Slow Tight-binding Inhibitor Research Articles

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Discovery of TP0597850: A Selective, Chemically Stable, and Slow Tight-Binding Matrix Metalloproteinase-2 Inhibitor with a Phenylbenzamide-Pentapeptide Hybrid Scaffold.

Matrix metalloproteinase-2 (MMP2) is a zinc-dependent endopeptidase and a promising target for various diseases, including cancer and fibrosis. Herein, we report the discovery of a novel MMP2-selective inhibitor with high chemical stability and slow tight-binding features. Based on the degradation mechanism of our small-molecule-peptide hybrid 1, the tripeptide linker {5-aminopentanoic acid [Ape(5)]-Glu-Asp} of 1 was replaced by a shorter linker (γ-D-Glu). Phenylbenzamide was suitable for the new generation of MMP2 inhibitors as an S1' pocket-binding group. The introduction of (4S)-aminoproline dramatically increased the chemical stability while maintaining high subtype selectivity because of its interaction with Glu130. TP0597850 (18) exhibited high stability over a wide range of pH values as well as potent MMP2 inhibition (Ki = 0.034 nM) and ≥2000-fold selectivity determined using the inhibition constants. A kinetic analysis revealed that it possesses slow tight-binding nature with a long MMP2 dissociative half-life (t1/2 = 265 min).

A Long Residence Time Enoyl-Reductase Inhibitor Explores an Extended Binding Region with Isoenzyme-Dependent Tautomer Adaptation and Differential Substrate-Binding Loop Closure.

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.

Functional Analysis of a Gene Cluster from Chitinophaga pinensis Involved in Biosynthesis of the Pyrrolidine Azasugar DAB-1.

Azasugars, "nitrogen in the ring" analogues of monosaccharides, are known to be distributed in select plant, fungal. and bacterial species. We identify Chitinophaga pinensis DSM 2588 as the first bacterial source of the plant pyrrolidine azasugar 1,4-dideoxy-1,4-aminoarabinitol (DAB-1). Comparative sequence analyses identified C. pinensis as a putative azasugar producer, via observation of a three-gene cluster coding for putative aminotransferase, alcohol dehydrogenase, and sugar phosphatase enzymes, similar to the previously reported azasugar biosynthetic signature identified in Bacillus amyloliquefaciens FZB42. Multistep fractionation of C. pinensis culture media guided by a maltase inhibition assay yielded a component with a mass consistent with the structure of DAB-1. Heterologous expression of the three-gene cluster in E. coli, a non-azasugar producer, led to the isolation of nectrisine, a biosynthetic precursor to DAB-1, which displayed potent slow tight binding inhibition of maltase. Reduction of nectrisine with NaBH4 removed the slow tight binding inhibition kinetics, and MS analysis provided evidence for the production of a compound matching that of the isolated DAB-1 from C. pinensis. 1H NMR analysis of the nectrisine produced in E. coli after NaBD4 reduction produced a spectrum consistent with DAB-1 deuterated at C-1, primarily at the pro-S position. These results support the idea that the azasugar three-gene cluster represents a general biosynthetic path leading to several different compounds, which may prove useful for the identification of other azasugar-producing organisms.

Molecular Mechanisms in the Selectivity of Nonsteroidal Anti-Inflammatory Drugs.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase (COX) 1 and 2 with varying degrees of selectivity. A group of COX-2 selective inhibitors-coxibs-binds in a time-dependent manner through a three-step mechanism, utilizing a side pocket in the binding site. Coxibs have been extensively probed to identify the structural features regulating the slow tight-binding mechanism responsible for COX-2 selectivity. In this study, we further probe a structurally and kinetically diverse data set of COX inhibitors in COX-2 by molecular dynamics and free energy simulations. We find that the features regulating the high affinities associated with time-dependency in COX depend on the inhibitor kinetics. In particular, most time-dependent inhibitors share a common structural binding mechanism, involving an induced-fit rotation of the side-chain of Leu531 in the main binding pocket. The high affinities of two-step slow tight-binding inhibitors and some slow reversible inhibitors can thus be explained by the increased space in the main binding pocket after this rotation. Coxibs that belong to a separate class of slow tight-binding inhibitors benefit more from the displacement of the neighboring side-chain of Arg513, exclusive to the COX-2 side-pocket. This displacement further stabilizes the aforementioned rotation of Leu531 and can explain the selectivity of coxibs for COX-2.

Probing the Time Dependency of Cyclooxygenase-1 Inhibitors by Computer Simulations.

