Human cathepsin L (hCatL), a lysosomal cysteine protease, facilitates endosomal entry for SARS-CoV-2, making it a critical therapeutic target for COVID-19. Here, we report irreversible tripeptidomimetic inactivators containing a 3-pyridyl alanine residue at the P1 position, modified with either electrophilic α,β-unsaturated ethyl ester or sulfone warheads. These inhibitors had Ki* values between 0.4 and 35 nM. The most potent inhibitors, VK-168 (Ki* = 0.58 nM) and VK-261 (Ki* = 0.38 nM), demonstrated robust anti-SARS-CoV-2 activity in Vero E6 (EC50 = 120-310 nM) and A549/ACE2 cells (EC50 = 78-120 nM). Additionally, VK-80 inhibited infection (EC50 < 156 nM) and showed partial efficacy in a murine COVID-19 model. Native mass spectrometry and dilution assays confirmed irreversible hCatL inactivation. Notably, these enoates acted as reversible inhibitors of the homologue cathepsin B (hCatB). Molecular modeling suggests that hCatL's active-site cysteine is better positioned for nucleophilic addition to the vinyl group than hCatB's.

The pathogen Mycobacterium tuberculosis (Mtb) relies on extensive metabolic remodeling and post-translational regulation of its proteins to retain persistence in its host, yet these biochemical functions remain largely uncharacterized, including the actions of lysine deacylases. Here, we characterized Rv1151c, the sole annotated NAD+-dependent deacylase in Mtb, which we designate Mt-Sirt. Using synthetic substrates, peptide libraries, and mutational analysis, we showed that Mt-Sirt exhibits strong selectivity for peptide substrates containing Nε-succinyl-lysine and long-chain fatty-acyl-lysine groups. Substitution of conserved active-site residues (Y53F and R56M) confirmed their roles in substrate recognition and acyl-group discrimination. Inhibition studies revealed that nicotinamide, NADH, ADP-ribose, and mechanism-based thioamide/thiourea analogs inhibited Mt-Sirt activity with low-micromolar to nanomolar potency. Initial-velocity, product-inhibition, and dead-end inhibition analyses confirmed that Mt-Sirt operates by an equilibrium-ordered kinetic mechanism in which NAD+ binds first, followed by the Nε-acylated peptide substrate. We further demonstrated that chemically succinylated Mtb isocitrate lyase 1 (ICL1), a key enzyme in Mtb metabolism, was a substrate of Mt-Sirt in vitro. Lastly, metabolic labeling with tetradec-13-ynoic acid (Alk-12) revealed widespread lysine and N-terminal fatty acylation in Mtb cells, suggesting that Mt-Sirt may regulate previously unrecognized fatty-acyl PTMs in vivo. Together, these findings provide a kinetic and substrate-specificity analysis of Mt-Sirt, and highlight its potential role in controlling reversible lysine acylation in Mtb.

The pathogen (Mtb) relies on extensive metabolic remodeling and post-translational regulation of its proteins to retain persistence in its host, yet these biochemical functions remain largely uncharacterized, including the actions of lysine deacylases. Here, we characterized Rv1151c, the sole annotated NAD+-dependent deacylase in Mtb, which we designate Mt-Sirt. Using synthetic substrates, peptide libraries, and mutational analysis, we showed that Mt-Sirt exhibits strong selectivity for peptide substrates containing N epsilon-succinyl-lysine and long-chain fatty-acyl-lysine groups. Substitution of conserved active-site residues (Y53F and R56M) confirmed their roles in substrate recognition and acyl-group discrimination. Inhibition studies revealed that nicotinamide, NADH, ADP-ribose, and mechanism-based thioamide/thiourea analogs inhibited Mt-Sirt activity with low-micromolar to nanomolar potency. Initial-velocity, product-inhibition, and dead-end inhibition analyses confirmed that Mt-Sirt operates by an equilibrium-ordered kinetic mechanism in which NAD+ binds first, followed by the N epsilon-acylated peptide substrate. We further demonstrated that chemically succinylated Mtb isocitrate lyase 1 (ICL1), a key enzyme in Mtb metabolism, was a substrate of Mt-Sirt in vitro. Lastly, metabolic labeling with tetradec-13-ynoic acid (Alk-12) revealed widespread lysine and N-terminal fatty acylation in Mtb cells, suggesting that Mt-Sirt may regulate previously unrecognized fatty-acyl PTMs in vivo. Together, these findings provide a kinetic and substrate-specificity analysis of Mt-Sirt, and highlight its potential role in controlling reversible lysine acylation in Mtb.

