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.

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.

Mental health in medical and biomedical doctoral students during the 2020 COVID-19 pandemic and racial protests.

Concerns about the mental health of students, trainees and staff at universities and medical schools have been growing for many years. Recently, these have been exacerbated by the COVID-19 pandemic and a period of heightened reckoning and protests about systemic racism in the United States in 2020. To better understand the mental health of medical students and biomedical doctoral students at the University of North Carolina at Chapel Hill during this challenging period, we performed a cross-sectional study (n=957) using institutional annual survey data on measures of depression, anxiety, hazardous alcohol use, problems related to substance use, and suicidal ideation. These data were collected in 2019 and 2020, and were analyzed by type of training program, race/ethnicity, gender, sexual orientation, and survey year. Results indicated significant differences for rates of depression, anxiety, and suicidal ideation, with biomedical doctoral students showing greater incidence than medical students, and historically excluded students (e.g., people of color, women, LGBQ+ trainees) showing greater incidence compared to their peers. Of note, mental health remained poor for biomedical doctoral students in 2020 and declined for those belonging to historically excluded populations. The high rates of depression, anxiety, and suicidal ideation reported suggest that training environments need to be improved and support for mental health increased.

Characterizing Protein-Ligand Binding Using Atomistic Simulation and Machine Learning: Application to Drug Resistance in HIV-1 Protease.

Over the past several decades, atomistic simulations of biomolecules, whether carried out using molecular dynamics or Monte Carlo techniques, have provided detailed insights into their function. Comparing the results of such simulations for a few closely related systems has guided our understanding of the mechanisms by which changes such as ligand binding or mutation can alter the function. The general problem of detecting and interpreting such mechanisms from simulations of many related systems, however, remains a challenge. This problem is addressed here by applying supervised and unsupervised machine learning techniques to a variety of thermodynamic observables extracted from molecular dynamics simulations of different systems. As an important test case, these methods are applied to understand the evasion by human immunodeficiency virus type-1 (HIV-1) protease of darunavir, a potent inhibitor to which resistance can develop via the simultaneous mutation of multiple amino acids. Complex mutational patterns have been observed among resistant strains, presenting a challenge to developing a mechanistic picture of resistance in the protease. In order to dissect these patterns and gain mechanistic insight into the role of specific mutations, molecular dynamics simulations were carried out on a collection of HIV-1 protease variants, chosen to include highly resistant strains and susceptible controls, in complex with darunavir. Using a machine learning approach that takes advantage of the hierarchical nature in the relationships among the sequence, structure, and function, an integrative analysis of these trajectories reveals key details of the resistance mechanism, including changes in the protein structure, hydrogen bonding, and protein-ligand contacts.

Interdependence of Inhibitor Recognition in HIV-1 Protease.

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

Elucidating the Interdependence of Drug Resistance from Combinations of Mutations.

HIV-1 protease is responsible for the cleavage of 12 nonhomologous sites within the Gag and Gag-Pro-Pol polyproteins in the viral genome. Under the selective pressure of protease inhibition, the virus evolves mutations within (primary) and outside of (secondary) the active site, allowing the protease to process substrates while simultaneously countering inhibition. The primary protease mutations impede inhibitor binding directly, while the secondary mutations are considered accessory mutations that compensate for a loss in fitness. However, the role of secondary mutations in conferring drug resistance remains a largely unresolved topic. We have shown previously that mutations distal to the active site are able to perturb binding of darunavir (DRV) via the protein's internal hydrogen-bonding network. In this study, we show that mutations distal to the active site, regardless of context, can play an interdependent role in drug resistance. Applying eigenvalue decomposition to collections of hydrogen bonding and van der Waals interactions from a series of molecular dynamics simulations of 15 diverse HIV-1 protease variants, we identify sites in the protease where amino acid substitutions lead to perturbations in nonbonded interactions with DRV and/or the hydrogen-bonding network of the protease itself. While primary mutations are known to drive resistance in HIV-1 protease, these findings delineate the significant contributions of accessory mutations to resistance. Identifying the variable positions in the protease that have the greatest impact on drug resistance may aid in future structure-based design of inhibitors.

A Direct Interaction with RNA Dramatically Enhances the Catalytic Activity of the HIV-1 Protease In Vitro.

Though the steps of human immunodeficiency virus type 1 (HIV-1) virion maturation are well documented, the mechanisms regulating the proteolysis of the Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) remain obscure. One proposed mechanism argues that the maturation intermediate p15NC must interact with RNA for efficient cleavage by the PR. We investigated this phenomenon and found that processing of multiple substrates by the HIV-1 PR was enhanced in the presence of RNA. The acceleration of proteolysis occurred independently from the substrate's ability to interact with nucleic acid, indicating that a direct interaction between substrate and RNA is not necessary for enhancement. Gel-shift assays demonstrated the HIV-1 PR is capable of interacting with nucleic acids, suggesting that RNA accelerates processing reactions by interacting with the PR rather than the substrate. All HIV-1 PRs examined have this ability; however, the HIV-2 PR does not interact with RNA and does not exhibit enhanced catalytic activity in the presence of RNA. No specific sequence or structure was required in the RNA for a productive interaction with the HIV-1 PR, which appears to be principally, though not exclusively, driven by electrostatic forces. For a peptide substrate, RNA increased the kinetic efficiency of the HIV-1 PR by an order of magnitude, affecting both turnover rate (k(cat)) and substrate affinity (K(m)). These results suggest that an allosteric binding site exists on the HIV-1 PR and that HIV-1 PR activity during maturation could be regulated in part by the juxtaposition of the enzyme with virion-packaged RNA.