Time-dependent inhibition of the cyclooxygenases (COX) by a range of nonsteroidal anti-inflammatory drugs has been described since the first experimental assays of COX were performed. Slow tight-binding inhibitors of COX-1 bind in a two-step mechanism in which the EI → EI* transition is slow and practically irreversible. Since then, various properties of the inhibitors have been proposed to cause or affect the time dependency. Conformational changes in the enzyme have also been proposed to cause the time dependency, but no particular structural feature has been identified. Here, we investigated a series of inhibitors of COX-1 that are either time-independent or time-dependent using a combination of molecular dynamics simulations, binding free energy calculations, and potential of mean force calculations. We find that the time-dependent inhibitors stabilize a conformational change in the enzyme mainly identified by the rotation of a leucine side chain adjacent to the binding pocket. The induced conformation has been previously shown to be essential for the high binding affinities of tight-binding inhibitors in COX-1. The results of this work show that the structural features of the enzyme involved in both time-dependent and tight-binding inhibition are identical and further identify a structural mechanism responsible for the transition between the two enzyme-inhibitor complexes characteristic of slow tight-binding COX-1 inhibitors.

Tebipenem, a New Carbapenem Antibiotic, Is a Slow Substrate That Inhibits the β-Lactamase from Mycobacterium tuberculosis

The genome of Mycobacterium tuberculosis contains a gene, blaC, which encodes a highly active β-lactamase (BlaC). We have previously shown that BlaC has an extremely broad spectrum of activity against penicillins and cephalosporins but weak activity against newer carbapenems. We have shown that carbapenems such as meropenem, doripenem, and ertapenem react with the enzyme to form enzyme–drug covalent complexes that are hydrolyzed extremely slowly. In the current study, we have determined apparent Km and kcat values of 0.8 μM and 0.03 min–1, respectively, for tebipenem, a novel carbapenem whose prodrug form, the pivalyl ester, is orally available. Tebipenem exhibits slow tight-binding inhibition at low micromolar concentrations versus the chromogenic substrate nitrocefin. FT-ICR mass spectrometry demonstrated that the tebipenem acyl–enzyme complex remains stable for greater than 90 min and exists as mixture of the covalently bound drug and the bound retro-aldol cleavage product. We have also determined the high-resolution crystal structures of the BlaC–tebipenem covalent acylated adduct (1.9 Å) with wild-type BlaC and the BlaC–tebipenem Michaelis–Menten complex (1.75 Å) with the K73A BlaC variant. These structures are compared to each other and to other carbapenem–BlaC structures.

Symmetric adamantyl-diureas as soluble epoxide hydrolase inhibitors

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing two urea groups has been developed. Inhibition potency of the described compounds ranges from 2.0μM to 0.4nM. 1,6-(Hexamethylene)bis[(adamant-1-yl)urea] (3b) was found to be a potent slow tight binding inhibitor (IC50=0.5nM) with a strong binding to sEH (Ki=3.1nM) and a moderately long residence time on the enzyme (koff=1.05×10−3s−1; t1/2=11min).

Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs

Myeloperoxidase (MPO) catalyzes the breakdown of hydrogen peroxide and the formation of the potent oxidant hypochlorous acid. We present the application of the fluorogenic peroxidase substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in steady-state and transient kinetic studies of MPO function. Using initial kinetic parameters for the MPO system, we characterized under the same conditions a number of gold standards for MPO inhibition, namely 4-amino benzoic acid hydrazide (4-ABAH), isoniazid and NaN3 before expanding our focus to isomers of 4-ABAH and benzoic acid hydrazide analogs. We determined that in the presence of hydrogen peroxide that 4-ABAH and its isomer 2-ABAH are both slow-tight binding inhibitors of MPO requiring at least two steps, whereas NaN3 and isoniazid-based inhibition has a single observable step. We also determined that MPO inhibition by benzoic acid hydrazide and 4-(trifluoromethyl) benzoic acid hydrazide was due to hydrolysis of the ester bond between MPO heavy chain Glu 242 residue and the heme pyrrole A ring, freeing the light chain and heme b fragment from the larger remaining MPO heavy chain. This new mechanism would essentially indicate that the benzoic acid hydrazide analogs impart inhibition through initial ejection of the heme catalytic moiety without prior loss of the active site iron.