SARS-CoV-2 3CL protease (Main protease) and human cathepsin L are proteases that play unique roles in the infection of human cells by SARS-CoV-2, the causative agent of COVID-19. Both proteases recognize leucine and other hydrophobic amino acids at the P2 position of a peptidomimetic inhibitor. At the P1 position, cathepsin L accepts many amino acid side chains, with a partial preference for phenylalanine, while 3CL-PR protease has a stringent specificity for glutamine or glutamine analogues. We have designed, synthesized, and evaluated peptidomimetic aldehyde dual-target (dual-acting) inhibitors using two peptide scaffolds based on those of two Pfizer 3CL-PR inhibitors, Nirmatrelvir, and PF-835321. Our inhibitors contain glutamine isosteres at the P1 position, including 2-pyridon-3-yl-alanine, 3-pyridinyl-alanine, and 1,3-oxazo-4-yl-alanine groups. Inhibition constants for these new inhibitors ranged from Ki = 0.6-18 nM (cathepsin L) and Ki = 2.6-124 nM (3CL-PR), for which inhibitors with the 2-pyridon-3-yl-alanal substituent were the most potent for 3CL-PR. The anti-CoV-2 activity of these inhibitors ranged from EC50 = 0.47-15 μM. X-ray structures of the peptidomimetic aldehyde inhibitors of 3CL-PR with similar scaffolds all demonstrated the formation of thiohemiacetals with Cys145, and hydrogen-bonding interactions with the heteroatoms of the pyridon-3-yl-alanyl group, as well as the nitrogen of the N-terminal indole and its appended carbonyl group at the P3 position. The absence of these hydrogen bonds for the inhibitors containing the 3-pyridinyl-alanyl and 1,3-oxazo-4-yl-alanyl groups was reflected in the less potent inhibition of the inhibitors with 3CL-PR. In summary, our studies demonstrate the value of a second generation of cysteine protease inhibitors that comprise a single agent that acts on both human cathepsin L and SARS-CoV-2 3CL protease. Such dual-target inhibitors will provide anti-COVID-19 drugs that remain active despite the development of resistance due to mutation of the viral protease. Such dual-target inhibitors are more likely to remain useful therapeutics despite the emergence of inactivating mutations in the viral protease because the human cathepsin L will not develop resistance. This particular dual-target approach is innovative since one of the targets is viral (3CL-PR) required for viral protein maturation and the other is human (hCatL) which enables viral infection.

The 3-chymotrypsin-like protease (3CL-PR; also known as Main protease) of SARS-CoV-2 is a cysteine protease that is the target of the COVID-19 drug, Paxlovid. Here, we report for 3CL-PR, the pH-rate profiles of a substrate, an inhibitor, affinity agents, and solvent kinetic isotope effects (sKIEs) obtained under both steady-state and pre-steady-state conditions. "Bell-shaped" plots of log(k cat/K a) vs pH for the substrate (Abz)SAVLQ*SGFRK(Dnp)-NH2 and pK i vs pH for a peptide aldehyde inhibitor demonstrated that essential acidic and basic groups of pK 2 = 8.2 +/- 0.4 and pK 1 = 6.2 +/- 0.3, respectively, are required for catalysis, and the pH-dependence of inactivation of 3CL-PR by iodoacetamide and diethylpyrocarbonate identified enzymatic groups of pK 2 = 7.8 +/- 0.1 and pK 1 = 6.05 +/- 0.07, which must be unprotonated for maximal inactivation. These data are most consistent with the presence of a neutral catalytic dyad (Cys-SH-His) in the 3CL-PR free enzyme, with respective pK values for the cysteine and histidine groups of pK 2 = 8.0 and pK 1 = 6.5. The steady-state sKIEs were D2O(k cat/K a) = 0.56 +/- 0.05 and D2O k cat = 1.0 +/- 0.1, and sKIEs indicated that the Cys-S--HisH+ tautomer was enriched in D2O. Presteady-state kinetic analysis of (Abz)SAVLQ*SGFRK(Dnp)-NH2 exhibited transient lags preceding steady-state rates, which were considerably faster in D2O than in H2O. The transient rates encompass steps that include substrate binding and acylation, and are faster in D2O wherein the more active Cys-S--HisH+ tautomer predominates. A full catalytic mechanism for 3CL-PR is proposed.

