-surface receptors for TcdB have been identified: CSPG4, NECTIN3, and FZD1/2/7. The sites on TcdB that mediate binding to each receptor are not defined. Furthermore, it is not known whether the combined repetitive oligopeptide (CROP) domain is involved in or required for receptor binding. Here, in a screen designed to identify sites in TcdB that are essential for target cell intoxication, we identified a region at the junction of the translocation and the CROP domains that is implicated in CSPG4 binding. Using a series of C-terminal truncations, we show that the CSPG4-binding site on TcdB extends into the CROP domain, requiring three short repeats for binding and for full toxicity on CSPG4-expressing cells. Consistent with the location of the CSPG4-binding site on TcdB, we show that the anti-TcdB antibody bezlotoxumab, which binds partially within the first three short repeats, prevents CSPG4 binding to TcdB. In addition to establishing the binding region for CSPG4, this work ascribes for the first time a role in TcdB CROPs in receptor binding and further clarifies the relative roles of host receptors in TcdB pathogenesis.

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.

Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro ß-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the ß-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore ß-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

Mechanism of action and epitopes of Clostridium difficile toxin B-neutralizing antibody bezlotoxumab revealed by X-ray crystallography.

The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a ß-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Broad coverage of genetically diverse strains of Clostridium difficile by actoxumab and bezlotoxumab predicted by in vitro neutralization and epitope modeling.

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.

Novel Pan-Coronavirus 3CL Protease Inhibitor MK-7845: Biological and Pharmacological Profiling.

Severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) continues to be a global threat due to its ability to evolve and generate new subvariants, leading to new waves of infection. Additionally, other coronaviruses like Middle East respiratory syndrome coronavirus (MERS-CoV, formerly known as hCoV-EMC), which first emerged in 2012, persist and continue to present a threat of severe illness to humans. The continued identification of novel coronaviruses, coupled with the potential for genetic recombination between different strains, raises the possibility of new coronavirus clades of global concern emerging. As a result, there is a pressing need for pan-CoV therapeutic drugs and vaccines. After the extensive optimization of an HCV protease inhibitor screening hit, a novel 3CLPro inhibitor (MK-7845) was discovered and subsequently profiled. MK-7845 exhibited nanomolar in vitro potency with broad spectrum activity against a panel of clinical SARS-CoV-2 subvariants and MERS-CoV. Furthermore, when administered orally, MK-7845 demonstrated a notable reduction in viral burdens by >6 log orders in the lungs of transgenic mice infected with SARS-CoV-2 (K18-hACE2 mice) and MERS-CoV (K18-hDDP4 mice).

Virologic Outcomes with Molnupiravir in Non-hospitalized Adult Patients with COVID-19 from the Randomized, Placebo-Controlled MOVe-OUT Trial.

INTRODUCTION: The randomized, placebo-controlled, double-blind MOVe-OUT trial demonstrated molnupiravir (800 mg every 12 h for 5 days) as safe and effective for outpatient treatment of mild-to-moderate COVID-19, significantly reducing the risk of hospitalization/death in high-risk adults. At the time of that report, virologic assessments from the trial were partially incomplete as a result of their time-intensive nature. Here we present final results from all prespecified virology endpoints in MOVe-OUT based on the full trial dataset. METHODS: Nasopharyngeal swabs were collected at baseline (day 1, prior to first dose) and days 3, 5 (end-of-treatment visit), 10, 15, and 29. From these samples, change from baseline in SARS-CoV-2 RNA titers (determined by quantitative PCR), detection of infectious SARS-CoV-2 (by plaque assay), and SARS-CoV-2 viral error induction (determined by whole genome next-generation sequencing) were assessed as exploratory endpoints. RESULTS: Molnupiravir was associated with greater mean reductions from baseline in SARS-CoV-2 RNA than placebo (including 50% relative reduction at end-of-treatment) through day 10. Among participants with infectious virus detected at baseline (n = 96 molnupiravir, n = 97 placebo) and evaluable post-baseline samples, no molnupiravir-treated participant had infectious SARS-CoV-2 by day 3, whereas infectious virus was recovered from 21% of placebo-arm participants on day 3 and 2% at end-of-treatment. Consistent with molnupiravir's mechanism of action, sequence analysis demonstrated that molnupiravir was associated with an increased number of low-frequency transition errors randomly distributed across the SARS-CoV-2 RNA genome compared with placebo (median 143.5 molnupiravir, 15 placebo), while transversion errors were infrequent overall (median 2 in both arms). Outcomes were consistent regardless of baseline SARS-CoV-2 clade, presence of SARS-CoV-2-specific immune response, or viral load. CONCLUSIONS: A 5-day course of orally administered molnupiravir demonstrated a consistently greater virologic effect than placebo, including rapidly eliminating infectious SARS-CoV-2, in high-risk outpatients with mild-to-moderate COVID-19. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04575597.