Comparative Kinetics of Qi Site Inhibitors of Cytochrome bc1 Complex: Picomolar Antimycin and Micromolar Cyazofamid

Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki =0.033±0.00027nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki =12.90±0.91μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

Histone Deacetylase (HDAC) Inhibitor Kinetic Rate Constants Correlate with Cellular Histone Acetylation but Not Transcription and Cell Viability

Histone deacetylases (HDACs) are critical in the control of gene expression, and dysregulation of their activity has been implicated in a broad range of diseases, including cancer, cardiovascular, and neurological diseases. HDAC inhibitors (HDACi) employing different zinc chelating functionalities such as hydroxamic acids and benzamides have shown promising results in cancer therapy. Although it has also been suggested that HDACi with increased isozyme selectivity and potency may broaden their clinical utility and minimize side effects, the translation of this idea to the clinic remains to be investigated. Moreover, a detailed understanding of how HDACi with different pharmacological properties affect biological functions in vitro and in vivo is still missing. Here, we show that a panel of benzamide-containing HDACi are slow tight-binding inhibitors with long residence times unlike the hydroxamate-containing HDACi vorinostat and trichostatin-A. Characterization of changes in H2BK5 and H4K14 acetylation following HDACi treatment in the neuroblastoma cell line SH-SY5Y revealed that the timing and magnitude of histone acetylation mirrored both the association and dissociation kinetic rates of the inhibitors. In contrast, cell viability and microarray gene expression analysis indicated that cell death induction and changes in transcriptional regulation do not correlate with the dissociation kinetic rates of the HDACi. Therefore, our study suggests that determining how the selective and kinetic inhibition properties of HDACi affect cell function will help to evaluate their therapeutic utility.

Design, Synthesis, and Kinetic Characterization of Protein N-Terminal Acetyltransferase Inhibitors

The N-termini of 80-90% of human proteins are acetylated by the N-terminal acetyltransferases (NATs), NatA-NatF. The major NAT complex, NatA, and particularly the catalytic subunit hNaa10 (ARD1) has been implicated in cancer development. For example, knockdown of hNaa10 results in cancer cell death and the arrest of cell proliferation. It also sensitized cancer cells to drug-induced cytotoxicity. Human NatE has a distinct substrate specificity and is essential for normal chromosome segregation. Thus, NAT inhibitors may potentially be valuable anticancer therapeutics, either directly or as adjuvants. Herein, we report the design and synthesis of the first inhibitors targeting these enzymes. Using the substrate specificity of the enzymes as a guide, we synthesized three bisubstrate analogues that potently and selectively inhibit the NatA complex (CoA-Ac-SES4; IC50 = 15.1 μM), hNaa10, the catalytic subunit of NatA (CoA-Ac-EEE4; Ki = 1.6 μM), and NatE/hNaa50 (CoA-Ac-MLG7; Ki* = 8 nM); CoA-Ac-EEE4 is a reversible competitive inhibitor of hNaa10, and CoA-Ac-MLG7 is a slow tight binding inhibitor of hNaa50. Our demonstration that it is possible to develop NAT selective inhibitors should assist future efforts to develop NAT inhibitors with more drug-like properties that can be used to chemically interrogate in vivo NAT function.

Slow-tight binding inhibition of pepsin by an aspartic protease inhibitor from Streptomyces sp. MBR04

The present article reports a low molecular weight aspartic protease inhibitor from a Streptomyces sp. MBR04 exhibiting a two-step inhibition mechanism against pepsin. The kinetic interactions revealed a reversible, competitive, slow-tight binding inhibition with an IC50 and Ki values of 4.5nM and 4nM respectively. The conformational changes induced upon inhibitor binding to pepsin was monitored by far and near UV analysis, demonstrated that the inhibitor binds to the active site and causes inactivation. Chemical modification of the inhibitor with WRK and TNBS abolished the antiproteolytic activity of the inhibitor.

1,3-Phenylene bis(ketoacid) derivatives as inhibitors of Escherichia coli dihydrodipicolinate synthase

Dihydrodipicolinate synthase is a key enzyme in the lysine biosynthesis pathway that catalyzes the condensation of pyruvate and aspartate semi-aldehyde. A series of phenolic ketoacid derivatives that mimic the proposed enzymatic intermediate were designed as potential inhibitors of this enzyme and were synthesized from simple precursors. The ketoacid derivatives were shown to act as slow and slow-tight binding inhibitors. Mass spectrometric experiments provided further evidence to support the proposed model of inhibition, demonstrating either an encounter complex or a condensation product for the slow and slow-tight binding inhibitors, respectively.

