CDK4/6 inhibitors have revolutionized treatment of hormone receptor positive (HR+), HER2 non-amplified (HER2-) breast cancer. Yet, all "dual" CDK4/6 inhibitors show common dose-limiting hematologic toxicities, foremost neutropenia. This poses challenges to provide these agents at concentrations necessary to extinguish cell cycling in tumors. HR+ breast cancer cells are highly dependent on CDK4 but not CDK6. By contrast, CDK4 is dispensable for human bone marrow derived cells, due to the primary and compensatory role of CDK6 in hematopoiesis. This prompted us to develop atirmociclib (PF-07220060), a next-generation CDK4 selective inhibitor. Atirmociclib's impact on circulating neutrophils was reduced, in proportion with its increase in CDK4 versus CDK6 selectivity. Realized dose intensification led to greater CDK4 inhibition and deeper anti-tumor responses, pointing to CDK4 target coverage as a limiting factor of CDK4/6 inhibitor efficacy. We also highlight combinatorial agents that may counter acquired resistance to CDK4 selective inhibition and widen its clinical application.

Table S1. Statistics for the crystallographic analysis; Table S2. Biochemical and cellular potencies of the second generation CDK-directed drug AG-024322; Table S3. In vitro analysis of binding potency of abemaciclib, dinaciclib, and palbociclib to non-kinase proteins; Table S4. Broad kinase selectivity of selective CDK4/6 drugs; Table S5. Biochemical dose-response follow-up was conducted for a subset of kinases inhibited by the three CDK4/6 drugs using a Km concentration of ATP (Carna Biosciences) (CDK proteins excluded); Table S6. Kinase selectivity toward endogenous human kinases using irreversible ATP analog target engagement assay; Table S7. Biochemical potencies of selective CDK4/6 drugs palbociclib (PD-0332991); Table S8. Human pharmacokinetic properties of CDK-targeted drugs; Table S9. Isothermal titration calorimetry results for CDK-directed drugs binding to CDK6 in the absence of cyclin D.

Detailed description of the methods used in the manuscript

The CDK4/6 inhibitor, palbociclib (PAL), significantly improves progression-free survival in HR+/HER2- breast cancer when combined with anti-hormonals. We sought to discover PAL resistance mechanisms in preclinical models and through analysis of clinical transcriptome specimens, which coalesced on induction of MYC oncogene and Cyclin E/CDK2 activity. We propose that targeting the G(1) kinases CDK2, CDK4, and CDK6 with a small-molecule overcomes resistance to CDK4/6 inhibition. We describe the pharmacodynamics and efficacy of PF-06873600 (PF3600), a pyridopyrimidine with potent inhibition of CDK2/4/6 activity and efficacy in multiple in vivo tumor models. Together with the clinical analysis, MYC activity predicts (PF3600) efficacy across multiple cell lineages. Finally, we find that CDK2/4/6 inhibition does not compromise tumor-specific immune checkpoint blockade responses in syngeneic models. We anticipate that (PF3600), currently in phase 1 clinical trials, offers a therapeutic option to cancer patients in whom CDK4/6 inhibition is insufficient to alter disease progression.

Control of the cell cycle through selective pharmacological inhibition of CDK4/6 has proven beneficial in the treatment of breast cancer. Extending this level of control to additional cell cycle CDK isoforms represents an opportunity to expand to additional tumor types and potentially provide benefits to patients that develop tumors resistant to selective CDK4/6 inhibitors. However, broad-spectrum CDK inhibitors have a long history of failure due to safety concerns. In this approach, we describe the use of structure-based drug design and Free-Wilson analysis to optimize a series of CDK2/4/6 inhibitors. Further, we detail the use of molecular dynamics simulations to provide insights into the basis for selectivity against CDK9. Based on overall potency, selectivity, and ADME profile, PF-06873600 (22) was identified as a candidate for the treatment of cancer and advanced to phase 1 clinical trials.

