Proof-of-concept studies with a computationally designed Mpro inhibitor as a synergistic combination regimen alternative to Paxlovid.

As the SARS-CoV-2 virus continues to spread and mutate, it remains important to focus not only on preventing spread through vaccination but also on treating infection with direct-acting antivirals (DAA). The approval of Paxlovid, a SARS-CoV-2 main protease (Mpro) DAA, has been significant for treatment of patients. A limitation of this DAA, however, is that the antiviral component, nirmatrelvir, is rapidly metabolized and requires inclusion of a CYP450 3A4 metabolic inhibitor, ritonavir, to boost levels of the active drug. Serious drug-drug interactions can occur with Paxlovid for patients who are also taking other medications metabolized by CYP4503A4, particularly transplant or otherwise immunocompromised patients who are most at risk for SARS-CoV-2 infection and the development of severe symptoms. Developing an alternative antiviral with improved pharmacological properties is critical for treatment of these patients. By using a computational and structure-guided approach, we were able to optimize a 100 to 250 µM screening hit to a potent nanomolar inhibitor and lead compound, Mpro61. In this study, we further evaluate Mpro61 as a lead compound, starting with examination of its mode of binding to SARS-CoV-2 Mpro. In vitro pharmacological profiling established a lack of off-target effects, particularly CYP450 3A4 inhibition, as well as potential for synergy with the currently approved alternate antiviral, molnupiravir. Development and subsequent testing of a capsule formulation for oral dosing of Mpro61 in B6-K18-hACE2 mice demonstrated favorable pharmacological properties, efficacy, and synergy with molnupiravir, and complete recovery from subsequent challenge by SARS-CoV-2, establishing Mpro61 as a promising potential preclinical candidate.

Covalent and noncovalent strategies for targeting Lys102 in HIV-1 reverse transcriptase.

Reverse transcriptase (RT) is one of three key proteins responsible for the replication cycle of HIV-1 in the host. Several classes of inhibitors have been developed to target the enzyme, with non-nucleoside reverse transcriptase inhibitors forming first-line treatment. Previously, covalent RT inhibitors have been identified and found to bind irreversibly to commonly mutated residues such as Y181C. In this work we aim to circumvent the issue of NNRTI resistance through targeting K102, which has not yet been identified to confer drug resistance. As reported here, 34 compounds were synthesized and characterized biochemically and structurally with wild-type (WT) HIV-1 RT. Two of these inhibitors demonstrate covalent inhibition as evidenced by protein crystallography, enzyme kinetics, mass spectrometry, and antiviral potency in HIV-1 infected human T-cell assays.

Exploring novel HIV-1 reverse transcriptase inhibitors with drug-resistant mutants: A double mutant surprise.

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.

Design, synthesis, and biological testing of biphenylmethyloxazole inhibitors targeting HIV-1 reverse transcriptase.

We report non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) using a biphenylmethyloxazole pharmacophore. A crystal structure of benzyloxazole 1 was obtained and suggested the potential viability of biphenyl analogues. In particular, 6a, 6b, and 7 turned out to be potent NNRTIs with low-nanomolar activity in enzyme inhibition and infected T-cell assays, and with low cytotoxicity. Though modeling further suggested that analogues with fluorosulfate and epoxide warheads might provide covalent modification of Tyr188, synthesis and testing did not find evidence for this outcome.

Structural Studies and Structure Activity Relationships for Novel Computationally Designed Non-nucleoside Inhibitors and Their Interactions With HIV-1 Reverse Transcriptase.

Reverse transcriptase (RT) from the human immunodeficiency virus continues to be an attractive drug target for antiretroviral therapy. June 2022 will commemorate the 30th anniversary of the first Human Immunodeficiency Virus (HIV) RT crystal structure complex that was solved with non-nucleoside reverse transcriptase inhibitor nevirapine. The release of this structure opened opportunities for designing many families of non-nucleoside reverse transcriptase inhibitors (NNRTIs). In paying tribute to the first RT-nevirapine structure, we have developed several compound classes targeting the non-nucleoside inhibitor binding pocket of HIV RT. Extensive analysis of crystal structures of RT in complex with the compounds informed iterations of structure-based drug design. Structures of seven additional complexes were determined and analyzed to summarize key interactions with residues in the non-nucleoside inhibitor binding pocket (NNIBP) of RT. Additional insights comparing structures with antiviral data and results from molecular dynamics simulations elucidate key interactions and dynamics between the nucleotide and non-nucleoside binding sites.

Long-acting and extended-release implant and nanoformulations with a synergistic antiretroviral two-drug combination controls HIV-1 infection in a humanized mouse model.