The main protease of SARS-CoV-2 (Mpro) is an essential enzyme for coronaviral maturation and is the target of Paxlovid, which is currently the standard-of-care treatment for COVID-19. There remains a need to identify new inhibitors of Mpro as viral resistance to Paxlovid emerges. Here, we report the use of native mass spectrometry coupled with 193-nm ultraviolet photodissociation (UVPD) to structurally characterize Mpro and its interactions with potential covalent inhibitors. Melting temperatures and the overall energy landscape were obtained using variable temperature nano-electrospray ionization (vT-nESI), thus providing quantitative evaluation of inhibitor binding on the stability of Mpro. The melting temperature was determined to be approximately 30°C for the dimer and 36°C for the monomer, suggesting an initial thermal dissociation pathway before subsequent unfolding of the monomer species. Thermodynamic parameters extracted from Van’t Hoff plots revealed that the dimeric complexes containing each inhibitor showed enhanced stability through increased melting temperatures as well as overall lower average charge states, giving insight into the basis for potential inhibition mechanisms.

Proteases, a subset of enzymes that catalyze the hydrolysis of peptide bonds within proteins or polypeptides, play a crucial role in the maintenance and regulation of a number of biological processes. The Renin-Angiotensin System (RAS), for example, contains a cascade of proteases. In the RAS, the protein angiotensinogen is processed to the decapeptide angiotensin 1 by the proteolytic enzyme renin, and further to the octapeptide angiotensin 2 by a second protease, angiotensin-converting enzyme (Scheme 13.1). A series of later hydrolytic steps produces various other peptide products including angiotensin 3. The physiological role of the RAS both in the circulatory system and as reported recently in various tissues is quite varied [1,2]. The RAS maintains salt and volume homeostasis in the body and regulates blood pressure [3]. This hormonal system is also important for cardiovascular regulation since angiotensin 2 is a potent vasoconstrictor. The use of peptide analog inhibitors as an approach to control the RAS cascade either by blocking renin or angiotensin-converting enzyme (ACE) has had strong clinical success against several physiological disorders such as hypertension and cardiovascular diseases [4,5].

Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine–guanine–xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities.

Chagas disease, caused by the parasitic protozoan Trypanosoma cruzi, affects over 8 million people worldwide. Current antiparasitic treatments for Chagas disease are ineffective in treating advanced, chronic stages of the disease, and are noted for their toxicity. Like most parasitic protozoa, T. cruzi is unable to synthesize purines de novo, and relies on the salvage of preformed purines from the host. Hypoxanthine-guanine phosphoribosyltransferases (HGPRTs) are enzymes that are critical for the salvage of preformed purines, catalyzing the formation of inosine monophosphate (IMP) and guanosine monophosphate (GMP) from the nucleobases hypoxanthine and guanine, respectively. Due to the central role of HGPRTs in purine salvage, these enzymes are promising targets for the development of new treatment methods for Chagas disease. In this study, we characterized two gene products in the T. cruzi CL Brener strain that encodes enzymes with functionally identical HGPRT activities in vitro: TcA (TcCLB.509693.70) and TcC (TcCLB.506457.30). The TcC isozyme was kinetically characterized to reveal mechanistic details on catalysis, including identification of the rate-limiting step(s) of catalysis. Furthermore, we identified and characterized inhibitors of T. cruzi HGPRTs originally developed as transition-state analogue inhibitors (TSAIs) of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase (PfHGXPRT), where the most potent compound bound to T. cruzi HGPRT with low nanomolar affinity. Our results validated the repurposing of TSAIs to serve as selective inhibitors for orthologous molecular targets, where primary and secondary structures as well as putatively common chemical mechanisms are conserved.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, 1), for which the P1 Phe group contains a 1′-hydroxy group, effectively, an o-tyrosinyl aldehyde (Cbz-Phe-o-Tyr-CHO, 2; (Li et al. (2021) J. Med. Chem. 64, 11,267–11,287)). Compound 2 and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of Trypanosoma cruzi, results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L (Ki = 11–60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott et al. (2021) ACS Chem. Biol. 16, 642–650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC50 = 2–8 μM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC50 = 0.3–0.6 μM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound (9) inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.

