Invention of MK-7845, a SARS-CoV-2 3CL Protease Inhibitor Employing a Novel Difluorinated Glutamine Mimic.

As SARS-CoV-2 continues to circulate, antiviral treatments are needed to complement vaccines. The virus's main protease, 3CLPro, is an attractive drug target in part because it recognizes a unique cleavage site, which features a glutamine residue at the P1 position and is not utilized by human proteases. Herein, we report the invention of MK-7845, a novel reversible covalent 3CLPro inhibitor. While most covalent inhibitors of SARS-CoV-2 3CLPro reported to date contain an amide as a Gln mimic at P1, MK-7845 bears a difluorobutyl substituent at this position. SAR analysis and X-ray crystallographic studies indicate that this group interacts with His163, the same residue that forms a hydrogen bond with the amide substituents typically found at P1. In addition to promising in vivo efficacy and an acceptable projected human dose with unboosted pharmacokinetics, MK-7845 exhibits favorable properties for both solubility and absorption that may be attributable to the unusual difluorobutyl substituent.

Erratum: Exploring APOBEC3A and APOBEC3B substrate specificity and their role in HPV positive head and neck cancer.

[This corrects the article DOI: 10.1016/j.isci.2022.105077.].

Exploring ABOBEC3A and APOBEC3B substrate specificity and their role in HPV positive head and neck cancer.

APOBEC3 family members are cytidine deaminases catalyzing conversion of cytidine to uracil. Many studies have established a link between APOBEC3 expression and cancer development and progression, especially APOBEC3A (A3A) and APOBEC3B (A3B). Preclinical studies with human papillomavirus positive (HPV+) head and neck squamous cell carcinoma (HNSCC) and clinical trial specimens revealed induction of A3B, but not A3A expression after demethylation. We examined the kinetic features of the cytidine deaminase activity for full length A3B and found that longer substrates and a purine at -2 position favored by A3B, whereas A3A prefers shorter substrates and an adenine or thymine at -2 position. The importance and biological significance of A3B catalytic activity rather than A3A and a preference for purine at the -2 position was also established in HPV+ HNSCCs. Our study explored factors influencing formation of A3A and A3B-related cancer mutations that are essential for understanding APOBEC3-related carcinogenesis and facilitating drug discovery.

APOBEC mutagenesis and selection for NFE2L2 contribute to the origin of lung squamous-cell carcinoma.

Lung squamous-cell carcinoma originates as a consequence of oncogenic molecular variants arising from diverse mutagenic processes such as tobacco, defective homologous recombination, aging, and cytidine deamination by APOBEC proteins. Only some of the many variants generated by these processes actually contribute to tumorigenesis. Therefore, molecular investigation of mutagenic processes such as cytidine deamination by APOBEC should also determine whether the mutations produced by these processes contribute substantially to the growth and survival of cancer. Here, we determine the processes that gave rise to mutations of 681 lung squamous-cell carcinomas, and quantify the probability that each mutation was the product of each process. We then calculate the contribution of each mutation to increases in cellular proliferation and survival. We performed in vitro experiments to determine cytidine deamination activity of APOBEC3B against oligonucleotides corresponding with genomic sequences that give rise to variants of high cancer effect size. The largest APOBEC-related cancer effects are attributable to mutations in PIK3CA and NFE2L2. We demonstrate that APOBEC effectively deaminates NFE2L2 at the locations that confer high cancer effect. Overall, we demonstrate that APOBEC activity can lead to mutations in NFE2L2 that have large contributions to cancer cell growth and survival, and that NFE2L2 is an attractive potential target for therapeutic intervention.

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.

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.

Structure-Guided Identification of DNMT3B Inhibitors.

