Inhibitors of Tripeptidyl Peptidase II. 2. Generation of the First Novel Lead Inhibitor of Cholecystokinin-8-Inactivating Peptidase:  A Strategy for the Design of Peptidase Inhibitors

Abstract

The cholecystokinin-8 (CCK-8)-inactivating peptidase is a serine peptidase which has been shown to be a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). It cleaves the neurotransmitter CCK-8 sulfate at the Met-Gly bond to give Asp-Tyr(SO(3)H)-Met-OH + Gly-Trp-Met-Asp-Phe-NH(2). In seeking a reversible inhibitor of this peptidase, the enzymatic binding subsites were characterized using a fluorimetric assay based on the hydrolysis of the artificial substrate Ala-Ala-Phe-amidomethylcoumarin. A series of di- and tripeptides having various alkyl or aryl side chains was studied to determine the accessible volume for binding and to probe the potential for hydrophobic interactions. From this initial study the tripeptides Ile-Pro-Ile-OH (K(i) = 1 microM) and Ala-Pro-Ala-OH (K(i) = 3 microM) and dipeptide amide Val-Nvl-NHBu (K(i) = 3 microM) emerged as leads. Comparison of these structures led to the synthesis of Val-Pro-NHBu (K(i) = 0.57 microM) which served for later optimization in the design of butabindide, a potent reversible competitive and selective inhibitor of the CCK-8-inactivating peptidase. The strategy for this work is explicitly described since it illustrates a possible general approach for peptidase inhibitor design.

Efficient Presentation of Both Cytosolic and Endogenous Transmembrane Protein Antigens on MHC Class II Is Dependent on Cytoplasmic Proteolysis

“…The identity and location of this essential TPP activity identified by AAF-cmk is still unclear. It is possible that TPP-II is not the only activity involved, since butabindide, an inhibitor of TPP-II (31,32), causes less drastic inhibition of the presentation of cytosolic protein on MHC class II. Whether other related enzymatic activities such as TPP-I, puromycin-sensitive aminopeptidase, or bleomycin hydrolase are also involved remains to be investigated.…”

Tripeptidyl peptidase II is dispensable for the generation of both proteasome‐dependent and proteasome‐independent ligands of HLA‐B27 and other class I molecules

“…BFA, which blocks egress of MHC‐peptide complexes from the ER 36, and AAF‐amc, a fluorogenic substrate for TPPII 37, were from Sigma‐Aldrich (St Louis, MO). Butabindide (Tocris Bioscience, Bristol, UK) is a potent, reversible, selective and competitive inhibitor of TPPII 38. AAF‐cmk, an irreversible serine and cysteine protease inhibitor with broad specificity, which inhibits TPPII 14, was purchased from Biomol.…”

Strategies in the Design of Small‐Molecule Fluorescent Probes for Peptidases

“…Fluorescent detection of peptidase activity primarily relies on a substrate containing an amide functionality formed with peptidase specific residue as the acid and a fluorophore as the amine. For instance, Leu‐AMC (leucine‐(4‐methyl‐7‐coumarinylamide)) is extensively used in APN and LAP (leucine aminopeptidase) activity assays, while tripeptidyl‐peptidase II inhibitors are based on Ala‐Ala‐Phe‐AMC, and so on …”

“…For instance, Leu-AMC (leucine-(4-methyl-7-coumarinylamide)) is extensively used in APN and LAP (leucine aminopeptidase) activity assays, while tripeptidyl-peptidase II inhibitors are based on Ala-Ala-Phe-AMC, and so on. [40][41][42] Because there are numerous classifications of enzymes in tissues, in the event that the binding-based probe is able to identify the activities and distributions of peptidases in vivo, many approaches should be considered to improve the selectivity of probes, and one feasible approach is to find specific substrates. For example, to prescribe a selective probe that could monitor DPP-I activity in living cells, a series of (dipeptidyl) 2 -rhodamine substrates were well designed and synthesized by Harris et al One of them, (NH 2 -aminobutyric-homophenylalanine) 2rhodamine, exhibited functional reactivity and selectivity in the fluorescence-activated cell sorting (FACS) analysis (Scheme 4).…”