Enzymes are extraordinary catalysts which humble the efforts of the best synthetic organic chemists. Consequently, the study of enzyme catalysis is woefully incomplete without an understanding of how it conducts catalysis to convert its chosen substrate(s) into product(s). The indispensable tool to achieve this is kinetic analysis, derived in part from the established rate laws of chemical catalysis, which when applied to the special case in which the template for this catalysis is a protein, results in a unique and venerable area of study. The well-established field of enzyme kinetics, especially in the past 60 years, has perhaps surpassed that of its formative antecedents: physical organic chemistry and bio-organic chemistry. This has resulted in an amicable demarcation of study comprising the assessment of enzymatic catalytic events in the millisecond timeframe of early binding and catalytic events, the so-called pre-steady-state, and the more widely studied area of steady-state kinetics, which reports on catalysis after multiple catalytic events. While steady-state enzyme kinetics may suffer a deficit in information that can be provided by the analysis of pre-steady-state kinetics, it may be more faithful to the events that occur from enzyme catalysis that occur in metabolic pathways in which multiple rounds of the conversion of substrates to products are relevant. Here we provide a brief overview of the rich history of steady-state enzyme kinetics, which, hopefully acquaints, or re-acquaints, a readership with the tools needed to evaluate the kinetics, and ultimately, the catalytic mechanisms of enzymes of study.

Phosphate groups play essential roles in biological processes, including retention inside biological membranes. Phosphodiesters link nucleic acids, and the reversible transfer of phosphate groups is essential in energy metabolism and cell-signalling processes. Phosphorylated metabolic intermediates are known targets for metabolic and disease-related disorders, and the enzymes involved in these pathways recognize phosphate groups in their catalytic sites. Therapeutics that target these enzymes can require charged (ionic) entities to capture the binding energy of ionic substrates. Such compounds are not cell-permeable and require pro-drug strategies for efficacy as therapeutics. Protozoan parasites such as Plasmodium and Trypanosoma spp. are unable to synthesise purines de novo and rely on the salvage of purines from the host cell to synthesise free purine bases. Purine phosphoribosyltransfereases (PPRTases) play a crucial role for purine salvage and are potential target for drug development. Here we present attempts to design inhibitors of PPRTases that are non-ionic and show affinity for the nucleotide 5'-phosphate binding site. Inhibitor design was based on known potent ionic inhibitors, reported phosphate mimics and computational modelling studies.

Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a Neglected Tropical Disease endemic to 36 African countries, with approximately 70 million people currently at risk for infection. Current therapeutics are suboptimal due to toxicity, adverse side effects, and emerging resistance. Thus, both effective and affordable treatments are urgently needed. The causative agent of HAT is the protozoan Trypanosoma brucei ssp. Annotation of its genome confirms previous observations that T. brucei is a purine auxotroph. Incapable of de novo purine synthesis, these protozoan parasites rely on purine phosphoribosyltransferases to salvage purines from their hosts for the synthesis of purine monophosphates. Complete and accurate genome annotations in combination with the identification and characterization of the catalytic activity of purine salvage enzymes enables the development of target-specific therapies in addition to providing a deeper understanding of purine metabolism in T. brucei. In trypanosomes, purine phosphoribosyltransferases represent promising drug targets due to their essential and central role in purine salvage. Enzymes involved in adenine and adenosine salvage, such as adenine phosphoribosyltransferases (APRTs, EC 2.4.2.7), are of particular interest for their potential role in the activation of adenine and adenosine-based pro-drugs. Analysis of the T. brucei genome shows two putative aprt genes: APRT1 (Tb927.7.1780) and APRT2 (Tb927.7.1790). Here we report studies of the catalytic activity of each putative APRT, revealing that of the two T. brucei putative APRTs, only APRT1 is kinetically active, thereby signifying a genomic misannotation of Tb927.7.1790 (putative APRT2). Reliable genome annotation is necessary to establish potential drug targets and identify enzymes involved in adenine and adenosine-based pro-drug activation.