The HIV pandemic has affected over 38 million people worldwide with close to 26 million currently accessing antiretroviral therapy (ART). A major challenge in the long-term treatment of HIV-1 infection is nonadherence to ART. Long-acting antiretroviral (LA-ARV) formulations, that reduce dosing frequency to less than once a day, are an urgent need that could tackle the adherence issue. Here, we have developed two LA-ART interventions, one an injectable nanoformulation, and the other, a removable implant, for the delivery of a synergistic two-drug ARV combination comprising a pre-clinical nonnucleoside reverse transcriptase inhibitor (NNRTI), Compound I, and the nucleoside reverse transcriptase inhibitor (NRTI), 4'-ethynyl-2-fluoro-2'-deoxyadenosine. The nanoformulation is poly(lactide-co-glycolide)-based and the implant is a copolymer of ω-pentadecalactone and p-dioxanone, poly(PDL-co-DO), a novel class of biocompatible, biodegradable materials. Both the interventions, packaged independently with each ARV, released sustained levels of the drugs, maintaining plasma therapeutic indices for over a month, and suppressed viremia in HIV-1-infected humanized mice for up to 42 days with maintenance of CD4+ T cells. These data suggest promise in the use of these new drugs as LA-ART formulations in subdermal implant and injectable mode.

Optimization of Triarylpyridinone Inhibitors of the Main Protease of SARS-CoV-2 to Low-Nanomolar Antiviral Potency.

Non-covalent inhibitors of the main protease (Mpro) of SARS-CoV-2 having a pyridinone core were previously reported with IC50 values as low as 0.018 µM for inhibition of enzymatic activity and EC50 values as low as 0.8 µM for inhibition of viral replication in Vero E6 cells. The series has now been further advanced by consideration of placement of substituted five-membered-ring heterocycles in the S4 pocket of Mpro and N-methylation of a uracil ring. Free energy perturbation calculations provided guidance on the choice of the heterocycles, and protein crystallography confirmed the desired S4 placement. Here we report inhibitors with EC50 values as low as 0.080 µM, while remdesivir yields values of 0.5-2 µM in side-by-side testing with infectious SARS-CoV-2. A key factor in the improvement is enhanced cell permeability, as reflected in PAMPA measurements. Compounds 19 and 21 are particularly promising as potential therapies for COVID-19, featuring IC50 values of 0.044-0.061 µM, EC50 values of ca. 0.1 µM, good aqueous solubility, and no cytotoxicity.

Potent Noncovalent Inhibitors of the Main Protease of SARS-CoV-2 from Molecular Sculpting of the Drug Perampanel Guided by Free Energy Perturbation Calculations.

Starting from our previous finding of 14 known drugs as inhibitors of the main protease (Mpro) of SARS-CoV-2, the virus responsible for COVID-19, we have redesigned the weak hit perampanel to yield multiple noncovalent, nonpeptidic inhibitors with ca. 20 nM IC50 values in a kinetic assay. Free-energy perturbation (FEP) calculations for Mpro-ligand complexes provided valuable guidance on beneficial modifications that rapidly delivered the potent analogues. The design efforts were confirmed and augmented by determination of high-resolution X-ray crystal structures for five analogues bound to Mpro. Results of cell-based antiviral assays further demonstrated the potential of the compounds for treatment of COVID-19. In addition to the possible therapeutic significance, the work clearly demonstrates the power of computational chemistry for drug discovery, especially FEP-guided lead optimization.

Covalent Inhibition of Wild-Type HIV-1 Reverse Transcriptase Using a Fluorosulfate Warhead.

Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether non-nucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC50 values for in vitro inhibition of WT RT and EC50 values for cytopathic protection of HIV-1-infected human T-cells in the 5-320 nM range.

Structural investigation of 2-naphthyl phenyl ether inhibitors bound to WT and Y181C reverse transcriptase highlights key features of the NNRTI binding site.

Human immunodeficiency virus (HIV)-1 remains as a global health issue that is primarily treated with highly active antiretroviral therapy, a combination of drugs that target the viral life cycle. One class of these drugs are non-nucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral reverse transcriptase (RT). First generation NNRTIs were troubled with poor pharmacological properties and drug resistance, incentivizing the development of improved compounds. One class of developed compounds are the 2-naphthyl phenyl ethers, showing promising efficacy against the Y181C RT mutation. Further biochemical and structural work demonstrated differences in potency against the Y181C mutation and binding mode of the compounds. This work aims to understand the relationship between the binding mode and ability to overcome drug resistance using macromolecular x-ray crystallography. Comparison of 2-naphthyl phenyl ethers bound to Y181C RT reveal that compounds that interact with the invariant W229 are more capable of retaining efficacy against the resistance mutation. Additional modifications to these compounds at the 4-position, computationally designed to compensate for the Y181C mutation, do not demonstrate improved potency. Ultimately, we highlight important considerations for the development of future HIV-1 drugs that are able to combat drug resistance.

