Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

Structural and enzyme activity studies demonstrate that aryl substituted 2,3-butadienamine analogs inactivate Arthrobacter globiformis amine oxidase (AGAO) by chemical derivatization of the 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor

Copper amine oxidases (CAOs) are a family of redox active enzymes containing a 2,4,5-trihydroxyphenylalanine quinone (TPQ) cofactor generated from post translational modification of an active site tyrosine residue. The Arthrobacter globiformis amine oxidase (AGAO) has been widely used as a model to guide the design and development of selective inhibitors of CAOs. In this study, two aryl 2,3-butadienamine analogs, racemic 5-phenoxy-2,3-pentadienylamine (POPDA) and racemic 6-phenyl-2,3-hexadienylamine (PHDA), were synthesized and evaluated as mechanism-based inactivators of AGAO. Crystal structures show that both compounds form a covalent adduct with the amino group of the substrate-reduced TPQ, and that the chemical structures of the rac-PHDA and rac-POPDA modified TPQ differ by the allenic carbon that is attached to the cofactor. A chemical mechanism accounting for the formation of the respective TPQ derivative is proposed. Under steady-state conditions, no recovery of enzyme activity is detected when AGAO pre-treated with rac-PHDA or rac-POPDA is diluted with excess amount of the benzylamine substrate (100-fold K(m)). Comparing the IC(50) values further reveals that the phenoxy substituent in POPDA offers an approximately 4-fold increase in inhibition potency, which can be attributed to a favourable binding interaction between the oxygen atom in the phenoxy group and the active site of AGAO as revealed by crystallographic studies. This hypothesis is corroborated by the observed >3-fold higher partition ratio of PHDA compared to POPDA. Taken together, the results presented in this study reveal the mechanism by which aryl 2,3-butadienamines act as mechanism-based inhibitors of AGAO, and the potency of enzyme inactivation could be fine-tuned by optimizing binding interaction between the aryl substituent and the enzyme active site.     

Inhibition of AmpC beta-lactamase through a destabilizing interaction in the active site.

Beta-lactamases hydrolyze beta-lactam antibiotics, including penicillins and cephalosporins; these enzymes are the most widespread resistance mechanism to these drugs and pose a growing threat to public health. beta-Lactams that contain a bulky 6(7)alpha substituent, such as imipenem and moxalactam, actually inhibit serine beta-lactamases and are widely used for this reason. Although mutant serine beta-lactamases have arisen that hydrolyze beta-lactamase resistant beta-lactams (e.g., ceftazidime) or avoid mechanism-based inhibitors (e.g., clavulanate), mutant serine beta-lactamases have not yet arisen in the clinic with imipenemase or moxalactamase activity. Structural and thermodynamic studies suggest that the 6(7)alpha substituents of these inhibitors form destabilizing contacts within the covalent adduct with the conserved Asn152 in class C beta-lactamases (Asn132 in class A beta-lactamases). This unfavorable interaction may be crucial to inhibition. To test this destabilization hypothesis, we replaced Asn152 with Ala in the class C beta-lactamase AmpC from Escherichia coli and examined the mutant enzyme's thermodynamic stability in complex with imipenem and moxalactam. Consistent with the hypothesis, the Asn152 --> Ala substitution relieved 0.44 and 1.10 kcal/mol of strain introduced by imipenem and moxalactam, respectively, relative to the wild-type complexes. However, the kinetic efficiency of AmpC N152A was reduced by 6300-fold relative to that of the wild-type enzyme. To further investigate the inhibitor's interaction with the mutant enzyme, the X-ray crystal structure of moxalactam in complex with N152A was determined to a resolution of 1.83 A. Moxalactam in the mutant complex is significantly displaced from its orientation in the wild-type complex; however, moxalactam does not adopt an orientation that would restore competence for hydrolysis. Although Asn152 forces beta-lactams with 6(7)alpha substituents out of a catalytically competent configuration, making them inhibitors, the residue is essential for orienting beta-lactam substrates and cannot simply be replaced with a much smaller residue to restore catalytic activity. Designing beta-lactam inhibitors that interact unfavorably with this conserved residue when in the covalent adduct merits further investigation.     

Inverse acyl phosph(on)ates: substrates or inhibitors of beta-lactam-recognizing enzymes?

