Structural and thermodynamic basis of amprenavir/darunavir and atazanavir resistance in HIV-1 protease with mutations at residue 50.

Drug resistance occurs through a series of subtle changes that maintain substrate recognition but no longer permit inhibitor binding. In HIV-1 protease, mutations at I50 are associated with such subtle changes that confer differential resistance to specific inhibitors. Residue I50 is located at the protease flap tips, closing the active site upon ligand binding. Under selective drug pressure, I50V/L substitutions emerge in patients, compromising drug susceptibility and leading to treatment failure. The I50V substitution is often associated with amprenavir (APV) and darunavir (DRV) resistance, while the I50L substitution is observed in patients failing atazanavir (ATV) therapy. To explain how APV, DRV, and ATV susceptibility are influenced by mutations at residue 50 in HIV-1 protease, structural and binding thermodynamics studies were carried out on I50V/L-substituted protease variants in the compensatory mutation A71V background. Reduced affinity to both I50V/A71V and I50L/A71V double mutants is largely due to decreased binding entropy, which is compensated for by enhanced enthalpy for ATV binding to I50V variants and APV binding to I50L variants, leading to hypersusceptibility in these two cases. Analysis of the crystal structures showed that the substitutions at residue 50 affect how APV, DRV, and ATV bind the protease with altered van der Waals interactions and that the selection of I50V versus I50L is greatly influenced by the chemical moieties at the P1 position for APV/DRV and the P2 position for ATV. Thus, the varied inhibitor susceptibilities of I50V/L protease variants are largely a direct consequence of the interdependent changes in protease inhibitor interactions.

Direct delivery of stabilized Cas-embedded base editors achieves efficient and accurate editing of clinically relevant targets.

Over the last 5 years, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBE's have the significant liabilities of off-target and bystander editing activities, in part due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study we engineeredhighly stabilized Cas-embedded CBEs (sCE_CBEs) that integrate several recent advances, andthat are highly expressible and soluble for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting sCE_CBE RNP complexes efficiently and specifically target TC dinucleotides with minimal off-target or bystander mutations. Additional uracil glycosylase inhibitor (UGI) protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation to untreated levels. A single electroporation was sufficient to effectively edit the therapeutically relevant locus for sickle cell disease in hematopoietic stem and progenitor cells (HSPC) in a dose dependent manner without cellular toxicity. Significantly, these sCE_CBE RNPs permitted for the transplantation of edited HSPCs confirming highly efficient editing in engrafting hematopoietic stem cells in mice. The success of the designed sCBE editors, with improved solubility and enhanced on-target editing, demonstrates promising agents for cytosine base editing at other disease-related sites in HSPCs and other cell types.

Systematic Analyses of the Resistance Potential of Drugs Targeting SARS-CoV-2 Main Protease.

Drugs that target the main protease (Mpro) of SARS-CoV-2 are effective therapeutics that have entered clinical use. Wide-scale use of these drugs will apply selection pressure for the evolution of resistance mutations. To understand resistance potential in Mpro, we performed comprehensive surveys of amino acid changes that can cause resistance to nirmatrelvir (Pfizer), and ensitrelvir (Xocova) in a yeast screen. We identified 142 resistance mutations for nirmatrelvir and 177 for ensitrelvir, many of which have not been previously reported. Ninety-nine mutations caused apparent resistance to both inhibitors, suggesting likelihood for the evolution of cross-resistance. The mutation with the strongest drug resistance score against nirmatrelvir in our study (E166V) was the most impactful resistance mutation recently reported in multiple viral passaging studies. Many mutations that exhibited inhibitor-specific resistance were consistent with the distinct interactions of each inhibitor in the substrate binding site. In addition, mutants with strong drug resistance scores tended to have reduced function. Our results indicate that strong pressure from nirmatrelvir or ensitrelvir will select for multiple distinct-resistant lineages that will include both primary resistance mutations that weaken interactions with drug while decreasing enzyme function and compensatory mutations that increase enzyme activity. The comprehensive identification of resistance mutations enables the design of inhibitors with reduced potential of developing resistance and aids in the surveillance of drug resistance in circulating viral populations.

