Abstract
Transition state analogs pepstatin methylester (PME) and L685458 have been shown to inhibit gamma-secretase non-competitively (Tian, G., Sobotka-Briner, C., Zysk, J., Liu, X., Birr, C., Sylvester, M. A., Edwards, P. D., Scott, C. W., and Greenberg, B. D. (2002) J. Biol. Chem. 277, 31499-31505). This unusual kinetics suggests physical separation of the sites for substrate binding and catalysis with binding of the transition state analogs to the catalytic site and not to the substrate binding site. Methods of inhibitor cross-competition kinetics and competition ligand binding were utilized to address whether non-transition state small molecule inhibitors, which also display non-competitive inhibition of gamma-secretase, inhibit the enzyme by binding to the catalytic site as well. Inhibitor cross-competition kinetics indicated competitive binding between the transition state analogs PME and L685458 and between small molecules arylsulfonamides and benzodiazepines, but non-competitive binding between the transition state analogs and the small molecule inhibitors. These results were indicative of two inhibitor binding sites, one for transition state analogs and the other for non-transition state small molecule inhibitors. The presence of two inhibitor binding sites for two different classes of inhibitors was corroborated by results from competition ligand binding using [3H]L685458 as the radioligand. Although L685458 and PME displaced the radioligand at the same concentrations as for enzyme inhibition, arylsulfonamides and benzodiazepines did not displace the radioligand at their Ki values, a result consistent with the presence of two inhibitor binding sites. These findings provide useful insights into the catalytic and regulatory mechanisms of gamma-secretase that may facilitate the design of novel gamma-secretase inhibitors.
Highlights
(34), and the lack of observed displacement of L685458 by C100 as shown in the present study, are consistent with a mechanism in which substrate binding and catalysis occur at different locations on the enzyme and the catalysis requires a substrate movement after binding at the substrate binding site that swings the scissile bond into the catalytic site [34]
As ␥-secretase may catalyze its reaction through separated substrate binding and catalytic sites (34, 36, 37, and this study), it is possible that inhibition may be achieved by blocking the movement of substrate into the catalytic site
Analogous to the mechanism of affecting catalysis, blocking substrate movement may be achieved in at least two ways: 1) through a physical mechanism, where the inhibitor occupies a site located between the substrate binding site and the catalytic site to form a physical blockage to the substrate motion or 2) by a conformational mechanism, in which a structural distortion in the region between the substrate binding and catalytic sites results from binding of inhibitor remotely (Fig. 8B)
Summary
A, -amyloid; APP, amyloid precursor protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydoxy-1-propanesulfonate; MES, 4-morpholineethanesulfonic acid; C99, APP C-terminal fragment of 99 amino acid residues; C100, recombinantly produced C99 containing an additional methionine residue at its metazoan species [27]. PME and L685458, which are transition state isosteres, have been shown to display non-competitive inhibition of ␥-secretase [34], suggesting an unprecedented enzyme kinetic mechanism that involves physical separation of substrate binding and catalysis [34]. Arylsulfonamides and benzodiazepines, which do not resemble transition state isosteres, display non-competitive inhibition of ␥-secretase [34] This raised the question of whether these small molecule inhibitors affect ␥-secretase activity by binding to the same site as the transition state isosteres or interact with ␥-secretase at some other as yet unknown binding sites. Data obtained from inhibitor cross-competition kinetics suggested the presence on ␥-secretase of two inhibitor binding sites, one for binding of transition state isosteres and the other for non-transition state small molecule inhibitors This observation was corroborated by results from competition ligand binding with the radioligand [3H]L685458. These findings provide useful insights into the catalytic and regulatory mechanisms of ␥-secretase that may facilitate the design of novel, and possibly substrate-selective, inhibitors
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