Modulation of glucokinase by glucose, small-molecule activator and glucokinase regulatory protein: steady-state kinetic and cell-based analysis

GK (glucokinase) is an enzyme central to glucose metabolism that displays positive co-operativity to substrate glucose. Small-molecule GKAs (GK activators) modulate GK catalytic activity and glucose affinity and are currently being pursued as a treatment for Type 2 diabetes. GK progress curves monitoring product formation are linear up to 1 mM glucose, but biphasic at 5 mM, with the transition from the lower initial velocity to the higher steady-state velocity being described by the rate constant kact. In the presence of a liver-specific GKA (compound A), progress curves at 1 mM glucose are similar to those at 5 mM, reflecting activation of GK by compound A. We show that GKRP (GK regulatory protein) is a slow tight-binding inhibitor of GK. Analysis of progress curves indicate that this inhibition is time dependent, with apparent initial and final Ki values being 113 and 12.8 nM respectively. When GK is pre-incubated with glucose and compound A, the inhibition observed by GKRP is time dependent, but independent of GKRP concentration, reflecting the GKA-controlled transition between closed and open GK conformations. These data are supported by cell-based imaging data from primary rat hepatocytes. This work characterizes the modulation of GK by a novel GKA that may enable the design of new and improved GKAs.

The Kunitz 1 and Kunitz 3 domains of tissue factor pathway inhibitor are required for efficient inhibition of factor Xa

Tissue factor pathway inhibitor (TFPI) is a slow tight-binding inhibitor that inhibits factor (F)Xa in a biphasic fashion: a rapid formation of loose FXa·TFPI encounter complex is followed by slow rearrangement into a tight FXa·TFPI* complex in which the Kunitz-2 (K2) domain of TFPI binds and inhibits FXa. In the current study, full-length TFPI (TFPIfl) and various truncated TFPI constructs were used to assess the importance of TFPI domains other than K2 in the inhibition of FXa. In the absence of Ca2+ ions, FXa was more effectively inhibited by TFPIfl than Gla-domain less FXa. In turn, Ca2+ ions impaired FXa inhibition by TFPIfl but not by TFPI constructs that lack the C-terminus. This suggests that, in absence of Ca2+ ions, interactions between the C-terminus of TFPI and the Gla-domain of FXa promote FXa-inhibition. TFPIfl and K2K3 had similar efficiencies for encounter complex formation. However, K2K3 showed monophasic inhibition instead of biphasic inhibition, indicating absence of rearrangement into a tight complex. K1K2 and TFPI1-161 showed biphasic inhibition, but had less efficient encounter complex formation than TFPIfl. Finally, K2K3 was a 10-fold more efficient FXa- inhibitor than K2. These results indicate that K3-C-terminus enhances the formation of encounter complex and that K1 is required for isomerisation of the encounter- into tight complex. Since TFPIfl has a 10-fold higher Ki than K2K3-C-terminus, we propose that K1 is not only required for the transition of the loose to the tight FXa·TFPI* complex, but also inhibits FXa·TFPI encounter complex formation. This inhibitory activity is counteracted by K3 and C-terminus.

Benzothiophene carboxamide derivatives as inhibitors of Plasmodium falciparum enoyl‐ACP reductase

Benzothiophene derivatives like benzothiophene sulphonamides, biphenyls, or carboxyls have been synthesized and have found wide pharmacological usage. Here we report, bromo-benzothiophene carboxamide derivatives as potent, slow tight binding inhibitors of Plasmodium enoyl-acyl carrier protein (ACP) reductase (PfENR). 3-Bromo-N-(4-fluorobenzyl)-benzo[b]thiophene-2-carboxamide (compound 6) is the most potent inhibitor with an IC50 of 115 nM for purified PfENR. The inhibition constant (Ki) of compound 6 was 18 nM with respect to the cofactor and 91 nM with respect to crotonoyl-CoA. These inhibitors showed competitive kinetics with cofactor and uncompetitive kinetics with the substrate. Thus, these compounds hold promise for the development of potent antimalarials.

Identification and Characterization of a Trypsin Inhibitor from Fagopyrum tataricumSeeds.