The novel coronavirus disease COVID-19 that emerged in 2019 is caused by the virus SARS CoV-2 and named for its close genetic similarity to SARS CoV-1 that caused severe acute respiratory syndrome (SARS) in 2002. Both SARS coronavirus genomes encode two overlapping large polyproteins which are cleaved at specific sites by a cysteine 3C-like protease (3CLpro) in a post-translational processing step that is critical for coronavirus replication. The 3CLpro sequences for CoV-1 and CoV-2 viruses are 100% identical in the catalytic domain that carries out protein cleavage. A research effort that focused on the discovery of reversible and irreversible ketone-based inhibitors of SARS CoV-1 3CLpro employing ligand-protease structures solved by X-ray crystallography led to the identification of 3 and 4. Preclinical experiments reveal 4 (PF-00835231) as a potent inhibitor of CoV-2 3CLpro with suitable pharmaceutical properties to warrant further development as an intravenous treatment for COVID-19.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.(1) Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double mutants T790M/L858R and T790M/Del), selectivity over wild-type EGFR, and desirable ADME properties. Compound 21 is currently being evaluated in phase-I clinical trials of mutant EGFR driven NSCLC.

Mutant epidermal growth factor receptor (EGFR) is a major driver of non-small-cell lung cancer (NSCLC). Marketed first generation inhibitors, such as erlotinib, effect a transient beneficial response in EGFR mutant NSCLC patients before resistance mechanisms render these inhibitors ineffective. Secondary oncogenic EGFR mutations account for approximately 50% of relapses, the most common being the gatekeeper T790M substitution that renders existing therapies ineffective. The discovery of PF-06459988 (1), an irreversible pyrrolopyrimidine inhibitor of EGFR T790M mutants, was recently disclosed.1 Herein, we describe our continued efforts to achieve potency across EGFR oncogenic mutations and improved kinome selectivity, resulting in the discovery of clinical candidate PF-06747775 (21), which provides potent EGFR activity against the four common mutants (exon 19 deletion (Del), L858R, and double …

Abstract Therapeutically targeting aberrant intracellular kinase signaling is attractive from a biological perspective but drug development is often hindered by toxicities and inadequate efficacy. Predicting drug behaviors using cellular and animal models is confounded by redundant kinase activities, a lack of unique substrates, and cell-specific signaling networks. Cyclin-dependent kinase (CDK) drugs exemplify this phenomenon because they are reported to target common processes yet have distinct clinical activities. Tumor cell studies of ATP-competitive CDK drugs (dinaciclib, AG-024322, abemaciclib, palbociclib, ribociclib) indicate similar pharmacology while analyses in untransformed cells illuminates significant differences. To resolve this apparent disconnect, drug behaviors are described at the molecular level. Nonkinase binding studies and kinome interaction analysis (recombinant and endogenous kinases) reveal that proteins outside of the CDK family appear to have little role in dinaciclib/palbociclib/ribociclib pharmacology, may contribute for abemaciclib, and confounds AG-024322 analysis. CDK2 and CDK6 cocrystal structures with the drugs identify the molecular interactions responsible for potency and kinase selectivity. Efficient drug binding to the unique hinge architecture of CDKs enables selectivity toward most of the human kinome. Selectivity between CDK family members is achieved through interactions with nonconserved elements of the ATP-binding pocket. Integrating clinical drug exposures into the analysis predicts that both palbociclib and ribociclib are CDK4/6 inhibitors, abemaciclib inhibits CDK4/6/9, and dinaciclib is a broad-spectrum CDK inhibitor (CDK2/3/4/6/9). Understanding the molecular components of potency and selectivity also facilitates rational design of future generations of kinase-directed drugs. Mol Cancer Ther; 15(10); 2273–81. ©2016 AACR.

First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (0 high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR

This article describes an integrated rate equation for the time course of covalent enzyme inhibition under the conditions where the substrate concentration is significantly lower than the corresponding Michaelis constant, for example, in the Omnia assays of epidermal growth factor receptor (EGFR) kinase. The newly described method is applicable to experimental conditions where the enzyme concentration is significantly lower than the dissociation constant of the initially formed reversible enzyme-inhibitor complex (no "tight binding"). A detailed comparison with the traditionally used rate equation for covalent inhibition is presented. The two methods produce approximately identical values of the first-order inactivation rate constant (kinact). However, the inhibition constant (Ki), and therefore also the second-order inactivation rate constant kinact/Ki, is underestimated by the traditional method by up to an order of magnitude.

