Design, synthesis, and kinetic evaluation of a unique class of elastase inhibitors, the peptidyl .alpha.-ketobenzoxazoles, and the x-ray crystal structure of the covalent complex between porcine pancreatic elastase and Ac-Ala-Pro-Val-2-benzoxazole

Edwards, Philip D.; Meyer, Edgar F.; Vijayalakshmi, J.; Tuthill, Paul A.; Andisik, D.; Gomes, Bruce; Strimpler, Anne M.

doi:10.1021/ja00031a046

Cited by 143 publications

(132 citation statements)

References 1 publication

(2 reference statements)

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“…Although the Pro-Arg motif of CtA was expected to interact in a manner similar to that of PPACK, as observed in the crystallographic structure of the PPACK-thrombin complex (11,12), the cyclic organization and additional substituents of CtA offered intriguing possibilities for new interactions. There has been a paucity of studies on the a-keto amide functionality in the design of enzyme inhibitors, although a-keto esters and other activated carbonyl derivatives have received substantial attention (13)(14)(15)(33)(34)(35). Besides the naturally occurring CtA described here, some a-keto amide inhibitors of proteases have recently been described (33)(34)(35).…”

Section: Discussionmentioning

confidence: 99%

“…la) with the hydroxyl group of Ser'95, part of the thrombin catalytic triad His57-Asp102-Ser'95 [chymotrypsinogen numbering (11,12)], to generate a tetrahedral intermediate (13)(14)(15). Also, CtA contains a Pro-Arg structural motif that correlates with the P2-P1 positions in the tripeptide class of thrombin inhibitors, represented by D-Phe-Pro-Arg-CH2Cl (PPACK) (16,17) and Me-D-Phe-Pro-Arg-H (GYKI-14766) (18,19) (Fig.…”

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confidence: 99%

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Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

Maryanoff

Qiu

Padmanabhan

et al. 1993

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

108

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The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human a-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the SI specificity pocket; formation of a hydrogen-bonded two-strand antiparallel 3-sheet with Ser214-Gly216) and the a-keto amide group of CtA are primarily responsible for binding to thrombin, with the a-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr6OA-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp6OD as part of an "aromatic stacking chain." Biochemical inhibition data (K, values at 37C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human ae-thrombin (K, = 0.18 ,IM) but a potent inhibitor of trypsin (Kj = 0.023 IAM) and streptokinase (K, = 0.035 ,AM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragmentcondensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

Maryanoff

Qiu

Padmanabhan

et al. 1993

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

108

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“…Since Edwards' disclosure of ␣-keto heterocycles as effective protease inhibitors, a number of enzyme inhibitors have been described on the basis of analogous design principles (60)(61)(62)(63)(64)(65)(66)(67)(68)(69)(70). The inhibitors developed herein define additional features that may contribute independently to binding affinity which, when Abbreviation: FAAH, fatty acid amide hydrolase.…”

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confidence: 99%

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

Boger

Sato

Lerner

et al. 2000

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

261

264

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The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features-an optimal C12-C8 chain length, -unsaturation introduction at the corresponding arachidonoyl ⌬ 8,9 ͞⌬ 11,12 and oleoyl ⌬ 9,10 location, and an ␣-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 10 2 -10 3 times more potent than the corresponding trifluoromethyl ketones.

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“…Protein inhibitor⅐PPE complexes include those with elafin (13), chymotrypsin elastase inhibitor (14), and a heptamer from ␤-casomorphin-7 (15). Small molecule inhibitor complexes include: peptidyl fluoromethyl ketones (16 -19), peptidyl chloromethyl ketones (20), peptidyl boronic acids (21), isocoumarin derivatives (22)(23)(24), peptidylketobenzoxazoles (25), and a peptidomimetic aminimide (26). In all cases the elastase active site maintains the same shape, and typical root mean square differences between backbone atoms are less than 0.3 Å.…”

Section: Novel Mechanism Of Inhibition Of Elastase By ␤-Lactamsmentioning

confidence: 99%

Novel Mechanism of Inhibition of Elastase by β-Lactams Is Defined by Two Inhibitor Crystal Complexes

Taylor¹,

Anderson²,

Dowden³

et al. 1999

Journal of Biological Chemistry

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Two structurally related ␤-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the ␤-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the ␤-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.␤-Lactam-related compounds are well established irreversible inhibitors of a wide range of serine proteases including elastase (1), ␤-lactamase (2), phospholipase A2 (3), and bacterial signal peptidases (4). Moreover, their efficacy as orally active inhibitors has led to their widespread use in the clinic. In all cases a first step in the inhibition process is acylation of the active site serine by the ␤-lactam ring to generate a covalently bound acyl-enzyme intermediate. Thereafter, the inhibitor may undergo a further fragmentation resulting in a more stable entity that is resistant to deacylation and hence regeneration of enzyme activity. In this study, we report the discovery of a novel mode of action of a class of monocyclic ␤-lactams, which results in a particularly stable acyl-enzyme intermediate. The proposed mechanism, which is based on the x-ray structures of the elastase-ligand complexes presented below, may be generally applicable to other related serine proteases.Human leukocyte elastase is an important therapeutic target in view of its implied role in diseases such as emphysema (5), cystic fibrosis (6), and rheumatoid arthritis (7). Two x-ray structures of ␤-lactams bound to porcine pancreatic elastase (PPE) 1 have been reported, the first involving a cephalosporin sulfone (8) and more recently an N-sulfonylaryl ␤-lactam (9). In the latter case, the monocyclic ␤-lactam simply acylates the active site serine residue without further fragmentation. The class of monocyclic ␤-lactams reported herein contain an aryloxy substituent at the C-4 position (Fig. 1) that departs subsequent to acylation of the serine residue. We have compared two related members of this class of ␤-lactam that differ primarily in the nature of the C-3 substituent. This difference results in distinct modes of action upon reaction with elastase. EXPERIMENTAL PROCEDURESThe crystals were grown by the hanging drop method at room temperature. Porcine pancreatic elastase at 10 mg/ml was incubated with a 3-fold molar excess of inhibitor for 1.5 h at room temperature before drops were laid. The well solution contained 0.1 M sodium acetate buffer at pH 5.1 and 55 mM sodium sulfate. X-ray data for the EI116-elastase crystal complex were collected at 100 K using a MAR300 image plate mount...

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Design, synthesis, and kinetic evaluation of a unique class of elastase inhibitors, the peptidyl .alpha.-ketobenzoxazoles, and the x-ray crystal structure of the covalent complex between porcine pancreatic elastase and Ac-Ala-Pro-Val-2-benzoxazole

Cited by 143 publications

References 1 publication

Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

Novel Mechanism of Inhibition of Elastase by β-Lactams Is Defined by Two Inhibitor Crystal Complexes

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