“…Chymotrypsin has been identified previously as a suitable target for aromatic haloenol lactones resulting in its mechanism-based inhibition as detailed by careful kinetic analyses by Katzenellenbogen (41)(42)(43)(44). In prior studies, (R)-BEL was determined to be a more efficient inhibitor of chymotrypsin than (S)-BEL in comparison to its chiral counterpart (36). To substantiate further the absolute stereochemistry of the BEL enantiomers resolved by chiral HPLC and to confirm the ability of the resolved enantiomers to inhibit selectively chymotrypsin activity, increasing concentrations of (R)-BEL, (S)-BEL, or rac-BEL were incubated with chymotrypsin and diluted in buffer as described under "Experimental Procedures."…”
Section: Resultsmentioning
confidence: 99%
“…Inhibition of ␣-Chymotrypsin by BEL-The kinetics of ␣-chymotrypsin inactivation by (R)-, (S)-, and rac-BEL were performed similar to methods described previously (36). The concentration of ␣-chymotrypsin (M r ϭ 25,000) was determined using an A 1% ϭ 20 (1-cm path length) at 280 nm (37).…”
The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, -cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A 2 (iPLA 2 s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA 2 s has been extended to include iPLA 2 ␥, which previously could not be pharmacologically distinguished from iPLA 2 . To determine whether iPLA 2  or iPLA 2 ␥ (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA 2  and iPLA 2 ␥ in intact cells. We hypothesized that the R-and S-enantiomers of BEL would possess different inhibitory potencies for iPLA 2  and iPLA 2 ␥. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA 2
“…Chymotrypsin has been identified previously as a suitable target for aromatic haloenol lactones resulting in its mechanism-based inhibition as detailed by careful kinetic analyses by Katzenellenbogen (41)(42)(43)(44). In prior studies, (R)-BEL was determined to be a more efficient inhibitor of chymotrypsin than (S)-BEL in comparison to its chiral counterpart (36). To substantiate further the absolute stereochemistry of the BEL enantiomers resolved by chiral HPLC and to confirm the ability of the resolved enantiomers to inhibit selectively chymotrypsin activity, increasing concentrations of (R)-BEL, (S)-BEL, or rac-BEL were incubated with chymotrypsin and diluted in buffer as described under "Experimental Procedures."…”
Section: Resultsmentioning
confidence: 99%
“…Inhibition of ␣-Chymotrypsin by BEL-The kinetics of ␣-chymotrypsin inactivation by (R)-, (S)-, and rac-BEL were performed similar to methods described previously (36). The concentration of ␣-chymotrypsin (M r ϭ 25,000) was determined using an A 1% ϭ 20 (1-cm path length) at 280 nm (37).…”
The agonist-stimulated release of arachidonic acid (AA) from cellular phospholipids in many cell types (e.g. myocytes, -cells, and neurons) has been demonstrated to be primarily mediated by calcium-independent phospholipases A 2 (iPLA 2 s) that are inhibited by the mechanism-based inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL). Recently, the family of mammalian iPLA 2 s has been extended to include iPLA 2 ␥, which previously could not be pharmacologically distinguished from iPLA 2 . To determine whether iPLA 2  or iPLA 2 ␥ (or both) were the enzymes responsible for arginine vasopressin (AVP)-induced AA release from A-10 cells, it became necessary to inhibit selectively iPLA 2  and iPLA 2 ␥ in intact cells. We hypothesized that the R-and S-enantiomers of BEL would possess different inhibitory potencies for iPLA 2  and iPLA 2 ␥. Accordingly, racemic BEL was separated into its enantiomeric constituents by chiral high pressure liquid chromatography. Remarkably, (S)-BEL was approximately an order of magnitude more selective for iPLA 2
“…Other strategies in inhibition that tie up catalytic residues in serinedependent enzymes have been described. [39][40][41][42][43][44][45][46] Either species 3 or 4 could account for the mechanism of inhibition by compound 2, both of which are unprecedented and novel for inhibition of cysteine proteases. 47,48 Synthesis of compound 2 is depicted in Scheme 2.…”
mentioning
confidence: 99%
“…By so doing, not only the histidine will be a poorer base for promotion of the water molecule for the “second step” of catalysis, but also the hydroxyl moiety that interacts with it is predisposed at the position of the incoming hydrolytic water molecule. Other strategies in inhibition that tie up catalytic residues in serine-dependent enzymes have been described. − 1 …”
Cathepsin B, a cysteine protease, is an important target in fighting cancer. This enzyme has been implicated in enhancing tumor invasiveness and metastasis, therefore inhibitors for cathepsin B are highly sought as potential anticancer and antimetastatic agents. A structure-based design effort was pursued in arriving at a template for inhibition of cathepsin B. Focused compound libraries were synthesized based on this template, which were screened for cathepsin B inhibitory properties. Compound 2, 1-(2(R)-[1(S)-acetoxy-2-[2(S)-(2,4-difluoro-benzoylamino)-3-phenyl-propionylaminooxy]-2-oxo-ethyl]-pentanoyl)-pyrrolidine-2(S)-carboxylic acid benzyl ester, is the prototype of this novel class of cysteine protease inhibitor that emerged from the search. The molecule modifies the active site of cathepsin B covalently, irreversibly, and efficiently, a process for which the kinetic parameters were evaluated. A set of three judiciously altered variants of compound 2 was also synthesized to explore the details of the proposed mechanism of action by this inhibitor. Compound 2 and its analogues may prove useful tools in reversing the deleterious effect of cathepsin B in fighting cancer.
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