Retrospective assessment of clonality of a legacy cell line by analytical subcloning of the master cell bank.

In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

The Invention of WM382, a Highly Potent PMIX/X Dual Inhibitor toward the Treatment of Malaria.

Drug resistance to first-line antimalarials-including artemisinin-is increasing, resulting in a critical need for the discovery of new agents with novel mechanisms of action. In collaboration with the Walter and Eliza Hall Institute and with funding from the Wellcome Trust, a phenotypic screen of Merck's aspartyl protease inhibitor library identified a series of plasmepsin X (PMX) hits that were more potent than chloroquine. Inspired by a PMX homology model, efforts to optimize the potency resulted in the discovery of leads that, in addition to potently inhibiting PMX, also inhibit another essential aspartic protease, plasmepsin IX (PMIX). Further potency and pharmacokinetic profile optimization efforts culminated in the discovery of WM382, a very potent dual PMIX/X inhibitor with robust in vivo efficacy at multiple stages of the malaria parasite life cycle and an excellent resistance profile.

Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax.

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.

Rapid Evolution of a Fragment-like Molecule to Pan-Metallo-Beta-Lactamase Inhibitors: Initial Leads toward Clinical Candidates.

With the emergence and rapid spreading of NDM-1 and existence of clinically relevant VIM-1 and IMP-1, discovery of pan inhibitors targeting metallo-beta-lactamases (MBLs) became critical in our battle against bacterial infection. Concurrent with our fragment and high-throughput screenings, we performed a knowledge-based search of known metallo-beta-lactamase inhibitors (MBLIs) to identify starting points for early engagement of medicinal chemistry. A class of compounds exemplified by 11, discovered earlier as B. fragilis metallo-beta-lactamase inhibitors, was selected for in silico virtual screening. From these efforts, compound 12 was identified with activity against NDM-1 only. Initial exploration on metal binding design followed by structure-guided optimization led to the discovery of a series of compounds represented by 23 with a pan MBL inhibition profile. In in vivo studies, compound 23 in combination with imipenem (IPM) robustly lowered the bacterial burden in a murine infection model and became the lead for the invention of MBLI clinical candidates.

Understanding the effect of increased cell specific productivity on galactosylation of monoclonal antibodies produced using Chinese hamster ovary cells.

Achieving optimal productivity and desired product quality of the therapeutic monoclonal antibody (mAb) is one of the primary goals of process development. Across the various mAb programs at our company, we observed that increasing the specific productivity (qp) results in a decrease in the % galactosylation (%Gal) level on the protein. In order to gain further insight into this correlation, cells were cultured under different process conditions such as pH or media osmolality or in the presence of supplements such as sodium butyrate. A range of qp and N-glycan profiles were obtained with the greatest changes observed under high pH (lower qp, higher %Gal), higher osmolality (higher qp, lower %Gal) or sodium butyrate (moderately higher qp, moderately lower %Gal) conditions. Abundance of individual glycan species highlighted different bottlenecks in the N-glycosylation pathway depending on the treatment condition. Transcriptomics analysis was performed to identify changes in gene expression profiles that correlate with the inverse relationship between qp and %Gal. Results showed downregulation of Beta-1,4-galactosyltransferase 1 (B4GalT1), UDP-GlcNAc and Mn2+ transporter (slc35a3 and slc39a8 respectively) for the high osmolality conditions. Significant downregulation of slc39a8 (Mn2+ transporter) was observed for the sodium butyrate condition. No significant differences were observed for any of the genes in the N-glycosylation pathway under the high pH condition even though this condition showed highest %Gal. Together, data suggests that different treatments have distinct complex mechanisms by which the overall glycan levels of a mAb are influenced. Further studies based on these results will help build the knowledge necessary to design strategies to obtain the desired productivity and product quality of mAbs.

Improving Antibody Production in Stably Transfected CHO Cells by CRISPR-Cas9-Mediated Inactivation of Genes Identified in a Large-Scale Screen with Chinese Hamster-Specific siRNAs.