Methyltransferase 3 beta (DNMT3B) inhibitors that interfere with cancer growth are emerging possibilities for treatment of melanoma. Herein we identify small molecule inhibitors of DNMT3B starting from a homology model based on a DNMT3A crystal structure. Virtual screening by docking led to purchase of 15 compounds, among which 5 were found to inhibit the activity of DNMT3B with IC50 values of 13-72 µM in a fluorogenic assay. Eight analogues of 7, 10, and 12 were purchased to provide 2 more active compounds. Compound 11 is particularly notable as it shows good selectivity with no inhibition of DNMT1 and 22 µM potency toward DNMT3B. Following additional de novo design, exploratory synthesis of 17 analogues of 11 delivered 5 additional inhibitors of DNMT3B with the most potent being 33h with an IC50 of 8.0 µM. This result was well confirmed in an ultrahigh-performance liquid chromatography (UHPLC)-based analytical assay, which yielded an IC50 of 4.8 µM. Structure-activity data are rationalized based on computed structures for DNMT3B complexes.

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.

Structural and pharmacological evaluation of a novel non-nucleoside reverse transcriptase inhibitor as a promising long acting nanoformulation for treating HIV.

Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

( R)- N-(1-Methyl-2-hydroxyethyl)-13-( S)-methyl-arachidonamide (AMG315): A Novel Chiral Potent Endocannabinoid Ligand with Stability to Metabolizing Enzymes.

The synthesis of potent metabolically stable endocannabinoids is challenging. Here we report a chiral arachidonoyl ethanolamide (AEA) analogue, namely, (13 S,1' R)-dimethylanandamide (AMG315, 3a), a high affinity ligand for the CB1 receptor ( Ki of 7.8 ± 1.4 nM) that behaves as a potent CB1 agonist in vitro (EC50 = 0.6 ± 0.2 nM). (13 S,1' R)-dimethylanandamide is the first potent AEA analogue with significant stability for all endocannabinoid hydrolyzing enzymes as well as the oxidative enzymes COX-2. When tested in vivo using the CFA-induced inflammatory pain model, 3a behaved as a more potent analgesic when compared to endogenous AEA or its hydrolytically stable analogue AM356. This novel analogue will serve as a very useful endocannabinoid probe.

DRONE: Direct Tracking of DNA Cytidine Deamination and Other DNA Modifying Activities.

Enzymes that catalyze DNA modifying activities including cytidine deamination and cytosine methylation play important biological roles and have been implicated pathologically in diseases such as cancer. Here, we report Direct Resolution of ONE dalton difference (DRONE), an ultra high performance liquid chromatography (UHPLC)-based analytical method to track a single dalton change in the cytosine-to-uracil conversion catalyzed by the human apolipoprotein B m-RNA editing catalytic polypeptide-like 3 (APOBEC3) cytidine deaminases, implicated in cancer and antiviral defense. Additionally, we demonstrate broad applicability by tracking other important DNA modifications and assessing epigenetic enzyme inhibition. We have extended our methodology to obtain data on two distinct deamination events in the same oligonucleotide substrate designed from a putative APOBEC substrate, diversifying the utility of the described method. DRONE provides an important foundation for in-depth analysis of DNA-modifying enzymes and versatile detection of novel DNA modifications of interest.

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.

Structural and Preclinical Studies of Computationally Designed Non-Nucleoside Reverse Transcriptase Inhibitors for Treating HIV infection.

