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.
Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified -ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K i ؍ 38 nM) and mast cell chymase (K i ؍ 2.3 nM). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 Å) and human chymase (1.90 Å) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S 1 and S 2 subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S 1 , the 1-naphthyl in S 2 , and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S 3 /S 4 subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy antiinflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1␣, interleukin-1, tissue necrosis factor-␣, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.
Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a K(i) value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ-56423 potently inhibits trypsin (K(i) = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-A resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75% blockade of the early response and complete ablation of the late response and airway hyperresponsiveness.
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