Drug Design Strategies to Avoid Resistance in Direct-Acting Antivirals and Beyond.

Drug resistance is prevalent across many diseases, rendering therapies ineffective with severe financial and health consequences. Rather than accepting resistance after the fact, proactive strategies need to be incorporated into the drug design and development process to minimize the impact of drug resistance. These strategies can be derived from our experience with viral disease targets where multiple generations of drugs had to be developed to combat resistance and avoid antiviral failure. Significant efforts including experimental and computational structural biology, medicinal chemistry, and machine learning have focused on understanding the mechanisms and structural basis of resistance against direct-acting antiviral (DAA) drugs. Integrated methods show promise for being predictive of resistance and potency. In this review, we give an overview of this research for human immunodeficiency virus type 1, hepatitis C virus, and influenza virus and the lessons learned from resistance mechanisms of DAAs. These lessons translate into rational strategies to avoid resistance in drug design, which can be generalized and applied beyond viral targets. While resistance may not be completely avoidable, rational drug design can and should incorporate strategies at the outset of drug development to decrease the prevalence of drug resistance.

Allosteric quinoxaline-based inhibitors of the flavivirus NS2B/NS3 protease.

Viruses from the Flavivirus genus infect millions of people worldwide and cause severe diseases, including recent epidemics of dengue virus (DENV), and Zika virus (ZIKV). There is currently no antiviral treatment against flavivirus infections, despite considerable efforts to develop inhibitors against essential viral enzymes including NS2B/NS3 protease. Targeting the flavivirus NS2B/NS3 protease proved to be challenging because of the conformational dynamics, topology, and electrostatic properties of the active site. Here, we report the identification of quinoxaline-based allosteric inhibitors by fragment-based drug discovery approach as a promising new drug-like scaffold to target the NS2B/NS3 protease. Enzymatic assays and mutational analysis of the allosteric site in ZIKV NS2B/NS3 protease support noncompetitive inhibition mechanism as well as engineered DENV protease construct indicating the compounds likely compete with the NS2B cofactor for binding to the protease domain. Furthermore, antiviral activity confirmed the therapeutic potential of this new inhibitor scaffold.

Structural basis of substrate specificity in human cytidine deaminase family APOBEC3s.

The human cytidine deaminase family of APOBEC3s (A3s) plays critical roles in both innate immunity and the development of cancers. A3s comprise seven functionally overlapping but distinct members that can be exploited as nucleotide base editors for treating genetic diseases. Although overall structurally similar, A3s have vastly varying deamination activity and substrate preferences. Recent crystal structures of ssDNA-bound A3s together with experimental studies have provided some insights into distinct substrate specificities among the family members. However, the molecular interactions responsible for their distinct biological functions and how structure regulates substrate specificity are not clear. In this study, we identified the structural basis of substrate specificities in three catalytically active A3 domains whose crystal structures have been previously characterized: A3A, A3B- CTD, and A3G-CTD. Through molecular modeling and dynamic simulations, we found an interdependency between ssDNA substrate binding conformation and nucleotide sequence specificity. In addition to the U-shaped conformation seen in the crystal structure with the CTC0 motif, A3A can accommodate the CCC0 motif when ssDNA is in a more linear (L) conformation. A3B can also bind both U- and L-shaped ssDNA, unlike A3G, which can stably recognize only linear ssDNA. These varied conformations are stabilized by sequence-specific interactions with active site loops 1 and 7, which are highly variable among A3s. Our results explain the molecular basis of previously observed substrate specificities in A3s and have implications for designing A3-specific inhibitors for cancer therapy as well as engineering base-editing systems for gene therapy.

Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode.

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.

Deciphering Antifungal Drug Resistance in Pneumocystis jirovecii DHFR with Molecular Dynamics and Machine Learning.

Drug resistance impacts the effectiveness of many new therapeutics. Mutations in the therapeutic target confer resistance; however, deciphering which mutations, often remote from the enzyme active site, drive resistance is challenging. In a series of Pneumocystis jirovecii dihydrofolate reductase variants, we elucidate which interactions are key bellwethers to confer resistance to trimethoprim using homology modeling, molecular dynamics, and machine learning. Six molecular features involving mainly residues that did not vary were the best indicators of resistance.