Acyl phosph(on)ates represent a new class of inhibitors of beta-lactam-recognizing enzymes. Previously described members of this class were aroyl phosph(on)ates. These compounds have been shown to acylate and/or phosphylate the active site serine residue, leading to either transient or essentially irreversible inhibition [Li, N., and Pratt, R. F. (1998) J. Am. Chem. Soc.120, 4264-4268]. The present paper describes the synthesis and evaluation as inhibitors of an inverse pair of acyl phosph(on)ates that incorporate the amido side chain that represents a major substrate specificity determinant of these enzymes. Thus, N-(phenylacetyl)glycyl phenyl phosphate and benzoyl N-(benzyloxycarbonyl)aminomethyl phosphonate were prepared. The former of these compounds was found to be a substrate of typical class A and C beta-lactamases and of the DD-peptidase of Streptomyces R61; it thus acylates the active site serine. In contrast, the latter compound was an irreversible inhibitor of the above enzymes, probably by phosphonylation of the active site serine. With each of these enzymes therefore, the amido side chain rather than the acyl group dictates the orientation of the bound phosph(on)ate and thus the mode of reaction.     

Mechanism-based inactivation of human leukocyte elastase via an enzyme-induced sulfonamide fragmentation process

We describe herein the design and in vitro biochemical evaluation of a novel class of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade. The inhibitors incorporate in their structure an appropriately functionalized saccharin scaffold. Furthermore, the inactivation of the enzyme by these inhibitors was found to be time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the formation of a stable acyl complex and is accompanied by the release of (L) phenylalanine methyl ester. The data are consistent with initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser(195)). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His(57)). It is also demonstrated herein that this class of compounds can be used in the design of inhibitors of serine proteases having either a neutral or basic primary substrate specificity. Thus, the results suggest that these inhibitors constitute a potential general class of mechanism-based inhibitors of (chymo)trypsin-like serine proteases.     

Quantitative structure-activity relationships for the pre-steady-state inhibition of cholesterol esterase by 4-nitrophenyl-N-substituted carbamates

4-Nitrophenyl-N-substituted carbamates (1-6) are the pseudo-substrate inhibitors of porcine pancreatic cholesterol esterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzyme inhibitor tetrahedral adduct and the second step of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzyme inhibitor tetrahedral adduct is further divided into two steps, the formation of the enzyme-inhibitor complex with the dissociation constant, KS, at the first step and the formation of the enzyme-inhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for the formation of the enzyme-inhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitative structure-activity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 1-6 indicate that values of -logKs and logk2/k-2 are correlated with the Taft substituent constant, sigma*, and the rho* values from these correlations are -0.33 and 0.1, respectively. The negative rho* value for the -logKS-sigma*-correlation indicates that the first step of the two-step formation of the enzyme-inhibitor tetrahedral adduct (KS step) is the formation of the positive enzyme inhibitor complex. The positive rho* value for the logk2/k-2 -sigma*-correlation indicates that the enzyme inhibitor tetrahedral adduct is more negative than the enzyme inhibitor complex. Finally, the two-step mechanism for the formation of the enzyme inhibitor tetrahedral adduct is proposed according to these results. Thus, the partially positive charge is developed at nitrogen of carbamates 1-6 in the enzyme-inhibitor complex probably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 1-6 and the amide hydrogen of the oxyanion hole of the enzyme. The second step of the two-step formation of the enzyme-inhibitor tetrahedral adduct is the nucleophilic attack of the serine of the enzyme to the carbonyl group of carbamates 1-6 in the enzyme-inhibitor complex and develops the negative-charged oxygen in the adduct.     

Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.     

New substrates for beta-lactam-recognizing enzymes: aryl malonamates

Aryl malonamates are demonstrated to be novel substrates of a broad range of beta-lactam-recognizing enzymes. These compounds are isomers of the aryl phenaceturates, which are well-known substrates of these enzymes, but the new compounds contain a retro-amide side chain. Several lines of evidence, including comparisons of steady-state kinetic parameters between enzymes and a detailed investigation of the methanolysis kinetics, solvent deuterium isotope effects, and pH-rate profile for turnover of a retro substrate by the Enterobacter cloacae P99 beta-lactamase, suggested that the new substrates are likely to be hydrolyzed by the same chemical mechanisms as "normal" substrates. Molecular modeling indicated that the retro-amide group fits snugly into the active site of the P99 beta-lactamase by hydrogen bonding to the conserved lysine-67 residue. The retro-amide side chain may represent a lead to novel mechanism-based and transition state analogue inhibitors.     