The clinical benefits of HIV-1 non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are hindered by their unsatisfactory pharmacokinetic (PK) properties along with the rapid development of drug-resistant variants. However, the clinical efficacy of these inhibitors can be improved by developing compounds with enhanced pharmacological profiles and heightened antiviral activity. We used computational and structure-guided design to develop two next-generation NNRTI drug candidates, compounds I and II, which are members of a class of catechol diethers. We evaluated the preclinical potential of these compounds in BALB/c mice because of their high solubility (510 µg/ml for compound I and 82.9 µg/ml for compound II), low cytotoxicity, and enhanced antiviral activity against wild-type (WT) HIV-1 RT and resistant variants. Additionally, crystal structures of compounds I and II with WT RT suggested an optimal binding to the NNRTI binding pocket favoring the high anti-viral potency. A single intraperitoneal dose of compounds I and II exhibited a prolonged serum residence time of 48 hours and concentration maximum (Cmax) of 4000- to 15,000-fold higher than their therapeutic/effective concentrations. These Cmax values were 4- to 15-fold lower than their cytotoxic concentrations observed in MT-2 cells. Compound II showed an enhanced area under the curve (0-last) and decreased plasma clearance over compound I and efavirenz, the standard of care NNRTI. Hence, the overall (PK) profile of compound II was excellent compared with that of compound I and efavirenz. Furthermore, both compounds were very well tolerated in BALB/c mice without any detectable acute toxicity. Taken together, these data suggest that compounds I and II possess improved anti-HIV-1 potency, remarkable in vivo safety, and prolonged in vivo circulation time, suggesting strong potential for further development as new NNRTIs for the potential treatment of HIV infection.

Assay of Endocannabinoid Oxidation by Cyclooxygenase-2.

The endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide (AEA), are endogenous ligands for the cannabinoid receptors (CB1 and CB2) and are implicated in a wide array of physiological processes. These neutral arachidonic acid (AA) derivatives have been identified as efficient substrates for the second isoform of the cyclooxygenase enzyme (COX-2). A diverse family of prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs) is generated by the action of COX-2 (and downstream prostaglandin synthases) on 2-AG and AEA. As the biological importance of the endocannabinoid system becomes more apparent, there is a tremendous need for robust, sensitive, and efficient analytical methodology for the endocannabinoids and their metabolites. In this chapter, we describe methodology suitable for carrying out oxygenation of endocannabinoids by COX-2, and analysis of products of endocannabinoid oxygenation by COX-2 and of endocannabinoids themselves from in vitro and cell assays.

Action at a distance: mutations of peripheral residues transform rapid reversible inhibitors to slow, tight binders of cyclooxygenase-2.

Cyclooxygenase enzymes (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin G2. The inhibitory activity of rapid, reversible COX inhibitors (ibuprofen, naproxen, mefenamic acid, and lumiracoxib) demonstrated a significant increase in potency and time dependence of inhibition against double tryptophan murine COX-2 mutants at the 89/90 and 89/119 positions. In contrast, the slow, time-dependent COX inhibitors (diclofenac, indomethacin, and flurbiprofen) were unaffected by those mutations. Further mutagenesis studies suggested that mutation at position 89 was principally responsible for the changes in inhibitory potency of rapid, reversible inhibitors, whereas mutation at position 90 may exert some effect on the potency of COX-2-selective diarylheterocycle inhibitors; no effect was observed with mutation at position 119. Several crystal structures with or without NSAIDs indicated that placement of a bulky residue at position 89 caused a closure of a gap at the lobby, and alteration of histidine to tryptophan at position 90 changed the electrostatic profile of the side pocket of COX-2. Thus, these two residues, especially Val-89 at the lobby region, are crucial for the entrance and exit of some NSAIDs from the COX active site.

13-Methylarachidonic acid is a positive allosteric modulator of endocannabinoid oxygenation by cyclooxygenase.