Mechanism for APOBEC3G catalytic exclusion of RNA and non-substrate DNA.

The potent antiretroviral protein APOBEC3G (A3G) specifically targets and deaminates deoxycytidine nucleotides, generating deoxyuridine, in single stranded DNA (ssDNA) intermediates produced during HIV replication. A non-catalytic domain in A3G binds strongly to RNA, an interaction crucial for recruitment of A3G to the virion; yet, A3G displays no deamination activity for cytidines in viral RNA. Here, we report NMR and molecular dynamics (MD) simulation analysis for interactions between A3Gctd and multiple substrate or non-substrate DNA and RNA, in combination with deamination assays. NMR ssDNA-binding experiments revealed that the interaction with residues in helix1 and loop1 (T201-L220) distinguishes the binding mode of substrate ssDNA from non-substrate. Using 2'-deoxy-2'-fluorine substituted cytidines, we show that a 2'-endo sugar conformation of the target deoxycytidine is favored for substrate binding and deamination. Trajectories of the MD simulation indicate that a ribose 2'-hydroxyl group destabilizes the π-π stacking of the target cytosine and H257, resulting in dislocation of the target cytosine base from the catalytic position. Interestingly, APOBEC3A, which can deaminate ribocytidines, retains the ribocytidine in the catalytic position throughout the MD simulation. Our results indicate that A3Gctd catalytic selectivity against RNA is dictated by both the sugar conformation and 2'-hydroxyl group.

Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody.

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

Molecular Determinants of Epistasis in HIV-1 Protease: Elucidating the Interdependence of L89V and L90M Mutations in Resistance.

Protease inhibitors have the highest potency among antiviral therapies against HIV-1 infections, yet the virus can evolve resistance. Darunavir (DRV), currently the most potent Food and Drug Administration-approved protease inhibitor, retains potency against single-site mutations. However, complex combinations of mutations can confer resistance to DRV. While the interdependence between mutations within HIV-1 protease is key for inhibitor potency, the molecular mechanisms that underlie this control remain largely unknown. In this study, we investigated the interdependence between the L89V and L90M mutations and their effects on DRV binding. These two mutations have been reported to be positively correlated with one another in HIV-1 patient-derived protease isolates, with the presence of one mutation making the probability of the occurrence of the second mutation more likely. The focus of our investigation is a patient-derived isolate, with 24 mutations that we call "KY"; this variant includes the L89V and L90M mutations. Three additional KY variants with back-mutations, KY(V89L), KY(M90L), and the KY(V89L/M90L) double mutation, were used to experimentally assess the individual and combined effects of these mutations on DRV inhibition and substrate processing. The enzymatic assays revealed that the KY(V89L) variant, with methionine at residue 90, is highly resistant, but its catalytic function is compromised. When a leucine to valine mutation at residue 89 is present simultaneously with the L90M mutation, a rescue of catalytic efficiency is observed. Molecular dynamics simulations of these DRV-bound protease variants reveal how the L90M mutation induces structural changes throughout the enzyme that undermine the binding interactions.

NMR and MD studies combined to elucidate inhibitor and water interactions of HIV-1 protease and their modulations with resistance mutations.

Over the last two decades, both the sensitivity of NMR and the time scale of molecular dynamics (MD) simulation have increased tremendously and have advanced the field of protein dynamics. HIV-1 protease has been extensively studied using these two methods, and has presented a framework for cross-evaluation of structural ensembles and internal dynamics by integrating the two methods. Here, we review studies from our laboratories over the last several years, to understand the mechanistic basis of protease drug-resistance mutations and inhibitor responses, using NMR and crystal structure-based parallel MD simulations. Our studies demonstrate that NMR relaxation experiments, together with crystal structures and MD simulations, significantly contributed to the current understanding of structural/dynamic changes due to HIV-1 protease drug resistance mutations.

Target-Specific Prediction of Ligand Affinity with Structure-Based Interaction Fingerprints.