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

The contributions of the phosphoacceptor and the catalytic domain context to protein kinase biology and inhibitor potency are routinely overlooked, which can lead to mischaracterization of inhibitor and receptor functions. The receptor tyrosine kinase vascular endothelial growth factor receptor-2 (VEGFR2) is studied as a model system using a series of phosphoacceptor substrates (k(cat)/K(m) 684-116,000 M(-1) s(-1)) to assess effects on catalysis and inhibitor binding. ATP-competitive inhibitor potency toward the VEGFR2 catalytic domain (VEGFR2-CD) varies with different phosphoacceptor substrates, which is unexpected because the phosphoacceptors do not affect K(m,ATP) values. Indazole-based inhibitors are up to 60-fold more potent with two substrates (gastrin, minigastrin) relative to the others. Thus there is a component of uncompetitive inhibition because a specific phosphoacceptor enhances potency but is not strictly required. This substrate-specific inhibitory potency enhancement correlates with phosphoacceptor active site saturation and is not observed with other related kinases. The effect is confined to a specific catalytic domain conformation because autophosphorylation eliminates the potency enhancement as does the addition of the juxtamembrane domain (20 amino acids). Indazole inhibitor structure-activity analysis reveals that the magnitude of potency enhancement correlates with the size of the substituent that binds in a regulatory region of the active site. VEGFR drugs profiled with VEGFR2-CD using minigastrin have potency well-correlated with inhibition of full-length, cellular VEGFR2 autophosphorylation, an indication that the minigastrin-induced conformation is biologically relevant. These findings raise the possibility that inhibitors directed toward a common target can have different biological effects based on the kinase-substrate complexes present in different cellular contexts.

Analyses of compounds in clinical development have shown that ligand efficient-molecules with privileged physical properties and low dose are less likely to fail in the various stages of clinical testing, have fewer postapproval withdrawals, and are less likely to receive black box safety warnings. However, detailed side-by-side examination of molecular interactions and properties within single drug classes are lacking. As a class, VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) have changed the landscape of how cancer is treated, particularly in clear cell renal cell carcinoma, which is molecularly linked to the VEGF signaling axis. Despite the clear role of the molecular target, member molecules of this validated drug class exhibit distinct clinical efficacy and safety profiles in comparable renal cell carcinoma clinical studies. The first head-to-head randomized phase III comparative study between active VEGFR TKIs has confirmed significant differences in clinical performance [Rini BI, et al. (2011) Lancet 378:193–1939]. To elucidate how fundamental drug potency–efficiency is achieved and impacts differentiation within the VEGFR TKI class, we determined potencies, time dependence, selectivities, and X-ray structures of the drug–kinase complexes using a VEGFR2 TK construct inclusive of the important juxtamembrane domain. Collectively, the studies elucidate unique drug–kinase interactions that are dependent on distinct juxtamembrane domain conformations, resulting in significant potency and ligand efficiency differences. The identified structural trends are consistent with in vitro measurements, which translate well to clinical performance, underscoring a principle that may be broadly applicable to prospective drug design for optimal in vivo performance.

The catalytic domains of protein kinases are commonly treated as independent modular units with distinct biological functions. Here, the interactions between the catalytic and juxtamembrane domains of VEGFR2 are studied. Highly purified preparations of the receptor tyrosine kinase VEGFR2 catalytic domain without (VEGFR2-CD) and with (VEGFR2-CD/JM) the juxtamembrane (JM) domain were characterized by kinetic, biophysical, and structural methods. Although the catalytic parameters for both constructs were similar, the autophosphorylation rate of VEGFR2-CD/JM was substantially faster than VEGFR2-CD. The first event in the autophosphorylation reaction was phosphorylation of JM residue Y801 followed by phosphorylation of activation loop residues in the CD. The rates of activation loop autophosphorylation for the two constructs were determined to be similar. The autophosphorylation rate of Y801 was invariant on enzyme concentration, which is consistent with an intramolecular reaction. In addition, the first biochemical characterization of the advanced clinical compound axitinib is reported. Axitinib was found to have 40-fold enhanced biochemical potency toward VEGFR2-CD/JM (K(i) = 28 pM) compared to VEGFR2-CD, which correlates better with cellular potency. Calorimetric studies, including a novel ITC compound displacement method, confirmed the potency and provided insight into the thermodynamic origin of the potency differences. A structural model for the VEGFR2-CD/JM is proposed based on the experimental findings reported here and on the JM position in c-Kit, FLT3, and CSF1/cFMS. The described studies identify potential functions of the VEGFR2 JM domain with implications to both receptor biology and inhibitor design.