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

The active form of transforming growth factor-β1 (TGF-β1) plays a key role in potentiating fibrosis. TGF-β1 is sequestered in an inactive state by a latency-associated glycopeptide (LAP). Sialidases (also called neuraminidases (NEU)) cleave terminal sialic acids from glycoconjugates. The sialidase NEU3 is upregulated in fibrosis, and mice lacking Neu3 show attenuated bleomycin-induced increases in active TGF-β1 in the lungs and attenuated pulmonary fibrosis. Here we observe that recombinant human NEU3 upregulates active human TGF-β1 by releasing active TGF-β1 from its latent inactive form by desialylating LAP. Based on the proposed mechanism of action of NEU3, we hypothesized that compounds with a ring structure resembling picolinic acid might be transition state analogs and thus possible NEU3 inhibitors. Some compounds in this class showed nanomolar IC50 for recombinant human NEU3 releasing active human TGF-β1 from the latent inactive form. The compounds given as daily 0.1-1-mg/kg injections starting at day 10 strongly attenuated lung inflammation, lung TGF-β1 upregulation, and pulmonary fibrosis at day 21 in a mouse bleomycin model of pulmonary fibrosis. These results suggest that NEU3 participates in fibrosis by desialylating LAP and releasing TGF-β1 and that the new class of NEU3 inhibitors are potential therapeutics for fibrosis. SIGNIFICANCE STATEMENT: The extracellular sialidase NEU3 appears to be a key driver of pulmonary fibrosis. The significance of this report is that 1) we show the mechanism (NEU3 desialylates the latency-associated glycopeptide protein that keeps the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in an inactive state, causing active TGF-β1 release), 2) we then use the predicted NEU3 mechanism to identify nM IC50 NEU3 inhibitors, and 3) these new NEU3 inhibitors are potent therapeutics in a mouse model of pulmonary fibrosis.

The isocitrate lyase paralogs of Mycobacterium tuberculosis (ICL1 and 2) are essential for mycobacterial persistence and constitute targets for the development of antituberculosis agents. We report that (2R,3S)-2-hydroxy-3-(nitromethyl)succinic acid (5-NIC) undergoes apparent retro-aldol cleavage as catalyzed by ICL1 to produce glyoxylate and 3-nitropropionic acid (3-NP), the latter of which is a covalent-inactivating agent of ICL1. Kinetic analysis of this reaction identified that 5-NIC serves as a robust and efficient mechanism-based inactivator of ICL1 (kinact/KI = (1.3 ± 0.1) × 103 M-1 s-1) with a partition ratio <1. Using enzyme kinetics, mass spectrometry, and X-ray crystallography, we identified that the reaction of the 5-NIC-derived 3-NP with the Cys191 thiolate of ICL1 results in formation of an ICL1-thiohydroxamate adduct as predicted. One aspect of the design of 5-NIC was to lower its overall charge compared to isocitrate to assist with cell permeability. Accordingly, the absence of the third carboxylate group will simplify the synthesis of pro-drug forms of 5-NIC for characterization in cell-infection models of M. tuberculosis.

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay.

The isocitrate lyases (ICL1/2) are essential enzymes of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. At present, no ICL1/2 inhibitors have progressed to clinical evaluation, despite extensive drug discovery efforts. Herein, we surveyed succinate analogs against ICL1 and found that dicarboxylic acids constrained in their synperiplanar conformations, such as maleic acid, comprise uncompetitive inhibitors of ICL1 and inhibit more potently than their trans-isomers. From this, we identified cis-2,3 epoxysuccinic acid (cis-EpS) as a selective, irreversible covalent inactivator of Mtb ICL1 (kinact/Kinact= (5.0 ± 1.4) × 104 M-1 s-1; Kinact = 200 ± 50 nM), the most potent inactivator of ICL1 yet characterized. Crystallographic and mass spectrometric analysis demonstrated that Cys191 of ICL1 was S-malylated by cis-EpS, and a crystallographic "snapshot" of inactivation lent insight into the chemical mechanism of this inactivation. Proteomic analysis of E. coli lysates showed that cis-EpS selectively labeled plasmid-expressed Mtb ICL1. Consistently, cis-EpS, but not its trans-isomer, inhibited the growth of Mtb under conditions in which ICL function is essential. These findings encourage the development of analogs of cis-2,3-epoxysuccinate as antituberculosis agents.

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 μM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 μM. There was no toxicity to any of the host cell lines at 10–100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.