Discovery and optimization of small molecule inhibitors as therapeutic drugs have immensely benefited from rational structure-based drug design. With recent advances in high-resolution structure determination, computational power, and machine learning methodology, it is becoming more tractable to elucidate the structural basis of drug potency. However, the applicability of machine learning models to drug design is limited by the interpretability of the resulting models in terms of feature importance. Here, we take advantage of the large number of available inhibitor-bound HIV-1 protease structures and associated potencies to evaluate inhibitor diversity and machine learning models to predict ligand affinity. First, using a hierarchical clustering approach, we grouped HIV-1 protease inhibitors and identified distinct core structures. Explicit features including protein-ligand interactions were extracted from high-resolution cocrystal structures as 3D-based fingerprints. We found that a gradient boosting machine learning model with this explicit feature attribution can predict binding affinity with high accuracy. Finally, Shapley values were derived to explain local feature importance. We found specific van der Waals (vdW) interactions of key protein residues are pivotal for the predicted potency. Protein-specific and interpretable prediction models can guide the optimization of many small molecule drugs for improved potency.

Probing Structural Changes among Analogous Inhibitor-Bound Forms of HIV-1 Protease and a Drug-Resistant Mutant in Solution by Nuclear Magnetic Resonance.

In the era of state-of-the-art inhibitor design and high-resolution structural studies, detection of significant but small protein structural differences in the inhibitor-bound forms is critical to further developing the inhibitor. Here, we probed differences in HIV-1 protease (PR) conformation among darunavir and four analogous inhibitor-bound forms and compared them with a drug-resistant mutant using nuclear magnetic resonance chemical shifts. Changes in amide chemical shifts of wild-type (WT) PR among these inhibitor-bound forms, ΔCSP, were subtle but detectable and extended >10 Å from the inhibitor-binding site, asymmetrically between the two subunits of PR. Molecular dynamics simulations revealed differential local hydrogen bonding as the molecular basis of this remote asymmetric change. Inhibitor-bound forms of the drug-resistant mutant also showed a similar long-range ΔCSP pattern. Differences in ΔCSP values of the WT and the mutant (ΔΔCSPs) were observed at the inhibitor-binding site and in the surrounding region. Comparing chemical shift changes among highly analogous inhibitors and ΔΔCSPs effectively eliminated local environmental effects stemming from different chemical groups and enabled exploitation of these sensitive parameters to detect subtle protein conformational changes and to elucidate asymmetric and remote conformational effects upon inhibitor interaction.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 µM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV.IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti, continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors provide novel scaffolds that enable exploiting the prime side of the protease active site, with the aim of achieving better specificity and lower hydrophilicity than those of current scaffolds in the design of antiflaviviral inhibitors.

Structural and molecular analysis of a protective epitope of Lyme disease antigen OspA and antibody interactions.

The murine monoclonal antibody LA-2 recognizes a clinically protective epitope on outer surface protein (OspA) of Borrelia burgdorferi, the causative agent of Lyme disease in North America. Human antibody equivalence to LA-2 is the best serologic correlate of protective antibody responses following OspA vaccination. Understanding the structural and functional basis of the LA-2 protective epitope is important for developing OspA-based vaccines and discovering prophylactic antibodies against Lyme disease. Here, we present a detailed structure-based analysis of the LA-2/OspA interaction interface and identification of residues mediating antibody recognition. Mutations were introduced into both OspA and LA-2 on the basis of computational predictions on the crystal structure of the complex and experimentally tested for in vitro binding and borreliacidal activity. We find that Y32 and H49 on the LA-2 light chain, N52 on the LA-2 heavy chain and residues A208, N228 and N251 on OspA were the key constituents of OspA/LA-2 interface. These results reveal specific residues that may be exploited to modulate recognition of the protective epitope of OspA and have implications for developing prophylactic passive antibodies.

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease.

Drug resistance mutations in response to HIV-1 protease inhibitors are selected not only in the drug target but elsewhere in the viral genome, especially at the protease cleavage sites in the precursor protein Gag. To understand the molecular basis of this protease-substrate coevolution, we solved the crystal structures of drug resistant I50V/A71V HIV-1 protease with p1-p6 substrates bearing coevolved mutations. Analyses of the protease-substrate interactions reveal that compensatory coevolved mutations in the substrate do not restore interactions lost due to protease mutations, but instead establish other interactions that are not restricted to the site of mutation. Mutation of a substrate residue has distal effects on other residues' interactions as well, including through the induction of a conformational change in the protease. Additionally, molecular dynamics simulations suggest that restoration of active site dynamics is an additional constraint in the selection of coevolved mutations. Hence, protease-substrate coevolution permits mutational, structural, and dynamic changes via molecular mechanisms that involve distal effects contributing to drug resistance.

