Active site directed irreversible inhibition of thermolysin

Rasnick, David; Powers, James C.

doi:10.1021/bi00614a002

Cited by 51 publications

(21 citation statements)

References 24 publications

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“…Other inhibitors utilize a leaving group, such as a halide, which upon nucleophilic attack by an active-site residue leaves the inhibitor, which then forms a covalent bond to the enzyme. These ligands and active groups are generally coupled to a peptide or amino acid conferring a specificity capable of interacting in a competitive manner with the substrate-binding sites of the enzyme [24,36,38,391. Owing to similarities in the mechanism of action of these zinc metalloproteases, an inhibitor of one of these enzymes may inhibit other enzymes of the same class when modified to conform to the enzymes' individual specificity requirements [9,11,40,411.…”

Section: Discussionmentioning

confidence: 99%

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

et al. 1986

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The proteolytic specificities of two zinc hemorrhagic toxins (Ht-c and Ht-d), isolated from Crotalus atrox venom, were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates. The enzymes cleaved the Ala14-Leu15 bond of the insulin B chain most rapidly and the T~r'~-Leu'' slightly more slowly. The His5-Leu6, His'O-Leu'l, and G l~~~-P h e '~ bonds were also cleaved but at condiserably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala14-Leu" bond were synthesized ranging in length from four to seven residues. The heptdpeptide NH2-Leu-Val-GluAla-Leu-Tyr-Leu-COOH was the best peptide substrate tested with the other peptides having decreasing k,,,/K,,, values with decreasing length. The tetrapeptide NH,-Ma-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. Furthermore, this peptide was shown to serve as a competitive inhibitor of the toxins. The N-acetylated pentapeptides and hexapeptides, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counterparts. The toxins had the highest k,,,/K, values for the acetylated peptide Ac-Val-Ala-Leu-Leu-Ala-COOH. The data suggest that the toxins may indeed have extended substratebinding sites, which may accomodate at least six amino acid residues. The best substrate examined thus far for the toxins is the fluorogenic peptide analog 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, suggestive of similarities between the toxins and mammalian collagenases as well as thermolysin. Mechanisms for inhibition of the enzymes were investigated using amino acid hydroxamates, chloromethyl esters, phosphoramidon and the peptide NH2-Ala-Leu-Tyr-Leu-COOH. All of these inhibitors had Ki values in the M range.

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Section: Discussionmentioning

confidence: 99%

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

et al. 1986

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“…As such, the design of electrophilic affinity-labeling reagents for MPs is not straightforward. Although select examples exist of electrophile-based covalent inhibitors of MPs (32), the potential conversion of these agents into ABPP probes is compromised by either weak potency (33) or restricted target selectivity (34).…”

Section: Design and Synthesis Of Abpp Probes For Mpsmentioning

confidence: 99%

Activity-based probes for the proteomic profiling of metalloproteases

Saghatelian

Jessani

Joseph

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

425

317

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Metalloproteases (MPs) are a large and diverse class of enzymes implicated in numerous physiological and pathological processes, including tissue remodeling, peptide hormone processing, and cancer. MPs are tightly regulated by multiple posttranslational mechanisms in vivo, hindering their functional analysis by conventional genomic and proteomic methods. Here we describe a general strategy for creating activity-based proteomic probes for MPs by coupling a zinc-chelating hydroxamate to a benzophenone photocrosslinker, which promote selective binding and modification of MP active sites, respectively. These probes labeled active MPs but not their zymogen or inhibitor-bound counterparts and were used to identify members of this enzyme class up-regulated in invasive cancer cells and to evaluate the selectivity of MP inhibitors in whole proteomes. Interestingly, the matrix metalloproteinase inhibitor GM6001 (ilomastat), which is currently in clinical development, was found to also target the neprilysin, aminopeptidase, and dipeptidylpeptidase clans of MPs. These results demonstrate that MPs can display overlapping inhibitor sensitivities despite lacking sequence homology and stress the need to evaluate MP inhibitors broadly across this enzyme class to develop agents with suitable target selectivities in vivo. Activitybased profiling offers a powerful means for conducting such screens, as this approach can be carried out directly in whole proteomes, thereby facilitating the discovery of disease-associated MPs concurrently with inhibitors that selectively target these proteins.