Cyclooxygenase-2 (COX-2) oxygenates arachidonic acid (AA) and the endocannabinoids 2-arachidonoylglycerol (2-AG) and arachidonylethanolamide to prostaglandins, prostaglandin glyceryl esters, and prostaglandin ethanolamides, respectively. A structural homodimer, COX-2 acts as a conformational heterodimer with a catalytic and an allosteric monomer. Prior studies have demonstrated substrate-selective negative allosteric regulation of 2-AG oxygenation. Here we describe AM-8138 (13(S)-methylarachidonic acid), a substrate-selective allosteric potentiator that augments 2-AG oxygenation by up to 3.5-fold with no effect on AA oxygenation. In the crystal structure of an AM-8138·COX-2 complex, AM-8138 adopts a conformation similar to the unproductive conformation of AA in the substrate binding site. Kinetic analysis suggests that binding of AM-8138 to the allosteric monomer of COX-2 increases 2-AG oxygenation by increasing kcat and preventing inhibitory binding of 2-AG. AM-8138 restored the activity of COX-2 mutants that exhibited very poor 2-AG oxygenating activity and increased the activity of COX-1 toward 2-AG. Competition of AM-8138 for the allosteric site prevented the inhibition of COX-2-dependent 2-AG oxygenation by substrate-selective inhibitors and blocked the inhibition of AA or 2-AG oxygenation by nonselective time-dependent inhibitors. AM-8138 selectively enhanced 2-AG oxygenation in intact RAW264.7 macrophage-like cells. Thus, AM-8138 is an important new tool compound for the exploration of allosteric modulation of COX enzymes and their role in endocannabinoid metabolism.

A role for catalase-peroxidase large loop 2 revealed by deletion mutagenesis: control of active site water and ferric enzyme reactivity.

Catalase-peroxidases (KatGs), the only catalase-active members of their superfamily, all possess a 35-residue interhelical loop called large loop 2 (LL2). It is essential for catalase activity, but little is known about its contribution to KatG function. LL2 shows weak sequence conservation; however, its length is nearly identical across KatGs, and its apex invariably makes contact with the KatG-unique C-terminal domain. We used site-directed and deletion mutagenesis to interrogate the role of LL2 and its interaction with the C-terminal domain in KatG structure and catalysis. Single and double substitutions of the LL2 apex had little impact on the active site heme [by magnetic circular dichroism or electron paramagnetic resonance (EPR)] and activity (catalase or peroxidase). Conversely, deletion of a single amino acid from the LL2 apex reduced catalase activity by 80%. Deletion of two or more apex amino acids or all of LL2 diminished catalase activity by 300-fold. Peroxide-dependent but not electron donor-dependent kcat/KM values for deletion variant peroxidase activity were reduced 20-200-fold, and kon for cyanide binding diminished by 3 orders of magnitude. EPR spectra for deletion variants were all consistent with an increase in the level of pentacoordinate high-spin heme at the expense of hexacoordinate high-spin states. Together, these data suggest a shift in the distribution of active site waters, altering the reactivity of the ferric state, toward, among other things, compound I formation. These results identify the importance of LL2 length conservation for maintaining an intersubunit interaction that is essential for an active site water distribution that facilitates KatG catalytic activity.

Enhancing the peroxidatic activity of KatG by deletion mutagenesis.

Catalase-peroxidase (KatG) enzymes use a peroxidase active site to facilitate robust catalase activity, an ability all other members of its superfamily lack. KatG's have a Met-Tyr-Trp covalent adduct that is essential for catalatic but not peroxidatic turnover. The tyrosine (Y226 in E. coli KatG) is supplied by a large loop (LL1) that is absent from all other plant peroxidases. Elimination of Y226 from the KatG structure, either by site directed mutagenesis (i.e., Y226F KatG) or by deletion of larger portions of LL1 invariably eliminates catalase activity, but deletion variants were substantially more active as peroxidases, up to an order of magnitude. Moreover, the deletion variants were more resistant to H(2)O(2)-dependent inactivation than Y226F KatG. Stopped-flow evaluation of reactions of H(2)O(2) with Y226F KatG and the most peroxidase active deletion variant (KatG[Δ209-228]) produced highly similar rate constants for formation of compounds I and II, and about a four-fold faster formation of compound III for the deletion variant as opposed to Y226F. Conversely, single turnover experiments showed a 60-fold slower return of Y226F KatG to its ferric state in the presence of the exogenous electron donor 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) than was determined for KatG(Δ209-228). Our data suggest that the peroxidatic output of KatG cannot be optimized simply by elimination of catalase activity alone, but also requires modifications that increase electron transfer between exogenous electron donors and the heme prosthetic group.