Molecular and cellular studies evaluating a potent 2-cyanoindolizine catechol diether NNRTI targeting wildtype and Y181C mutant HIV-1 reverse transcriptase.

The development of efficacious NNRTIs for HIV/AIDS therapy is commonly met with the emergence of drug resistant strains, including the Y181C variant. Using a computationally-guided approach, we synthesized the catechol diether series of NNRTIs, which display sub-nanomolar potency in cellular assays. Among the most potent were a series of 2-cyanoindolizine substituted catechol diethers, including Compound 1. We present here a thorough evaluation of this compound, including biochemical, cellular, and structural studies. The compound demonstrates low nanomolar potency against both WT and Y181C HIV-1 RT in in vitro and cellular assays. Our crystal structures of both the wildtype and mutant forms of RT in complex with Compound 1 allow the interrogation of this compound's features that allow it to maintain strong efficacy against the drug resistant mutant. Among these are compensatory shifts in the NNRTI binding pocket, persistence of multiple hydrogen bonds, and van der Waals contacts throughout the binding site. Further, the fluorine at the C6 position of the indolizine moiety makes multiple favorable interactions with both RT forms. The present study highlights the indolizine-substituted catechol diether class of NNRTIs as promising therapeutic candidates possessing optimal pharmacological properties and significant potency against multiple RT variants.

From in silico hit to long-acting late-stage preclinical candidate to combat HIV-1 infection.

The HIV-1 pandemic affecting over 37 million people worldwide continues, with nearly one-half of the infected population on highly active antiretroviral therapy (HAART). Major therapeutic challenges remain because of the emergence of drug-resistant HIV-1 strains, limitations because of safety and toxicity with current HIV-1 drugs, and patient compliance for lifelong, daily treatment regimens. Nonnucleoside reverse transcriptase inhibitors (NNRTIs) that target the viral polymerase have been a key component of the current HIV-1 combination drug regimens; however, these issues hamper them. Thus, the development of novel more effective NNRTIs as anti-HIV-1 agents with fewer long-term liabilities, efficacy on new drug-resistant HIV-1 strains, and less frequent dosing is crucial. Using a computational and structure-based design strategy to guide lead optimization, a 5 µM virtual screening hit was transformed to a series of very potent nanomolar to picomolar catechol diethers. One representative, compound I, was shown to have nanomolar activity in HIV-1-infected T cells, potency on clinically relevant HIV-1 drug-resistant strains, lack of cytotoxicity and off-target effects, and excellent in vivo pharmacokinetic behavior. In this report, we show the feasibility of compound I as a late-stage preclinical candidate by establishing synergistic antiviral activity with existing HIV-1 drugs and clinical candidates and efficacy in HIV-1-infected humanized [human peripheral blood lymphocyte (Hu-PBL)] mice by completely suppressing viral loads and preventing human CD4+ T-cell loss. Moreover, a long-acting nanoformulation of compound I [compound I nanoparticle (compound I-NP)] in poly(lactide-coglycolide) (PLGA) was developed that shows sustained maintenance of plasma drug concentrations and drug efficacy for almost 3 weeks after a single dose.

Covalent inhibitors for eradication of drug-resistant HIV-1 reverse transcriptase: From design to protein crystallography.

Development of resistance remains a major challenge for drugs to treat HIV-1 infections, including those targeting the essential viral polymerase, HIV-1 reverse transcriptase (RT). Resistance associated with the Tyr181Cys mutation in HIV-1 RT has been a key roadblock in the discovery of nonnucleoside RT inhibitors (NNRTIs). It is the principal point mutation that arises from treatment of HIV-infected patients with nevirapine, the first-in-class drug still widely used, especially in developing countries. We report covalent inhibitors of Tyr181Cys RT (CRTIs) that can completely knock out activity of the resistant mutant and of the particularly challenging Lys103Asn/Tyr181Cys variant. Conclusive evidence for the covalent modification of Cys181 is provided from enzyme inhibition kinetics, mass spectrometry, protein crystallography, and antiviral activity in infected human T-cell assays. The CRTIs are also shown to be selective for Cys181 and have lower cytotoxicity than the approved NNRTI drugs efavirenz and rilpivirine.

