Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors.

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.

Kinetic Characterization and Inhibition of Trypanosoma cruzi Hypoxanthine-Guanine Phosphoribosyltransferases.

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.

The design of protozoan phosphoribosyltransferase inhibitors containing non-charged phosphate mimic residues.

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.

Mechanism-Based Inactivation of Mycobacterium tuberculosis Isocitrate Lyase 1 by (2R,3S)-2-Hydroxy-3-(nitromethyl)succinic acid.

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.

Inhibiting Sialidase-Induced TGF-ß1 Activation Attenuates Pulmonary Fibrosis in Mice.

The active form of transforming growth factor-ß1 (TGF-ß1) plays a key role in potentiating fibrosis. TGF-ß1 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-ß1 in the lungs and attenuated pulmonary fibrosis. Here we observe that recombinant human NEU3 upregulates active human TGF-ß1 by releasing active TGF-ß1 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-ß1 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-ß1 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-ß1 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-ß1 (TGF-ß1) in an inactive state, causing active TGF-ß1 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.

Mechanism-based inactivator of isocitrate lyases 1 and 2 from Mycobacterium tuberculosis.

Isocitrate lyase (ICL, types 1 and 2) is the first enzyme of the glyoxylate shunt, an essential pathway for Mycobacterium tuberculosis (Mtb) during the persistent phase of human TB infection. Here, we report 2-vinyl-d-isocitrate (2-VIC) as a mechanism-based inactivator of Mtb ICL1 and ICL2. The enzyme-catalyzed retro-aldol cleavage of 2-VIC unmasks a Michael substrate, 2-vinylglyoxylate, which then forms a slowly reversible, covalent adduct with the thiolate form of active-site Cys191 2-VIC displayed kinetic properties consistent with covalent, mechanism-based inactivation of ICL1 and ICL2 with high efficiency (partition ratio, <1). Analysis of a complex of ICL1:2-VIC by electrospray ionization mass spectrometry and X-ray crystallography confirmed the formation of the predicted covalent S-homopyruvoyl adduct of the active-site Cys191.

Transition state for the NSD2-catalyzed methylation of histone H3 lysine 36.

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

Catalytic Mechanism of Cruzain from Trypanosoma cruzi As Determined from Solvent Kinetic Isotope Effects of Steady-State and Pre-Steady-State Kinetics.

Cruzain, an important drug target for Chagas disease, is a member of clan CA of the cysteine proteases. Understanding the catalytic mechanism of cruzain is vital to the design of new inhibitors. To this end, we have determined pH-rate profiles for substrates and affinity agents and solvent kinetic isotope effects in pre-steady-state and steady-state modes using three substrates: Cbz-Phe-Arg-AMC, Cbz-Arg-Arg-AMC, and Cbz-Arg-Ala-AMC. The pH-rate profile of kcat/ Km for Cbz-Arg-Arg-AMC indicated p K1 = 6.6 (unprotonated) and p K2 ∼ 9.6 (protonated) groups were required for catalysis. The temperature dependence of the p K = 6.2-6.6 group exhibited a Δ Hion value of 8.4 kcal/mol, typical of histidine. The pH-rate profile of inactivation by iodoacetamide confirmed that the catalytic cysteine possesses a p Ka of 9.8. Normal solvent kinetic isotope effects were observed for both D2O kcat (1.6-2.1) and D2O kcat/ Km (1.1-1.4) for all three substrates. Pre-steady-state kinetics revealed exponential bursts of AMC production for Cbz-Phe-Arg-AMC and Cbz-Arg-Arg-AMC, but not for Cbz-Arg-Ala-AMC. The overall solvent isotope effect on kcat can be attributed to the solvent isotope effect on the deacylation step. Our results suggest that cruzain is unique among papain-like cysteine proteases in that the catalytic cysteine and histidine have neutral charges in the free enzyme. The generation of the active thiolate of the catalytic cysteine is likely preceded (and possibly triggered) by a ligand-induced conformational change, which could bring the catalytic dyad into the proximity to effect proton transfer.

On the catalytic mechanism of human ATP citrate lyase.

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The ß,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.

The amino-acid substituents of dipeptide substrates of cathepsin C can determine the rate-limiting steps of catalysis.