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“…The non-Michaelis-Menten kinetics of the hydrolysis of fMet-Leu-Phe at inflammatory doses, as well as the apparent two affinity states of the Met-Leu-Phe receptor [31,33], could be accounted for by the five-node cycle (receptor-NEP cycle). These features result from the existence of two uneven routes by which Met-Leu-Phe can reach the hydrolyzable fMet-LeuPhe -receptor -NEP complex.…”

Section: Discussionmentioning

confidence: 99%

“…leupeptin and pepstatin, which are not NEP inhibitors, did not affect Ant-Ala-GlyLeu-Ala-NH-Nb hydrolysis. By contrast, 1 ,I O-phenanthroline, phosphoramidon and the synthetic antagonist chloromethylcarboxy-N-hydroxylimine-DLphenylalanylalanylalaninamide ( a high-affinity labeling agent that binds irreversibly to the NEP active site) [31] abrogated Ant-Ala-Gly-Leu-Ala-NH-Nb hydrolysis. The NEP substrate Cbz-Gly-LeuNH2 inhibited Ant-Ala-Gly-Leu-Ala-NH-Nb hydrolysis by more than go%, while the non-NEP-substrate Cbz-Gly-GlyNH2 was totally ineffective (Fig.…”

Section: Chaructrrixtion Of Pmn Rizetizbrune Nep Activitymentioning

confidence: 99%

“…the ordinary enzymatic cleavage of specific NEP substrates 11 31, the degradation of Met-Leu-Phe in the chemotactic/ bacteriocidal relevant dose range of 10 -1000 nM, displayed an autocatalytic-like pattern 131. The dependence of the initial proteolysis rate on the bulk substrate concentration of fMet-L e~-[~H l P h e exceeded the theoretical boundary of MichaelisMenten kinetics.…”

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confidence: 99%

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Neutral endopeptidase activity in the interaction ofN‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine with human polymorphonuclear leukocytes

Yuli

Lelkes²

1991

European Journal of Biochemistry

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Human polymorphonuclear leukocytes (PMN) hydrolyze the synthetic chemoattractant N-formyl-Lmethionly-L-leucyl-L-phenylalanine (fMet-Leu-Phe) at nanomolar concentrations in an autocatalytic-like manner that deviates from classical Michaelis-Menten kinetics [Yuli, I. & Snyderman, R. (1986) J . B i d . Chem. ,761,4902-49081. By using inhibitors of distinct classes of endoproteases, this particular Wet-Leu-Phe degradation was attributed exclusively to an exoplasmic metalloendoprotease that matches the ubiquitous neutral endopeptidase (NEP). Membrane-bound NEP hydrolyzes non-chemotactic substrates according to a classic Michaelis-Menten mechanism. By competitive inhibition with non-chemotactic substrates, fMet-Leu-Phe was found to interact with membrane NEP through a single active site, in a non-cooperative mode with an apparent K,, in the order of 1 mM. The discrepancy between the ordinary hydrolysis of the micromolar and millimolar concentrations of Met-Leu-Phe, reported by others, and the particular degradation of the nanomolar met-Leu-Phe, could not be accounted for by any coherent correlation between NEP activity/inhibition and modulation of met-Leu-Phe binding to its receptor, and/or induction of Met-Leu-Phe-receptor-mediated inflammatory responses. Based on these and previously reported results, a novel model is proposed in which the Met-Leu-Phe-induced inflammatory stimulation of PMN involves both NEP and the fMet-Leu-Phe receptor. By this model, NEP and the Met-Leu-Phe receptor are distinct membrane entities which can form dynamic binary and tertiary complexes; thus accounting for the unusual kinetic features of Wet-Leu-Phe degradation, as well as the two receptor states. The complex of NEP and the Met-Leu-Phe receptor might be conceived as a chemotactic-perception mechanism that combines the high affinity of the receptor and the rapid turnover of NEP.The interaction of the model chemoattractant N-formyl-L-methionyl-L-leucyl-L-phenylalanine (Met-Leu-Phe) [l] with human polymorphonuclear leukocytes (PMN) involves rapid and extensive degradation of the chemoattractant [2]. This effect consists of seuqential proteolytic cleavages of, first, Nformylniethionine. then leucine residues [ 3 ] . Each of these steps suffices to revoke the chemotactic competence of the oligopeptide [4]. and may thus serve as a mechanism for signal termination.The degradation of the Met-Leu-Phe by PMN has been attributed to a plasma-membrane metalloendoprotease [2, 5, 61, which is immunologically related to the well-characterized renal neutral endopeptidase (NEP) [7, 81 and the common acute lymphocytic leukemia antigen [9-121. In contrast to

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Active site directed irreversible inhibition of thermolysin

Cited by 51 publications

References 24 publications

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

Substrate specificities and inhibition of two hemorrhagic zinc proteases Ht-c and Ht-d from Crotalus atrox venom

Activity-based probes for the proteomic profiling of metalloproteases

Neutral endopeptidase activity in the interaction ofN‐formyl‐l‐methionyl‐l‐leucyl‐l‐phenylalanine with human polymorphonuclear leukocytes

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