The active site of the P99 beta-lactamase from Enterobacter cloacae.

Labelling the beta-lactamase of Enterobacter cloacae P99 with a poor substrate or a mechanism-based inactivator points to an active-site serine residue in a sequence closely resembling that of the ampC beta-lactamase. These results establish the P99 enzyme as a class-C beta-lactamase, and the concurrence of the two approaches helps to confirm the reliability of determining active-site sequences with the aid of mechanism-based inactivators.     

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.     

Inhibition of class D beta-lactamases by diaroyl phosphates

The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.     

Mechanism of inactivation of monoamine oxidase by 1-phenylcyclopropylamine

1-Phenylcyclopropylamine (1-PCPA) is shown to be a mechanism-based inactivator of mitochondrial monoamine oxidase (MAO). The strained cyclopropyl ring is important to inactivation since alpha,alpha-dimethylbenzylamine, the acyclic analogue of 1-PCPA, is neither an inactivator nor a substrate of MAO. Two different pathways occur during inactivation by 1-PCPA, both believed to be derived from a common intermediate. One pathway leads to irreversible inactivation of the enzyme and a 1:1 stoichiometry of radioactivity to the active site when 1-[phenyl-14C]PCPA is used as the inactivator; the other pathway results in a covalent reversible adduct. Three organic reactions are carried out on the irreversibly labeled enzyme in order to determine the structure of the active site adduct. Sodium boro[3H]hydride reduction results in the incorporation of 0.73 equiv of tritium, suggesting a carbonyl functionality. Baeyer-Villiger oxidation followed by saponification gives 0.8 equiv of phenol, indicating the presence of a phenyl ketone. Treatment of the labeled enzyme with hydroxide produces acrylophenone, as would be expected from the retro-Michael reaction of beta-X-propiophenone. The identity of X is determined in two ways. The optical spectrum of the flavin cofactor is reduced during inactivation; no reoxidation occurs upon denaturation. Pronase treatment of the radioactively labeled enzyme produces fragments that contain both the radioactivity and the flavin. The X group, therefore, is the flavin. The results of two tests designed to differentiate N5 from C4a attachment to the flavin suggest an N5 adduct. In addition to formation of this stable covalent adduct, another pathway occurs 7 times as often. This alternate reaction of 1-[phenyl-14C]PCPA with MAO produces 7 equiv of [14C]acrylophenone during the course of irreversible inactivation and is believed to arise from formation of the same type of adduct as described above except that X is something other than the N5-flavin (Y). Upon denaturation of this labeled enzyme, the flavin is completely oxidized when most of the radioactivity is still bound to the enzyme. This indicates that Y is not a C4a-flavin adduct and suggests attachment to an active site amino acid residue. More facile elimination of Y from this beta-substituted propiophenone adduct would give acrylophenone on the time scale of the inactivation. Treatment of the reversible adduct with sodium borohydride prior to denaturation prevents release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.     

Thermodynamic evaluation of a covalently bonded transition state analogue inhibitor: inhibition of beta-lactamases by phosphonates

Serine beta-lactamases are inhibited by phosphonate monoesters in a reaction that involves phosphonylation of the active site serine residue. This reaction is much more rapid than the hydrolysis of these inhibitors in solution under the same conditions. The beta-lactamase active site therefore must have the ability to stabilize not only the anionic tetrahedral transition states of the acyl transfer reactions of substrates but also the pentacoordinated transition state(s) of phosphyl transfer reactions. A series of p-nitrophenyl arylphosphonates have been synthesized and the rate constants for their inhibition of the class C beta-lactamase of Enterobacter cloacae P99 determined. There is no direct correlation between these rate constants and the dissociation constants of analogous aryl boronic acids, where the latter are believed to generate good tetrahedral transition state analogue structures. Thus, the mode of stabilization of pentacoordinated phosphorus transition states by the beta-lactamase active site is qualitatively different from that of tetrahedral transition states. Molecular modeling suggests that the difference arises from different positioning of the side chain and of one of the oxygen ligands. In principle, the quality of the stable tetrahedral phosphonate complex as a transition state analogue structure can be assessed from the effect of its formation on the stability of the protein. Phosphonylation of the P99 beta-lactamase, however, had little effect on the stability of the protein, as measured both by thermal and guanidine hydrochloride denaturation. Consideration of the results of similar experiments with the Staphylococcus aureus PC1 beta-lactamase, where considerable stabilization is observed in thermal melting and, to a lesser degree, in formation of the molten globule in guanidine hydrochloride, but not in the complete unfolding transition in guanidine, suggests that results from the method may be strongly influenced by the interactions of the ligand with its environment in the unfolded state of the protein. Thus, quantitative estimates of the quality of a covalently bonded transition state analogue cannot generally be achieved by this method.     