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CLpro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CLpro to characterize the reaction mechanism. We show that 3CLpro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CLpro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000-fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CLpro mechanism that utilizes an "electrostatic" trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P1, and a general base-catalyzed deacylation reaction.

3992 PF-00337210 is a potent, selective, and orally bioavailable small molecule inhibitor of VEGFRs. It is an ATP-competitive compound that is selective for VEGFR-2 when biochemically profiled against >100 diverse tyrosine and serine-threonine kinases. PF-00337210 preferentially binds to the unactivated kinase (Ki=0.7 nM) relative to the fully phosphorylated form (Ki= 8.8 nM). Crystallographic evidence suggests that its selectivity is likely a result of its ability to bind to a DFG-out conformation of VEGFR-2. In the cell, PF-00337210 inhibits the autophosphorylation of human and murine VEGFR-2 with IC50s of 0.87 ± 0.11 nM and 0.83 ± 0.29 nM, respectively. In cell-based assays, the compound is less potent against other split kinase family members: KIT (9.6 ± 3.4 nM), CSF1-R (13 ± 3 nM), PDGFR-α (11 ± 1 nM) and PDGFR-β (29 ± 8 nM), and does not inhibit Flt-3 (IC50 >10 μM). It also demonstrates greater than 60 fold selectivity for VEGFR-2 versus FGFR-1 in a growth factor stimulated HUVEC survival assay. PF-00337210 shows low to moderate in vivo clearance in several preclinical species (13, 22 and 25 mL/min/kg in rats, dogs and monkeys, respectively). It has high oral bioavailability in rats (>80%) and moderate oral bioavailability in dogs (>30%). In preclinical tumor models, the compound shows dose-dependent anti-tumor efficacy that is associated with significant reduction of microvessel density and vascular permeability in the tumor. The pharmacologically efficacious concentration of PF-00337210 is estimated to be

Liquid chromatography–mass spectrometry (LC–MS) has been used extensively in determination of the molecular weights of proteins, as well as covalent protein–ligand complexes. We have successfully developed LC–MS method for protein molecular weight measurement using small-bore and capillary LC–MS under acidic and basic conditions. A high pH method was critical in studying complexes that were unstable under acidic conditions. Microgram sensitivity was achieved using both methods. A protocol to study the binding mode of protein–ligand complexes under denaturing conditions was developed. These methods were applied to CP88 (a proprietary cysteine protease) inhibitors and revealed different binding modes of inhibitors to proteins that had similar non-reversible behavior in biochemical activity assays. The method also confirmed that one inhibitor studied binds to CP88 in a reversible covalent manner.

