1. Synthesis and biological activity of a series of potent fluoromethyl ketone inhibitors of recombinant human calpain I
S Chatterjee, et al. J Med Chem. 1997 Nov 7;40(23):3820-8. doi: 10.1021/jm970197e.
Calpain I, an intracellular cysteine protease, has been implicated in the neurodegeneration following an episode of stroke. In this paper, we report on a series of potent dipeptide fluoromethyl ketone inhibitors of recombinant human calpain I (rh calpain I). SAR studies revealed that while calpain I tolerates a variety of hydrophobic groups at the P1 site, Leu at P2 is preferred. However, the nature of the N-terminal capping group has a significant effect on the inhibitory activity of this series of compounds. Compound 4e [(1,2,3,4-tetrahydroisoquinolin-2-yl)carbonyl-Leu-D,L-Phe-CH2F+ ++], having a tetrahydroisoquinoline containing urea as the N-terminal capping group, is the most potent dipeptide fluoromethyl ketone inhibitor of calpain I (with a second-order rate constant for inactivation of 276,000 M-1 s-1) yet reported; tripeptide 4k (Cbz-Leu-Leu-D,L-Phe-CH2F) is equipotent. A number of compounds presented in this study displayed excellent selectivity for calpain I over cathepsins B and L, two related cysteine proteases. Compounds which exhibited good inhibitory activity in the assay against isolated rh calpain I also inhibited intracellular calpain I in a human cell line. Thus, in an intact cell assay, compounds 4e and 4k inhibited calpain I with IC50 values of 0.2 and 0.1 microM, respectively. Finally, we also disclose the first example of fluorination of a dipeptide enol silyl ether to generate the corresponding dipeptide fluoromethyl ketone.
2. The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B
C Crawford, R W Mason, P Wikstrom, E Shaw Biochem J. 1988 Aug 1;253(3):751-8. doi: 10.1042/bj2530751.
A series of peptidyldiazomethanes was synthesized and tested as inactivators of the cysteine proteinases calpain II, cathepsin L and cathepsin B. Inactivators that react rapidly and that show a degree of selectivity between the enzymes were identified. Z-Tyr(I)-Ala-CHN2 (where Z represents benzyloxycarbonyl) reacts rapidly with cathepsin L and more slowly with cathepsin B, but does not inhibit calpain II. Z-Leu-Leu-Tyr-CHN2 reacts rapidly with cathepsin L and calpain II but very slowly with cathepsin B. Boc-Val-Lys(epsilon-Z)Leu-Tyr-CHN2 (where Boc represents t-butyloxycarbonyl) reacts more rapidly with calpain II than with cathepsin L or cathepsin B. The discriminating inhibitory effects of these compounds make them potentially useful for investigation of enzyme functions in vivo. The data presented also provide insights into the subsite specificity of calpain.
3. The affinity-labelling of cathepsin S with peptidyl diazomethyl ketones. Comparison with the inhibition of cathepsin L and calpain
E Shaw, S Mohanty, A Colic, V Stoka, V Turk FEBS Lett. 1993 Nov 22;334(3):340-2. doi: 10.1016/0014-5793(93)80707-2.
Since peptidyl diazomethyl ketones are useful irreversible inhibitors for inactivating cysteinyl proteinases in vitro and in vivo and in order to reveal their role, we set out to obtain selective and effective reagents for cathepsin S. A number of such derivatives with hydrophobic amino acid residues, such as valine, leucine and tryptophane in positions adjacent to the primary specificity site were synthesized and these provided inhibitors rapidly acting at high dilution. For example, 1 nM Z-Leu-Leu-Nle-CHN2 inactivates cathepsin S with k2nd = 4.6 x 10(6) M-1 x s-1 at pH 6.5, 25 degrees C. Similarities to the specificities of cathepsin L and calpain were evident. However, Z-Val-Val-NleCHN2 is over 300 times more effective in inactivating S than L. On the other hand, Z-Phe-Tyr(t-Bu)CHN2 is about 10(4) more effective against L than S. Reagents are thus now available for a clear discrimination between these proteases.