Kinetics and mechanism of inhibition of a serine beta-lactamase by O-aryloxycarbonyl hydroxamates

The class C serine beta-lactamase of Enterobacter cloacae P99 is irreversibly inhibited by O-aryloxycarbonyl hydroxamates. A series of these new inhibitors has been prepared to investigate the kinetics and mechanism of the inactivation reaction. A pH-rate profile for the reaction indicated that the reactive form of the inhibitor is neutral rather than anionic. The reaction rate is enhanced by electron-withdrawing aryloxy substituents and by hydrophobic substitution on both aryloxy and hydroxamate groups. Kinetics studies show that the rates of loss of the two possible leaving groups, aryloxide and hydroxamate, are essentially the same as the rate of enzyme inactivation. Nucleophilic trapping experiments prove, however, that the aryl oxide is the first to leave. It is likely, therefore, that the rate-determining step of inactivation is the initial acylation reaction, most likely of the active site serine, yielding a hydroxamoyl-enzyme intermediate. This then partitions between hydrolysis and aminolysis by Lys 315, the latter to form an inactive, cross-linked active site. A previously described crystal structure of the inactivated enzyme shows a carbamate cross-link of Ser 64 and Lys 315. Structure-activity studies of the reported compounds suggest that they do not react at the enzyme active site in the same way as normal substrates. In particular, it appears that the initial acylation by these compounds does not involve the oxyanion hole, an unprecedented departure from known and presumed reactivity. Molecular modeling suggests that an alternative oxyanion hole may have been recruited, consisting of the side chain functional groups of Tyr 150 and Lys 315. Such an alternative mode of reaction may lead to the design of novel inhibitors.     

Indolequinone inhibitors of NRH:quinone oxidoreductase 2. Characterization of the mechanism of inhibition in both cell-free and cellular systems

We describe a series of indolequinones as efficient mechanism-based inhibitors of NRH:quinone oxidoreductase 2 (NQO2) for use either in cellular or cell-free systems. Compounds were designed to be reduced in the active site of the enzyme leading to loss of a substituted phenol leaving group and generation of a reactive iminium electrophile. Inhibition of NQO2 activity was assessed in both cell-free systems and the human leukemia K562 cell line. Inhibition of recombinant human NQO2 by the indolequinones was NRH-dependent, with kinetic parameters characteristic of mechanism-based inhibition and partition ratios as low as 2.0. Indolequinones inhibited NQO2 activity in K562 cells at nanomolar concentrations that did not inhibit NQO1 and were nontoxic to cells. Computation-based molecular modeling simulations demonstrated favorable conformations of indolequinones positioned directly above and in parallel with the isoalloxazine ring of FAD, and mass spectrometry extended our previous finding of adduction of the FAD in the active site of NQO2 by an indolequinone-derived iminium electrophile to the wider series of indolequinone inhibitors. Modeling combined with biochemical testing identified key structural parameters for effective inhibition, including a 5-aminoalkylamino side chain. Hydrogen bonding of the terminal amine nitrogen in the aminoalkylamino side chain was found to be critical for the correct orientation of the inhibitors in the active site. These indolequinones were irreversible inhibitors and were found to be at least 1 order of magnitude more potent than any previously documented competitive inhibitors of NQO2 and represent the first mechanism-based inhibitors of NQO2 to be characterized in cellular systems.     

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.     

Glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of γ-glutamyl transpeptidase for probing cysteinyl-glycine binding site

γ-Glutamyl transpeptidase (GGT) catalyzing the cleavage of γ-glutamyl bond of glutathione and its S-conjugates is involved in a number of physiological and pathological processes through glutathione homeostasis. Defining its Cys-Gly binding site is extremely important not only in defining the physiological function of GGT, but also in designing specific and effective inhibitors for pharmaceutical purposes. Here we report the synthesis and evaluation of a series of glutathione-analogous peptidyl phosphorus esters as mechanism-based inhibitors of human and Escherichia coli GGTs to probe the structural and stereochemical preferences in the Cys-Gly binding site. Both enzymes were inhibited strongly and irreversibly by the peptidyl phosphorus esters with a good leaving group (phenoxide). Human GGT was highly selective for l-aliphatic amino acid such as l-2-aminobutyrate (l-Cys mimic) at the Cys binding site, whereas E. coli GGT significantly preferred l-Phe mimic at this site. The C-terminal Gly and a l-amino acid analogue at the Cys binding site were necessary for inhibition, suggesting that human GGT was highly selective for glutathione (γ-Glu-l-Cys-Gly), whereas E. coli GGT are not selective for glutathione, but still retained the dipeptide (l-AA-Gly) binding site. The diastereoisomers with respect to the chiral phosphorus were separated. Both GGTs were inactivated by only one of the stereoisomers with the same stereochemistry at phosphorus. The strict recognition of phosphorus stereochemistry gave insights into the stereochemical course of the catalyzed reaction. Ion-spray mass analysis of the inhibited E. coli GGT confirmed the formation of a 1:1 covalent adduct with the catalytic subunit (small subunit) with concomitant loss of phenoxide, leaving the peptidyl moiety that presumably occupies the Cys-Gly binding site. The peptidyl phosphonate inhibitors are highly useful as a ligand for X-ray structural analysis of GGT for defining hitherto unidentified Cys-Gly binding site to design specific inhibitors.     

Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.     

Design, synthesis, and evaluation of gamma-phosphono diester analogues of glutamate as highly potent inhibitors and active site probes of gamma-glutamyl transpeptidase

Gamma-glutamyl transpeptidase (GGT, EC 2.3.2.2) catalyzes the transfer of the gamma-glutamyl group of glutathione and related gamma-glutamyl amides to water (hydrolysis) or to amino acids and peptides (transpeptidation) and plays a central role in glutathione metabolism. GGT is involved in a number of biological events, such as drug resistance and metastasis of cancer cells by detoxification of xenobiotics and reactive oxygen species through glutathione metabolism, and is also implicated in physiological disorders, such as Parkinson's disease, neurodegerative disease, diabetes, and cardiovascular diseases. In this study, we designed, synthesized, and evaluated a series of gamma-phosphono diester analogues of glutamate as transition-state mimic inhibitors of GGT. The electrophilic phosphonate diesters served as highly potent mechanism-based inhibitors that caused the time-dependent and irreversible inhibition of both the E. coli and human enzymes, probably by phosphonylating the catalytic Thr residue of the enzyme. In particular, one of the inhibitors exhibited more than 6000 times higher activity toward human GGT than acivicin, a classical but nonselective inhibitor of GGT. The dependence of the inactivation rate on the leaving group ability of the phosphonates (Br?nsted plot) revealed that the phosphonylation of the catalytic Thr residue proceeded via a dissociative transition-state with substantial bond cleavage between the phosphorus and the leaving group for both E. coli and human GGTs. The binding site of GGT for the Cys-Gly moiety of glutathione or for the acceptor molecules was probed by the phosphonate diesters to reveal a significant difference in the mechanism of substrate recognition between E. coli and human GGT. Thus, in the human enzyme, a specific residue in the Cys-Gly binding site played a critical role in recognizing the Cys-Gly moiety or the acceptor molecules by interacting with the C-terminal carboxy group, whereas the Cys side chain and the Cys-Gly amide bond were not recognized significantly. In contrast, the E. coli enzyme was a nonselective enzyme that accommodated substrates without specifically recognizing the C-terminal carboxy group of the Cys-Gly moiety of gamma-glutamyl compounds or the acceptor molecules. The phosphonate diester-based GGT inhibitors shown here should serve as a blue print for the future design of highly selective GGT inhibitors for use as drug leads and biological probes that gain insight into the hitherto undefined physiological roles of GGT and the relationships between GGT and a variety of diseases.