Severe acute respiratory syndrome (SARS) was a worldwide epidemic caused by a coronavirus that has a cysteine protease (3CL pro) essential to its life cycle. Steady-state and pre-steady-state kinetic methods were used with highly active 3CL pro to characterize the reaction mechanism. We show that 3CL pro has mechanistic features common and disparate to the archetypical proteases papain and chymotrypsin. The kinetic mechanism for 3CL pro-mediated ester hydrolysis, including the individual rate constants, is consistent with a simple double displacement mechanism. The pre-steady-state burst rate was independent of ester substrate concentration indicating a high commitment to catalysis. When homologous peptidic amide and ester substrates were compared, a series of interesting observations emerged. Despite a 2000fold difference in nonenzymatic reactivity, highly related amide and ester substrates were found to have similar kinetic parameters in both the steady-state and pre-steady-state. Steady-state solvent isotope effect (SIE) studies showed an inverse SIE for the amide but not ester substrates. Evaluation of the SIE in the pre-steady-state revealed normal SIEs for both amide and ester burst rates. Proton inventory (PI) studies on amide peptide hydrolysis were consistent with two proton-transfer reactions in the transition state while the ester data was consistent with a single proton-transfer reaction. Finally, the pH-inactivation profile of 3CLpro with iodoacetamide is indicative of an ion-pair mechanism. Taken together, the data are consistent with a 3CL pro mechanism that utilizes an “electrostatic” trigger to initiate the acylation reaction, a cysteine-histidine catalytic dyad ion pair, an enzyme-facilitated release of P 1, and a general basecatalyzed deacylation reaction. Severe acute respiratory syndrome (SARS) 1 was a worldwide epidemic that appeared in November of 2002. The overall mortality rate was estimated by the World Health Organization to have reached 15% of all patients and up to 50% for patients over 65 years of age. In the wake of significant media attention, the research community applied intense and productive attention to this new viral disease. Four months after the first SARS case was identified, a new coronavirus was shown to be the causative agent: SARSCoV (1). Within 1 month, the entire genome of SARS-CoV had been sequenced ( 2, 3). The SARS-CoV was shown to be phylogenetically distinct from the three previously identified coronavirus groups ( 4). The main protease responsible for maturation of the polyproteins and central to the lifecycle was identified as a cysteine protease similar to the 3C family of cysteine proteases: 3CL pro (also known as Mpro). The 3CLpro has been cloned, expressed in Escherichia coli , and purified ( 5, 6). Its enzymatic activity has been measured by the development of both discontinuous and continuous enzymatic assays ( 6-11). Initial kinetic studies showed that the 3CLpro had modest activity ( kcat/Km of 177 M-1 s-1) (6), while other reports had high protease activity ( kcat/Km >10000 M-1 s-1) (8, 12). Differences in enzymatic activity and dimerization constants may be attributed to the 3CL pro construct design, the substrate, or the handling/storage conditions ( 11, 13). The crystal structure of the protease has been released (PDB entry 1q2w by Bonnano et al. 2003) with other structures published subsequently ( 14, 15). 3CLpro has been characterized as having a chymotrypsin-fold ( 14, 16). The structure revealed that the full-length protease contains a catalytic domain (aa1 -184), a linker region (aa185 -200), and a putative regulatory domain (aa201 -303). Structural studies have provided some insight into the 3CL pro reaction mechanism. The active protease is a homodimer with the Cys145-His41 catalytic dyad located in the cleft between the first two domains ( 7, 14-16). Detailed studies of the * To whom correspondence should be addressed. Tel: 858-622-6038. Fax: 858-526-4240. E-mail: brion.murray@pfizer.com. ‡ Present address: Accelagen, 11585 Sorrento Valley Road, Suite 107, San Diego, CA 92121. 1 Abbreviations: SARS, severe acute respiratory syndrome; SARSCoV, severe acute respiratory syndrome coronavirus; 3CL pro, 3C-like protease; D2O, deuterium oxide; SIE, solvent isotope effect; PI, proton inventory; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; BTP, bis-tris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane; MES, 2-morpholinoethanesulfonic acid; DTT, dithiothreitol; pNA, p-nitroaniline; pNP, p-nitrophenol; FRET, fluorescence resonance energy transfer; ITC, isothermal titration calorimetry; EDTA, ethylenediaminetetraacetic acid; DMSO, dimethylsulfoxide; Dabcyl, [4-(4dimethylaminophenylazo)benzoic acid]; IA, iodoacetamide; Tamra, 5-carboxytetramethylrhodamine; Edans, 5-[2 ′-(aminoethyl)amino]naphthalenesulfonic acid; IPTG, isopropylâ-thiogalactopyranoside; PCR, polymerase chain reaction; TFA, trifluoroacetic acid; RFU, relative fluorescence units. 