HIV-1 protease inhibitors with a P1 phosphonate modification maintain potency against drug-resistant variants by increased interactions with flap residues.

Protease inhibitors are the most potent antivirals against HIV-1, but they still lose efficacy against resistant variants. Improving the resistance profile is key to developing more robust inhibitors, which may be promising candidates for simplified next-generation antiretroviral therapies. In this study, we explored analogs of darunavir with a P1 phosphonate modification in combination with increasing size of the P1' hydrophobic group and various P2' moieties to improve potency against resistant variants. The phosphonate moiety substantially improved potency against highly mutated and resistant HIV-1 protease variants, but only when combined with more hydrophobic moieties at the P1' and P2' positions. Phosphonate analogs with a larger hydrophobic P1' moiety maintained excellent antiviral potency against a panel of highly resistant HIV-1 variants, with significantly improved resistance profiles. The cocrystal structures indicate that the phosphonate moiety makes extensive hydrophobic interactions with the protease, especially with the flap residues. Many residues involved in these protease-inhibitor interactions are conserved, enabling the inhibitors to maintain potency against highly resistant variants. These results highlight the need to balance inhibitor physicochemical properties by simultaneous modification of chemical groups to further improve resistance profiles.

Selection of HIV-1 for resistance to fifth-generation protease inhibitors reveals two independent pathways to high-level resistance.

Darunavir (DRV) is exceptional among potent HIV-1 protease inhibitors (PIs) in high drug concentrations that are achieved in vivo. Little is known about the de novo resistance pathway for DRV. We selected for resistance to high drug concentrations against 10 PIs and their structural precursor DRV. Mutations accumulated through two pathways (anchored by protease mutations I50V or I84V). Small changes in the inhibitor P1'-equivalent position led to preferential use of one pathway over the other. Changes in the inhibitor P2'-equivalent position determined differences in potency that were retained in the resistant viruses and that impacted the selected mutations. Viral variants from the two pathways showed differential selection of compensatory mutations in Gag cleavage sites. These results reveal the high level of selective pressure that is attainable with fifth-generation PIs and how features of the inhibitor affect both the resistance pathway and the residual potency in the face of resistance.

Comprehensive fitness landscape of SARS-CoV-2 Mpro reveals insights into viral resistance mechanisms.

With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.

Mucosal nanobody IgA as inhalable and affordable prophylactic and therapeutic treatment against SARS-CoV-2 and emerging variants.

Anti-COVID antibody therapeutics have been developed but not widely used due to their high cost and escape of neutralization from the emerging variants. Here, we describe the development of VHH-IgA1.1, a nanobody IgA fusion molecule as an inhalable, affordable and less invasive prophylactic and therapeutic treatment against SARS-CoV-2 Omicron variants. VHH-IgA1.1 recognizes a conserved epitope of SARS-CoV-2 spike protein Receptor Binding Domain (RBD) and potently neutralizes major global SARS-CoV-2 variants of concern (VOC) including the Omicron variant and its sub lineages BA.1.1, BA.2 and BA.2.12.1. VHH-IgA1.1 is also much more potent against Omicron variants as compared to an IgG Fc fusion construct, demonstrating the importance of IgA mediated mucosal protection for Omicron infection. Intranasal administration of VHH-IgA1.1 prior to or after challenge conferred significant protection from severe respiratory disease in K18-ACE2 transgenic mice infected with SARS-CoV-2 VOC. More importantly, for cost-effective production, VHH-IgA1.1 produced in Pichia pastoris had comparable potency to mammalian produced antibodies. Our study demonstrates that intranasal administration of affordably produced VHH-IgA fusion protein provides effective mucosal immunity against infection of SARS-CoV-2 including emerging variants.