This study was aimed at investigating the purification and identification of serine protease inhibitors, F. tataricum trypsin inhibitor (FtTI) from tartary buckwheat (Fagopyrum tataricum) seeds. The FtTI was isolated by anion exchange chromatography, affinity chromatography, and centrifugal ultrafiltration. Under reducing and nonreducing conditions, an SDS-PAGE analysis showed that the isolated protein consists of a single polypeptide chain with a molecular mass of approximately 14kDa. The two isoforms of FtTI were confirmed by the mass spectrometric profile where the two peaks corresponded to 11.487 and 13.838kDa. The complete amino acid sequence of FtTI has been established by automatic Edman degradation and mass spectrometry. The molecule of FtTI consists of 86 amino acid residues containing two disulfide bonds which connect Cys8 to Cys65 and Cys49 to Cys58. The active site of FtTI contains an Asp66-Arg67 bond. The Ki value was calculated using the equation for slow tight binding inhibition which was 1.6nM for trypsin. FtTI retained its inhibitory activity over a wide range of pH (3-10) and temperature (20-80°C). FtTI can be rapidly inactivated by the combination of high temperature and high pressure. An analysis of the amino acid sequence suggests that FtTI is a member of the protease inhibitor Ι family. Furthermore, FtTI exhibited a strong inhibitory activity against phytopathogenic fungi.

Synthesis, stability, biochemical and pharmacokinetic properties of a new potent and selective 4-oxo-β-lactam inhibitor of human leukocyte elastase

The 4-oxo-β-lactams (azetidine-2,4-diones) are potent acylating agents of the human leukocyte elastase (HLE), a neutrophil serine protease that plays a key role in several inflammatory diseases. A novel 4-oxo-β-lactam containing a N-(4-(phenylsulphonylmethyl)phenyl) group, 3, was designed as a potential mechanism-based inhibitor capable of undergoing elimination of phenylsulphinate upon Ser-195 acylation. Compound 3 was found to be a potent slow-tight binding inhibitor of HLE, presenting a remarkable second-order rate constant of 1.46 × 106 M−1s−1 and displaying selectivity over the proteinase 3 and cathepsin G. However, liberation of phenylsulphinate was not observed in the hydrolysis of 3 in both pH 7.4 phosphate buffer and human plasma. The Cmax values of 1207 μg/total blood, 179 μg/g spleen and 106 μg/g lung were determined by HPLC, following a single 30 mg/kg dose of 3 given intraperitoneally to NMRI mice, suggesting that the inhibitor distributes well into tissues. Although being a powerful selective inhibitor of HLE, 4-oxo-β-lactam 3 has a limited stability, being susceptible to off-target reactions (plasma and liver enzymes).

A novel bifunctional peptidic aspartic protease inhibitor inhibits chitinase A from Serratia marcescens: Kinetic analysis of inhibition and binding affinity

Background Chitinase inhibitors have chemotherapeutic potential as fungicides, pesticides and antiasthmatics. The majority of chitinase inhibitors reported are natural products like argifin, argifin linear fragments, argadin, allosamidin and disulfide-cyclized peptides. Here, we report a novel peptidic inhibitor API ( Aspartic Protease Inhibitor), isolated from Bacillus licheniformis that inhibits chitinase A (ChiA) from Serratia marcescens. Methods The binding affinity of API with ChiA and type of inhibition was determined by the inhibition kinetics assays. Fluorescence and CD spectroscopic analysis and chemical modification of API with different affinity reagents elucidated the mechanism of binding of API with ChiA. Results and conclusions The peptide has an amino acid sequence N-Ile 1-Cys 2-Glu 3-Ala 4-Glu 5-His 6-Lys 7-Trp 8-Gly 9-Asp 10-Tyr 11-Leu 12-Asp 13-C. The ChiA–API kinetic interactions reveal noncompetitive, irreversible and tight binding nature of API with I 50 = 600 nM and K i = 510 nM in the presence of chromogenic substrate p-nitrophenyl-N,N′-diacetyl-β-chitobioside[p-NP-(GlcNAc) 2]. The inhibition progress curves show a two-step slow tight binding inhibition mechanism with the rate constant k 5 = 8.7 ± 1 × 10 − 3 s − 1 and k 6 = 7.3 ± 0.6 × 10 − 5 s − 1 . CD-spectra and tryptophanyl fluorescence analysis of ChiA incubated with increasing API concentrations confirms conformational changes in enzyme structure which may be due to irreversible denaturation of enzyme upon binding of API. Chemical modifications by WRK abolished the anti-chitinase activity of API and revealed the involvement of carboxyl groups in the enzyme inactivation. Abolished isoindole fluorescence of OPTA-labeled ChiA demonstrates the irreversible denaturation of ChiA upon incubation with API for prolonged time and distortion of active site of the enzyme. General significance The data provide useful information that could lead to the generation of drug-like, natural product-based chitinase inhibitors.