The Chinese hamster ovary (CHO) cell line is commonly used for the production of biotherapeutics. As cell productivity directly affects the cost of production, methods are developed to manipulate the expression of specific genes that are known to be involved in protein synthesis, folding, and secretion to increase productivity. However, there are no large-scale CHO-specific functional screens to identify novel gene targets that impact the production of secreted recombinant proteins. Here, a large-scale, CHO cell-specific small interfering RNA screen is performed to identify genes that consistently enhance antibody production when silenced in a panel of seven CHO cell lines. Four genes, namely, Cyp1a2, Atp5s, Dgki, and P3h2, are identified, and then selected for CRISPR-Cas9 knockout validation in recombinant CHO cell lines. Single knockout of Cyp1a2, Atp5s, or Dgki, but not P3h2, results in a more than 90% increase in specific antibody productivity. Overall, the knockout of Cyp1a2 demonstrates the most significant improvement of antibody production, with a minimal impact on cell growth.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) affects gene expression pathways beyond cholesterol metabolism in liver cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) induces degradation of low-density lipoprotein receptor (LDLR) in the liver. It is being pursued as a therapeutic target for LDL-cholesterol reduction. Earlier genome-wide gene expression studies showed that PCSK9 over-expression in HepG2 cells resulted in up-regulation of genes in cholesterol biosynthesis and down-regulation of genes in stress response pathways; however, it was not known whether these changes were directly regulated by PCSK9 or were secondary to PCSK9-induced changes to the intracellular environment. In order to further understand the biological function of PCSK9 we treated HepG2 cells with purified recombinant wild type (WT) and D374Y gain-of-function PCSK9 proteins for 8, 24, and 48 h, and used microarray analysis to identify genome-wide expression changes and pathways. These results were compared to the changes induced by culturing HepG2 cells in cholesterol-free medium, mimicking the intracellular environment of cholesterol starvation. We determined that PCSK9-induced up-regulation of cholesterol biosynthesis genes resulted from intracellular cholesterol starvation. In addition, we identified novel pathways that are presumably regulated by PCSK9 and are independent of its effects on cholesterol uptake. These pathways included "protein ubiquitination," "xenobiotic metabolism," "cell cycle," and "inflammation and stress response." Our results indicate that PCSK9 affects metabolic pathways beyond cholesterol metabolism in HepG2 cells.

Structure-function analysis of human immunodeficiency virus type 1 gp120 amino acid mutations associated with resistance to the CCR5 coreceptor antagonist vicriviroc.

Vicriviroc (VCV) is a small-molecule CCR5 coreceptor antagonist currently in clinical trials for treatment of R5-tropic human immunodeficiency virus type 1 (HIV-1) infection. With this drug in development, identification of resistance mechanisms to VCV is needed to allow optimal outcomes in clinical practice. In this study we further characterized VCV resistance in a lab-adapted, VCV-resistant RU570 virus (RU570-VCV(res)). We show that K305R, R315Q, and K319T amino acid changes in the V3 loop, along with P437S in C4, completely reproduced the resistance phenotype in a chimeric ADA envelope containing the C2-V5 region from RU570 passage control gp120. The K305R amino acid change primarily impacted the degree of resistance, whereas K319T contributed to both resistance and virus infectivity. The P437S mutation in C4 had more influence on the relative degree of virus infectivity, while the R315Q mutation contributed to the virus concentration-dependent phenotypic resistance pattern observed for RU570-VCV(res). RU570-VCV(res) pseudovirus entry with VCV-bound CCR5 was dramatically reduced by Y10A, D11A, Y14A, and Y15A mutations in the N terminus of CCR5, whereas these mutations had less impact on entry in the absence of VCV. Notably, an additional Q315E/I317F substitution in the crown region of the V3 loop enhanced resistance to VCV, resulting in a stronger dependence on the N terminus for viral entry. By fitting the envelope mutations to a molecular model of a recently described docked N-terminal CCR5 peptide consisting of residues 2 to 15 in complex with HIV-1 gp120 CD4, potential new interactions in gp120 with the N terminus of CCR5 were uncovered. The cumulative results of this study suggest that as the RU570 VCV-resistant virus adapted to use the drug-bound receptor, it also developed an increased reliance on the N terminus of CCR5.

Dual Plasmepsin-Targeting Antimalarial Agents Disrupt Multiple Stages of the Malaria Parasite Life Cycle.

Artemisin combination therapy (ACT) is the main treatment option for malaria, which is caused by the intracellular parasite Plasmodium. However, increased resistance to ACT highlights the importance of finding new drugs. Recently, the aspartic proteases Plasmepsin IX and X (PMIX and PMX) were identified as promising drug targets. In this study, we describe dual inhibitors of PMIX and PMX, including WM382, that block multiple stages of the Plasmodium life cycle. We demonstrate that PMX is a master modulator of merozoite invasion and direct maturation of proteins required for invasion, parasite development, and egress. Oral administration of WM382 cured mice of P. berghei and prevented blood infection from the liver. In addition, WM382 was efficacious against P. falciparum asexual infection in humanized mice and prevented transmission to mosquitoes. Selection of resistant P. falciparum in vitro was not achievable. Together, these show that dual PMIX and PMX inhibitors are promising candidates for malaria treatment and prevention.