Genome-scale in vivo CRISPR screen identifies RNLS as a target for beta cell protection in type 1 diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Pluripotent stem cells can now be differentiated into beta cells, thus raising the prospect of a cell replacement therapy for T1D. However, autoimmunity would rapidly destroy newly transplanted beta cells. Using a genome-scale CRISPR screen in a mouse model for T1D, we show that deleting RNLS, a genome-wide association study candidate gene for T1D, made beta cells resistant to autoimmune killing. Structure-based modelling identified the U.S. Food and Drug Administration-approved drug pargyline as a potential RNLS inhibitor. Oral pargyline treatment protected transplanted beta cells in diabetic mice, thus leading to disease reversal. Furthermore, pargyline prevented or delayed diabetes onset in several mouse models for T1D. Our results identify RNLS as a modifier of beta cell vulnerability and as a potential therapeutic target to avert beta cell loss in T1D.

IgA MAb blocks SARS-CoV-2 Spike-ACE2 interaction providing mucosal immunity.

COVID-19 caused by SARS-CoV-2 has become a global pandemic requiring the development of interventions for the prevention or treatment to curtail mortality and morbidity. No vaccine to boost mucosal immunity or as a therapeutic has yet been developed to SARS-CoV-2. In this study we discover and characterize a cross-reactive human IgA monoclonal antibody, MAb362. MAb362 binds to both SARS-CoV and SARS-CoV-2 spike proteins and competitively blocks hACE2 receptor binding, by completely overlapping the hACE2 structural binding epitope. Furthermore, MAb362 IgA neutralizes both pseudotyped SARS-CoV and SARS-CoV-2 in human epithelial cells expressing hACE2. SARS-CoV-2 specific IgA antibodies, such as MAb362, may provide effective immunity against SARS-CoV-2 by inducing mucosal immunity within the respiratory system, a potentially critical feature of an effective vaccine.

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2' Ligands To Optimize Hydrogen Bonding in the Substrate Envelope.

A structure-guided design strategy was used to improve the resistance profile of HIV-1 protease inhibitors by optimizing hydrogen bonding and van der Waals interactions with the protease while staying within the substrate envelope. Stereoisomers of 4-(1-hydroxyethyl)benzene and 4-(1,2-dihydroxyethyl)benzene moieties were explored as P2' ligands providing pairs of diastereoisomers epimeric at P2', which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1' group. While compounds with the 4-(1-hydroxyethyl)benzene P2' moiety maintained excellent antiviral potency against a panel of multidrug-resistant HIV-1 strains, analogues with the polar 4-(1,2-dihydroxyethyl)benzene moiety were less potent, and only the (R)-epimer incorporating a larger 2-ethylbutyl P1' group showed improved potency. Crystal structures of protease-inhibitor complexes revealed strong hydrogen bonding interactions of both (R)- and (S)-stereoisomers of the hydroxyethyl group with Asp30'. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29' and Asp30'. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors.

Structural Adaptation of Darunavir Analogues against Primary Mutations in HIV-1 Protease.

HIV-1 protease is one of the prime targets of agents used in antiretroviral therapy against HIV. However, under selective pressure of protease inhibitors, primary mutations at the active site weaken inhibitor binding to confer resistance. Darunavir (DRV) is the most potent HIV-1 protease inhibitor in clinic; resistance is limited, as DRV fits well within the substrate envelope. Nevertheless, resistance is observed due to hydrophobic changes at residues including I50, V82, and I84 that line the S1/S1' pocket within the active site. Through enzyme inhibition assays and a series of 12 crystal structures, we interrogated susceptibility of DRV and two potent analogues to primary S1' mutations. The analogues had modifications at the hydrophobic P1' moiety compared to DRV to better occupy the unexploited space in the S1' pocket where the primary mutations were located. Considerable losses of potency were observed against protease variants with I84V and I50V mutations for all three inhibitors. The crystal structures revealed an unexpected conformational change in the flap region of I50V protease bound to the analogue with the largest P1' moiety, indicating interdependency between the S1' subsite and the flap region. Collective analysis of protease-inhibitor interactions in the crystal structures using principle component analysis was able to distinguish inhibitor identity and relative potency solely based on van der Waals contacts. Our results reveal the complexity of the interplay between inhibitor P1' moiety and S1' mutations and validate principle component analyses as a useful tool for distinguishing resistance and inhibitor potency.