Design, Conformation, and Crystallography of 2-Naphthyl Phenyl Ethers as Potent Anti-HIV Agents.

Catechol diethers that incorporate a 7-cyano-2-naphthyl substituent are reported as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Many of the compounds have 1-10 nM potencies toward wild-type HIV-1. An interesting conformational effect allows two unique conformers for the naphthyl group in complexes with HIV-RT. X-ray crystal structures for 4a and 4f illustrate the alternatives.

Potent Inhibitors Active against HIV Reverse Transcriptase with K101P, a Mutation Conferring Rilpivirine Resistance.

Catechol diether compounds have nanomolar antiviral and enzymatic activity against HIV with reverse transcriptase (RT) variants containing K101P, a mutation that confers high-level resistance to FDA-approved non-nucleoside inhibitors efavirenz and rilpivirine. Kinetic data suggests that RT (K101P) variants are as catalytically fit as wild-type and thus can potentially increase in the viral population as more antiviral regimens include efavirenz or rilpivirine. Comparison of wild-type structures and a new crystal structure of RT (K101P) in complex with a leading compound confirms that the K101P mutation is not a liability for the catechol diethers while suggesting that key interactions are lost with efavirenz and rilpivirine.

Discovery and crystallography of bicyclic arylaminoazines as potent inhibitors of HIV-1 reverse transcriptase.

Non-nucleoside inhibitors of HIV-1 reverse transcriptase (HIV-RT) are reported that incorporate a 7-indolizinylamino or 2-naphthylamino substituent on a pyrimidine or 1,3,5-triazine core. The most potent compounds show below 10 nanomolar activity towards wild-type HIV-1 and variants bearing Tyr181Cys and Lys103Asn/Tyr181Cys resistance mutations. The compounds also feature good aqueous solubility. Crystal structures for two complexes enhance the analysis of the structure-activity data.

Structure-based evaluation of non-nucleoside inhibitors with improved potency and solubility that target HIV reverse transcriptase variants.

The development of novel non-nucleoside inhibitors (NNRTIs) with activity against variants of HIV reverse transcriptase (RT) is crucial for overcoming treatment failure. The NNRTIs bind in an allosteric pocket in RT ∼10 Å away from the active site. Earlier analogues of the catechol diether compound series have picomolar activity against HIV strains with wild-type RT but lose potency against variants with single Y181C and double K103N/Y181C mutations. As guided by structure-based and computational studies, removal of the 5-Cl substitution of compound 1 on the catechol aryl ring system led to a new analogue compound 2 that maintains greater potency against Y181C and K103N/Y181C variants and better solubility (510 µg/mL). Crystal structures were determined for wild-type, Y181C, and K103N/Y181C RT in complex with both compounds 1 and 2 to understand the structural basis for these findings. Comparison of the structures reveals that the Y181C mutation destabilizes the binding mode of compound 1 and disrupts the interactions with residues in the pocket. Compound 2 maintains the same conformation in wild-type and mutant structures, in addition to several interactions with the NNRTI binding pocket. Comparison of the six crystal structures will assist in the understanding of compound binding modes and future optimization of the catechol diether series.

Illuminating HIV gp120-Ligand Recognition through Computationally-Driven Optimization of Antibody-Recruiting Molecules.

Here we report on the structure-based optimization of antibody-recruiting molecules targeting HIV gp120 (ARM-H). These studies have leveraged a combination of medicinal chemistry, biochemical and cellular assay analysis, and computation. Our findings have afforded an optimized analog of ARM-H, which is ~1000 fold more potent in gp120-binding and MT-2 antiviral assays than our previously reported derivative. Furthermore, computational analysis, taken together with experimental data, provides evidence that azaindole- and indole-based attachment inhibitors bind gp120 at an accessory hydrophobic pocket beneath the CD4-binding site and can also adopt multiple unique binding modes in interacting with gp120. These results are likely to prove highly enabling in the development of novel HIV attachment inhibitors, and more broadly, they suggest novel applications for ARMs as probes of conformationally flexible systems.

Picomolar Inhibitors of HIV-1 Reverse Transcriptase: Design and Crystallography of Naphthyl Phenyl Ethers.

Catechol diethers that incorporate a 6-cyano-1-naphthyl substituent have been explored as non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs). Promising compounds are reported that show midpicomolar activity against the wild-type virus and sub-20 nM activity against viral variants bearing Tyr181Cys and Lys103Asn mutations in HIV-RT. An X-ray crystal structure at 2.49 Å resolution is also reported for the key compound 6e with HIV-RT.