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\u27s high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a Ki value of 2.9 muM 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 Analysis of Potent Hybrid HIV-1 Protease Inhibitors Containing Bis-Tetrahydrofuran in a Pseudo-Symmetric Dipeptide Isostere

The design, synthesis, and X-ray structural analysis of hybrid HIV-1 protease inhibitors (PIs) containing bis-tetrahydrofuran (bis-THF) in a pseudo-C2-symmetric dipeptide isostere are described. A series of PIs were synthesized by incorporating bis-THF of darunavir on either side of the Phe-Phe isostere of lopinavir in combination with hydrophobic amino acids on the opposite P2/P2\u27 position. Structure-activity relationship studies indicated that the bis-THF moiety can be attached at either the P2 or P2\u27 position without significantly affecting potency. However, the group on the opposite P2/P2\u27 position had a dramatic effect on potency depending on the size and shape of the side chain. Cocrystal structures of inhibitors with wild-type HIV-1 protease revealed that the bis-THF moiety retained similar interactions as observed in the darunavir-protease complex regardless of position on the Phe-Phe isostere. Analyses of cocrystal structures and molecular dynamics simulations provide insights for optimizing HIV-1 PIs containing bis-THF in non-sulfonamide dipeptide isosteres

Design and synthesis of potent macrocyclic HIV-1 protease inhibitors and non-nucleoside dengue virus mRNA methyltransferase inhibitors

The first part of this thesis describes a novel class of macrocyclic HIV-1 protease inhibitors. A series of macrocyclic inhibitors containing aromatic P2 ligand have been designed, synthesized and evaluated. The inhibitors have been designed by incorporating 16-18 membered macrocyclic rings between the aromatic P2 ligand, and a tyrosine P1 ligand attached to (hydroxyethylamino)sulfonamide isostere resembling the P1\u27-P 2\u27 ligand in TMC-126. The macrocyclic compounds were more potent than the corresponding acyclic compounds. All macrocyclic compounds and their acyclic precursors exhibited high enzyme inhibitory potency, displaying K i values between 0.2 nM and 45 nM. Ring-closing metathesis using Grubbs 1st generation catalyst furnished the cis/trans unsaturated macrocycles. The synthesis of the aromatic P2 ligand was carried out using a 1,6-electrocyclization reaction. The isosteres containing two stereocenters were conveniently prepared through a series of stereo- and regioselective transformations including copper cyanide catalyzed SN 2 epoxide opening, Sharpless asymmetric epoxidation, and regioselective epoxide opening with TMSN3 in the presence of Ti(O-iPr) 4. A series of slightly modified 15- and 16-membered macrocyclic inhibitors containing meta-substituted aromatic P1 ligand have also been prepared. The inhibitors displayed high enzyme inhibitory potency with Ki values between 0.05 nM and 2.26 nM. Among these was also a macrocycle containing a 3-hydroxysalicylic acid P2 ligand, which exhibited comparable potency. The second part of this thesis describes the synthesis of a novel class of methyltransferase inhibitors. N-alkyl substituted diaminoquinoline ureas were synthesized and examined for N-7 methylation inhibition in dengue virus methyltransferase-catalyzed mRNA cap formation. This series of inhibitors was derived from a lead identified by high throughput screening. All compounds showed high potency, displaying IC50 values as low as 0.2 &mgr;M. The urea-containing inhibitors were formed by coupling their respective diaminoquinoline monomers using triphosgene. A series of novel squaramide analogs of the urea-based inhibitors were also synthesized. These inhibitors were relatively less potent than the corresponding urea compounds. Adenine containing urea compounds were also prepared. However, these compounds did not show any significant methyltransferase inhibitory activity

Quinoxaline-Based Linear HCV NS3/4A Protease Inhibitors Exhibit Potent Activity against Drug Resistant Variants

A series of linear HCV NS3/4A protease inhibitors was designed by eliminating the P2-P4 macrocyclic linker in grazoprevir, which, in addition to conferring conformational flexibility, allowed structure-activity relationship (SAR) exploration of diverse quinoxalines at the P2 position. Biochemical and replicon data indicated preference for small hydrophobic groups at the 3-position of P2 quinoxaline for maintaining potency against resistant variants R155K, A156T, and D168A/V. The linear inhibitors, though generally less potent than the corresponding macrocyclic analogues, were relatively easier to synthesize and less susceptible to drug resistance. Three inhibitor cocrystal structures bound to wild-type NS3/4A protease revealed a conformation with subtle changes in the binding of P2 quinoxaline, depending on the 3-position substituent, likely impacting both inhibitor potency and resistance profile. The SAR and structural analysis highlight inhibitor features that strengthen interactions of the P2 moiety with the catalytic triad residues, providing valuable insights to improve potency against resistant variants

HIV-1 Protease Inhibitors Incorporating Stereochemically Defined P2\u27 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\u27 ligands providing pairs of diastereoisomers epimeric at P2\u27, which exhibited distinct potency profiles depending on the configuration of the hydroxyl group and size of the P1\u27 group. While compounds with the 4-(1-hydroxyethyl)benzene P2\u27 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\u27 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\u27. Notably, the (R)-dihydroxyethyl group was involved in a unique pattern of direct hydrogen bonding interactions with the backbone amides of Asp29\u27 and Asp30\u27. The SAR data and analysis of crystal structures provide insights for optimizing these promising HIV-1 protease inhibitors

Inhibiting HTLV-1 Protease: A Viable Antiviral Target

Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that can cause severe paralytic neurologic disease and immune disorders as well as cancer. An estimated 20 million people worldwide are infected with HTLV-1, with prevalence reaching 30% in some parts of the world. In stark contrast to HIV-1, no direct acting antivirals (DAAs) exist against HTLV-1. The aspartyl protease of HTLV-1 is a dimer similar to that of HIV-1 and processes the viral polyprotein to permit viral maturation. We report that the FDA-approved HIV-1 protease inhibitor darunavir (DRV) inhibits the enzyme with 0.8 muM potency and provides a scaffold for drug design against HTLV-1. Analogs of DRV that we designed and synthesized achieved submicromolar inhibition against HTLV-1 protease and inhibited Gag processing in viral maturation assays and in a chronically HTLV-1 infected cell line. Cocrystal structures of these inhibitors with HTLV-1 protease highlight opportunities for future inhibitor design. Our results show promise toward developing highly potent HTLV-1 protease inhibitors as therapeutic agents against HTLV-1 infections

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

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