Molecular and Dynamic Mechanism Underlying Drug Resistance in Genotype 3 Hepatitis C NS3/4A Protease.

Hepatitis C virus (HCV), affecting an estimated 150 million people worldwide, is the leading cause of viral hepatitis, cirrhosis and hepatocellular carcinoma. HCV is genetically diverse with six genotypes (GTs) and multiple subtypes of different global distribution and prevalence. Recent development of direct-acting antivirals against HCV including NS3/4A protease inhibitors (PIs) has greatly improved treatment outcomes for GT-1. However, all current PIs exhibit significantly lower potency against GT-3. Lack of structural data on GT-3 protease has limited our ability to understand PI failure in GT-3. In this study the molecular basis for reduced potency of current inhibitors against GT-3 NS3/4A protease is elucidated with structure determination, molecular dynamics simulations and inhibition assays. A chimeric GT-1a3a NS3/4A protease amenable to crystallization was engineered to recapitulate decreased sensitivity of GT-3 protease to PIs. High-resolution crystal structures of this GT-1a3a bound to 3 PIs, asunaprevir, danoprevir and vaniprevir, had only subtle differences relative to GT-1 despite orders of magnitude loss in affinity. In contrast, hydrogen-bonding interactions within and with the protease active site and dynamic fluctuations of the PIs were drastically altered. The correlation between loss of intermolecular dynamics and inhibitor potency suggests a mechanism where polymorphisms between genotypes (or selected mutations) in the drug target confer resistance through altering the intermolecular dynamics of the protein-inhibitor complex.

HIV-1 protease-substrate coevolution in nelfinavir resistance.

UNLABELLED: Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. IMPORTANCE: Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations.

Burial of nonpolar surface area and thermodynamic stabilization of globins as a function of chain elongation.

Proteins are biosynthesized from N to C terminus before they depart from the ribosome and reach their bioactive state in the cell. At present, very little is known about the evolution of conformation and the free energy of the nascent protein with chain elongation. These parameters critically affect the extent of folding during ribosome-assisted biosynthesis. Here, we address the impact of vectorial amino acid addition on the burial of nonpolar surface area and on the free energy of native-like structure formation in the absence of the ribosomal machinery. We focus on computational predictions on proteins bearing the globin fold, which is known to encompass the 3/3, 2/2, and archaeal subclasses. We find that the burial of nonpolar surface increases progressively with chain elongation, leading to native-like conformations upon addition of the last C-terminal residues, corresponding to incorporation of the last two helices. Additionally, the predicted folding entropy for generating native-like structures becomes less unfavorable at nearly complete chain lengths, suggesting a link between the late burial of nonpolar surface and water release. Finally, the predicted folding free energy takes a progressive favorable dip toward more negative values, as the chain gets longer. These results suggest that thermodynamic stabilization of the native structure of newly synthesized globins during translation in the cell is significantly enhanced as the chain elongates. This is especially true upon departure of the last C-terminal residues from the ribosomal tunnel, which hosts ca., 30-40 amino acids. Hence, we propose that release from the ribosome is a crucial step in the life of single-domain proteins in the cell.

Drug resistance conferred by mutations outside the active site through alterations in the dynamic and structural ensemble of HIV-1 protease.

HIV-1 protease inhibitors are part of the highly active antiretroviral therapy effectively used in the treatment of HIV infection and AIDS. Darunavir (DRV) is the most potent of these inhibitors, soliciting drug resistance only when a complex combination of mutations occur both inside and outside the protease active site. With few exceptions, the role of mutations outside the active site in conferring resistance remains largely elusive. Through a series of DRV-protease complex crystal structures, inhibition assays, and molecular dynamics simulations, we find that single and double site mutations outside the active site often associated with DRV resistance alter the structure and dynamic ensemble of HIV-1 protease active site. These alterations correlate with the observed inhibitor binding affinities for the mutants, and suggest a network hypothesis on how the effect of distal mutations are propagated to pivotal residues at the active site and may contribute to conferring drug resistance.

Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance.

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.