We examined the cathepsin C-catalyzed hydrolysis of dipeptide substrates of the form Yaa-Xaa-AMC, using steady-state and pre-steady-state kinetic methods. The substrates group into three kinetic profiles based upon the broad range observed for k(cat)/K(a) and k(cat) values, pre-steady-state time courses, and solvent kinetic isotope effects (sKIEs). The dipeptide substrate Gly-Arg-AMC displayed large values for k(cat)/K(a) (1.6 ± 0.09 µM(-1) s(-1)) and k(cat) (255 ± 6 s(-1)), an inverse sKIE on k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.6 ± 0.15), a modest, normal sKIE on k(cat) ((D)k(cat) = 1.6 ± 0.2), and immeasurable pre-steady-state kinetics, indicating an extremely fast pre-steady-state rate (>400 s(-1)). (Errors on fitted values are omitted in the text for clarity but may be found in Table 2.) These results conformed to a kinetic model where the acylation (k(ac)) and deacylation (k(dac)) half-reactions are very fast and similar in value. The second substrate type, Gly-Tyr-AMC and Ser-Tyr-AMC, the latter the subject of a comprehensive kinetic study (Schneck et al. (2008) Biochemistry 47, 8697-8710), were found to be less active substrates compared to Gly-Arg-AMC, with respective k(cat)/K(a) values of 0.49 ± 0.07 µM(-1 )s(-1) and 5.3 ± 0.5 µM(-1 )s(-1), and k(cat) values of 28 ± 1 s(-1) and 25 ± 0.5 s(-1). Solvent kinetic isotope effects for Ser-Tyr-AMC were found to be inverse for k(cat)/K(a) ((D)(k(cat)/K(a)) = 0.74 ± 0.05) and normal for k(cat) ((D)k(cat) = 2.3 ± 0.1) but unlike Gly-Arg-AMC, pre-steady-state kinetics of Gly-Tyr-AMC and Ser-Tyr-AMC were measurable and characterized by a single-exponential burst, with fast transient rates (490 s(-1) and 390 s(-1), respectively), from which it was determined that k(ac) ≫ k(dac) ∼ k(cat). The third substrate type, Gly-Ile-AMC, gave very low values of k(cat)/K(a) (0.0015 ± 0.0001 µM(-1) s(-1)) and k(cat) (0.33 ± 0.02 s(-1)), no sKIEs, ((D)(k(cat)/K(a)) = 1.05 ± 0.5 and (D)k(cat) = 1.06 ± 0.4), and pre-steady-state kinetics exhibited a discernible, but negligible, transient phase. For this third class of substrate, kinetic modeling was consistent with a mechanism in which k(dac) > k(ac) ∼ k(cat), and for which an isotope-insensitive step in the acylation half-reaction is the slowest. The combined results of these studies suggested that the identity of the amino acid at the P(1) position of the substrate is the main determinant of catalysis. On the basis of these kinetic data, together with crystallographic studies of substrate analogues and molecular dynamics analysis with models of acyl-enzyme intermediates, we present a catalytic model derived from the relative rates of the acylation vs deacylation half-reactions of cathepsin C. The chemical steps of catalysis are proposed to be dependent upon the conformational freedom of the amino acid substituents for optimal alignment for thiolation (acylation) or hydrolysis (deacylation). These studies suggest ideas for inhibitor design for papain-family cysteine proteases and strategies to progress drug discovery for other classes of disease-relevant cysteine proteases.

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active.

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.

Self-Masked Aldehyde Inhibitors of Human Cathepsin L Are Potent Anti-CoV-2 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 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 (K i = 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.

Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L.

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.

Understanding the origins of time-dependent inhibition by polypeptide deformylase inhibitors.

The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.

Dinitrosyl iron complexes (DNICs) as inhibitors of the SARS-CoV-2 main protease.

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. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(µ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-ß-d-glucose tetraacetate) and TG-RRE, [(µ-S-TG)Fe(NO)2]2 (TG = 1-thio-ß-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 µM for TGTA-RRE and 33 ± 2 µM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.

Covalent Inactivation of Mycobacterium tuberculosis Isocitrate Lyase by cis-2,3-Epoxy-Succinic Acid.

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.

Self-Masked Aldehyde Inhibitors: A Novel Strategy for Inhibiting Cysteine Proteases.

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.

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells.

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.

Kinetic mechanism and rate-limiting steps of focal adhesion kinase-1.

Steady-state kinetic analysis of focal adhesion kinase-1 (FAK1) was performed using radiometric measurement of phosphorylation of a synthetic peptide substrate (Ac-RRRRRRSETDDYAEIID-NH(2), FAK-tide) which corresponds to the sequence of an autophosphorylation site in FAK1. Initial velocity studies were consistent with a sequential kinetic mechanism, for which apparent kinetic values k(cat) (0.052 +/- 0.001 s(-1)), K(MgATP) (1.2 +/- 0.1 microM), K(iMgATP) (1.3 +/- 0.2 microM), K(FAK-tide) (5.6 +/- 0.4 microM), and K(iFAK-tide) (6.1 +/- 1.1 microM) were obtained. Product and dead-end inhibition data indicated that enzymatic phosphorylation of FAK-tide by FAK1 was best described by a random bi bi kinetic mechanism, for which both E-MgADP-FAK-tide and E-MgATP-P-FAK-tide dead-end complexes form. FAK1 catalyzed the betagamma-bridge:beta-nonbridge positional oxygen exchange of [gamma-(18)O(4)]ATP in the presence of 1 mM [gamma-(18)O(4)]ATP and 1.5 mM FAK-tide with a progressive time course which was commensurate with catalysis, resulting in a rate of exchange to catalysis of k(x)/k(cat) = 0.14 +/- 0.01. These results indicate that phosphoryl transfer is reversible and that a slow kinetic step follows formation of the E-MgADP-P-FAK-tide complex. Further kinetic studies performed in the presence of the microscopic viscosogen sucrose revealed that solvent viscosity had no effect on k(cat)/K(FAK-tide), while k(cat) and k(cat)/K(MgATP) were both decreased linearly at increasing solvent viscosity. Crystallographic characterization of inactive versus AMP-PNP-liganded structures of FAK1 showed that a large conformational motion of the activation loop upon ATP binding may be an essential step during catalysis and would explain the viscosity effect observed on k(cat)/K(m) for MgATP but not on k(cat)/K(m) for FAK-tide. From the positional isotope exchange, viscosity, and structural data it may be concluded that enzyme turnover (k(cat)) is rate-limited by both reversible phosphoryl group transfer (k(forward) approximately 0.2 s(-1) and k(reverse) approximately 0.04 s(-1)) and a slow step (k(conf) approximately 0.1 s(-1)) which is probably the opening of the activation loop after phosphoryl group transfer but preceding product release.