2617 Biochemistry2008,47, 2617-2630 10.1021/bi702107v CCC: $40.75 © 2008 American Chemical Society Published on Web 02/01/2008 functions of the individual domains have revealed that the carboxy-terminal domain (aa201 -303) has a critical role in the formation of the active dimer ( 17). While at least eight superfamilies of cysteine proteases exist, our understanding of cysteine protease mechanism is derived primarily from studies of the papain family of cysteine proteases or inferred from related serine proteases (18). Papain is the prototypical cysteine protease, which catalyzes peptide hydrolysis with a catalytic dyad ion pair (Cys25-His159), which is oriented by a third residue (Asn175) (18). For the acylation reaction of papain, the ionpair dyad is the catalytic device responsible for the nucleophilic attack on the substrate’s scissile amide carbonyl, and it also is involved in the facilitation of the leaving group stabilization ( 18-21). For papain, His159 acts as a general base to facilitate the activation of an active site water in the deacylation half-reaction ( 19). With 3CLpro, an analogous catalytic dyad (Cys145 -His41) has been observed in the crystal structure. But unlike papain, the histidine residue of the 3CLpro catalytic dyad is oriented by a water molecule not an asparagine residue ( 16). The other common enzymatic mechanism for proteolytic peptide cleavage is general base catalysis. The fundamental feature of a general base mechanism is that the catalytic group participates in a protontransfer that stabilizes the transition state. General base catalysis is the commonly accepted mechanism for chymotrypsin (22). Chymotrypsin has a catalytic triad (Ser195/ His57/Asp102) that acts as a charge relay system to increase the nucleophilicity of Ser195 through the abstraction of the hydroxyl proton in a concerted attack of the substrate carbonyl of the scissile bond. The minimal model for cysteine protease catalysis (Scheme 1) is a three-step reaction that first involves the binding of the substrate to form a Michaelis complex (ES). Next, an acylation reaction occurs with the simultaneous formation of the covalent enzyme acyl intermediate (ES ′) and the alcohol or amine product (P 1). Finally, a deacylation reaction occurs through the hydrolysis of the ES ′ acyl intermediate to generate the second product, P 2. In this model, the acylation rate is described by k2 and the deacylation rate by k3. An alternative model (five step) has been proposed that has an additional conformational change to facilitate the release of P1 (Scheme 2) ( 23). The (ES′‚P1)* complex represents the enzyme conformation from which the alcohol or amine P1 rapidly diffuses to leave (ES ′). The enzymatic mechanism for 3CL pro has been proposed to utilize chymotrypsin-like general base catalysis and not a papain-like thiolate -imidazolium ion-pair mechanism ( 10). To support this assignment, the authors presented multiple lines of evidence. They showed that the mutation of the catalytic cysteine to serine (C145S) was active with a 40fold reduction of activity. An inverse solvent isotope effect (SIE) was observed for the wild-type 3CL pro, while a normal SIE was observed for the C145S mutant. Last, the measured pKa values were reported to be consistent with a general base mechanism. This first reported kinetic study used a 3CL pro construct that had an additional N-terminal residue, which significantly reduced enzymatic activity. In this paper, we have further studied the mechanism of the 3CLpro protease using steady-state and pre-steady-state methods on amide and ester substrate hydrolysis reactions. The rate constants that define the kinetic mechanism of ester hydrolysis were characterized. Included in this report are solvent isotope effect and proton inventory studies utilizing amide and ester substrates. Although proton inventory studies have been widely used for serine proteases, they have not been widely used with cysteine proteases ( 20). The proton inventory studies coupled with pre-steady-state isotope effect evaluation provide a deeper level of understanding of the recently discovered cysteine protease 3CL pro. We also used the mechanistic approach for characterizing the catalytic mechanism of a cysteine protease: pH dependence of iodoacetamide inactivation. The inactivation profile is more consistent with an ion-pair mechanism. Taken as a whole, our data are more consistent with the 3CL pro catalysis of the acylation reaction occurring through an ion-pair mechanism and the catalysis of the deacylation reaction by a general base mechanism. Evidence is provided for an “electrostatic switch” to trigger the acylation reaction and an enzymefacilitated release of the first product. MATERIALS AND METHODS Materials. Peptide 1, peptide 2, and peptide 3 were purchased from SynPep (Dublin, CA) and had the following purities: >97%, >93%, and>91%, respectively. Peptide 4, peptide 5, and peptide 6 were purchased f