Dengue Protease Substrate Recognition: Binding of the Prime Side.

Dengue virus (DENV), transmitted predominantly in tropical and subtropical regions by the mosquito Aedes aegypti, infects millions of people and leads to dengue fever and thousands of deaths each year. There are no direct-acting antivirals to combat DENV, and molecular and structural knowledge is required to develop such compounds. The dengue NS2B/NS3 protease is a promising target for direct-acting antivirals, as viral polyprotein cleavage during replication is required for the maturation of the viral particle. The NS2B/NS3 protease processes 8 of the 13 viral polyprotein cleavage sites to allow viral maturation. Although these sites share little sequence homology beyond the P1 and P2 positions, most are well conserved among the serotypes. How the other substrate residues, especially at the P' side, affect substrate recognition remains unclear. We exploited the tight-binding general serine protease inhibitor aprotinin to investigate protease-substrate interactions at the molecular level. We engineered aprotinin's binding loop with sequences mimicking the P' side of DENV substrates. P' residues significantly modulate substrate affinity to protease, with inhibition constants varying from nanomolar to sub-millimolar. Structural and dynamic analysis revealed the molecular basis of this modulation and allowed identifying optimal residues for each of the P' positions. In addition, isothermal titration calorimetry showed binding to be solely entropy driven for all constructs. Potential flaviviral P' side inhibitors could benefit from mimicking the optimal residues at P' positions and incorporate hydrophobicity and rigidity to maintain entropic advantage for potency.

Substrate envelope-designed potent HIV-1 protease inhibitors to avoid drug resistance.

The rapid evolution of HIV under selective drug pressure has led to multidrug resistant (MDR) strains that evade standard therapies. We designed highly potent HIV-1 protease inhibitors (PIs) using the substrate envelope model, which confines inhibitors within the consensus volume of natural substrates, providing inhibitors less susceptible to resistance because a mutation affecting such inhibitors will simultaneously affect viral substrate processing. The designed PIs share a common chemical scaffold but utilize various moieties that optimally fill the substrate envelope, as confirmed by crystal structures. The designed PIs retain robust binding to MDR protease variants and display exceptional antiviral potencies against different clades of HIV as well as a panel of 12 drug-resistant viral strains. The substrate envelope model proves to be a powerful strategy to develop potent and robust inhibitors that avoid drug resistance.

Differential Flap Dynamics in Wild-type and a Drug Resistant Variant of HIV-1 Protease Revealed by Molecular Dynamics and NMR Relaxation.

In the rapidly evolving disease of HIV drug resistance readily emerges, nullifying the effectiveness of therapy. Drug resistance has been extensively studied in HIV-1 protease where resistance occurs when the balance between enzyme inhibition and substrate recognition and turn-over is perturbed to favor catalytic activity. Mutations which confer drug resistance can impact the dynamics and structure of both the bound and unbound forms of the enzyme. Flap+ is a multi-drug-resistant variant of HIV-1 protease with a combination of mutations at the edge of the active site, within the active site, and in the flaps (L10I, G48V, I54V, V82A). The impact of these mutations on the dynamics in the unliganded form in comparison with the wild-type protease was elucidated with Molecular Dynamic simulations and NMR relaxation experiments. The comparative analyses from both methods concur in showing that the enzyme's dynamics are impacted by the drug resistance mutations in Flap+ protease. These alterations in the enzyme dynamics, particularly within the flaps, likely modulate the balance between substrate turn-over and drug binding, thereby conferring drug resistance.

Extreme entropy-enthalpy compensation in a drug-resistant variant of HIV-1 protease.

The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.