Pepstatin inhibition

First, the presence of a pepsinogen intermediate was indicated because pepstatin inhibited intramolecular pepsinogen activation. In a typical experiment, conditions which led to 57% activation gave rise to only 10% activation when the solution was saturated with pepstatin. In these studies the production of pepsin was evaluated from the appearance of its Ile-Gly- NH2-terminal sequence (12). 

When the molar ratio of pepstatin/pepsinogen was 0.9 1, the composition of the crude peptides obtained was much more similar to the expected composition from residues 1-44 (Table IV). Several NH2-termini were obtained similar to the result obtained on activation of pepsinogen in the absence of pepstatin. Presumably the pepstatin inhibits 90% of the activating pepsinogen but the remaining 10% can convert itself all the way into pepsin. This active pepsin could then split off the remaining activation segments exposed on the inhibited molecules so that a mix of 90% inhibited pepsin plus 10% active pepsin is obtained. 

Since pepstatin inhibits all acid proteases, but not the proteases of other groups, a common transition state which is a result of a common catalytic mechanism can obviously be suggested for this group of proteases. We know from the work of Umerzawa elt al., however, that the inhibitory potency of pepstatin differs for various acid proteases (Ref.l, Table X). We confirmed this for pepstatin inhibition of a group of acid proteases (Table II). The inhibitory potency of N-acetyl-Val-Sta-Ala-Sta also was found to vary widely. 

D. H. Rich, and E. T. Sun, Mechanism of inhibition of pepsin by pepstatin. Effect of inhibitor structure on dissociation constant and time-dependent inhibition, Biochem. Pharmacol. 29 2205 (1980). 

D. H. Rich, M. S. Bematowicz, N. S. Agarwal, M. Kawai, F. G. Salituro, and P. G. Schmidt, Inhibition of aspartic proteases by pepstatin and 3-methylstatine derivatives of pepstatin. Evidence for collected-substrate enzyme inhibition, Biochemistry 24 3165... 

The syndecan family of heparin sulfate proteoglycans (HSPGs) plays critical roles in several signal transduction pathways, and syndecan 3 intramembrane proteolysis is presenilin/y-secretase dependent (357). COX2 and COXl potentiate ABP formation through mechanisms that involve y-secretase activity. Sulindac sulfide and other NSAIDs (ibuprofen, indomethacin, R-flurbiprofen) selectively decrease the secretion of ABP independently of COX activity, probably via y-secretase inhibition (358-360). Pepstatin A methylester, sulfonamides, and benzodiazepines can also act as potent, noncompetitive, y-secretase inhibitors (335). These are but a few examples of the potential repercussions and biochemical consequences that the pharmacological manipulation of secretases in AD may bring about. 

Tian, G., Sobotka-Biiner, C.D., Zysk, J., et al. (2002) Linear non-competitive inhibition of solubilized human y-secretase by pepstatin A methylesper, L685458, sulfonamides, and benzodiazepines. J. Biol. Chem., 277, 31499-31505. 

Table I. Inhibition of Aspartyl Proteases by Pepstatin Analogs... 

Figure 1. Schematic representation of the relationships between proposed catalytic and inhibitory mechanisms. A. Postulated general acid-general base catalyzed mechanism for substrate hydrolysis by an aspartyl protease. The water molecule indicated is extensively hydrogen bonded to both aspartic acid residues plus other sites in the active site (see Reference 16 for details). Hydrogen bonds to water are omitted here. B. Kinetic events associated with the inhibition of pepsin by pepstatin. The pro-S hydroxyl group of statine displaces the enzyme immobilized water molecule shown in Figure lA. Variable aspartyl sequence numbers refer to penicillopepsin (pepsin, Rhizopus pepsin), respectively.

We synthesized the ketomethylene, , and hydroxyethylene,8, isosteres of a Leu-Ala dipeptide sequence in order to explore the importance of the two extra atoms in statine relative either to substrate or to the tetrahedral intermediate (Figure 1) in another aspartyl protease system. The compounds were synthesized by the routes outlined in Scheme I. This route was chosen so as to provide steric control at C-2 and C-5 of both 7 and 8 as well as to provide ready access to C-4 labeled analogs. Details of the synthesis have been described else-where.(23.24) Inhibitors were synthesized in which Leu-Ala dipeptide Isosteres replaced either Sta or Sta-Ala in known pepstatin analogs. Inhibition of porcine pepsin was determined using the reported spectrophotometric assay (Table I).(25)... 

S,4S)-4,8-Diamino-3-hydroxyoctanoic acid (DAHOA) derivatives were synthesized by the route shown in Scheme II using the known aldehyde Boc-Lys(Z)-CH0 (29) and were converted to the DAHOA peptide analogs shown. Inhibition of penicillopepsin by all statine and DAHOA pepstatin analogs was measured using substrate 21. The results of these determinations are shown in Table I. 

All of the aspartic proteases are inhibited by pepstatin, a peptide produced by some species of Actinomyces and which contains two residues of the unusual amino acid statine (sta).w Pepstatin has the sequence Isovaleryl-L-Val-L-Val-Sta-L-Ala-Sta. 

Oda, K., Fukuda, Y., Murao, S., Uchida, K., and Kainosho, M. (1989). A novel proteinase inhibitor, tyrostatin, inhibiting some pepstatin-insensitive carboxyl proteinases. Agric. Biol. Chem., 53, 405-415. 

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