Structure-activity studies with high-affinity inhibitors of pyroglutamyl-peptidase II

Abstract

Inhibitors of PPII (pyroglutamyl-peptidase II) (EC 3.4.19.6) have potential applications as investigative and therapeutic agents. The rational design of inhibitors is hindered, however, by the lack of an experimental structure for PPII. Previous studies have demonstrated that replacement of histidine in TRH (thyrotropin-releasing hormone) with asparagine produces a competitive PPII inhibitor (Ki 17.5 microM). To gain further insight into which functional groups are significant for inhibitory activity, we investigated the effects on inhibition of structural modifications to Glp-Asn-ProNH2 (pyroglutamyl-asparaginyl-prolineamide). Synthesis and kinetic analysis of a diverse series of carboxamide and C-terminally extended Glp-Asn-ProNH2 analogues were undertaken. Extensive quantitative structure-activity relationships were generated, which indicated that key functionalities in the basic molecular structure of the inhibitors combine in a unique way to cause PPII inhibition. Data from kinetic and molecular modelling studies suggest that hydrogen bonding between the asparagine side chain and PPII may provide a basis for the inhibitory properties of the asparagine-containing peptides. Prolineamide appeared to be important for interaction with the S2' subsite, but some modifications were tolerated. Extension of Glp-Asn-ProNH2 with hydrophobic amino acids at the C-terminus led to a novel set of PPII inhibitors active in vitro at nanomolar concentrations. Such inhibitors were shown to enhance recovery of TRH released from rat brain slices. Glp-Asn-Pro-Tyr-Trp-Trp-7-amido-4-methylcoumarin displayed a Ki of 1 nM, making it the most potent competitive PPII inhibitor described to date. PPII inhibitors with this level of potency should find application in exploring the biological functions of TRH and PPII, and potentially provide a basis for development of novel therapeutics.

Figure 1. Inhibition of PPII hydrolysis of TRH-AMC by Glp-Asn-Pro-Tyr-Trp-Trp-AMC

Initial rates were determined using the continuous fluorometric assay. Initial rates against substrate concentration for increasing inhibitor concentration were fitted to a rectangular hyperbola (Michaelis–Menten plot) by non-linear regression analysis as shown in the upper graph. All kinetic parameters stated in the text were determined by non-linear regression analysis of data using the computer program Prism (GraphPad Software). Linear regression analysis was used to fit data to a Lineweaver–Burk plot to illustrate the type of inhibition observed. The results shown for Glp-Asn-Pro-Tyr-Trp-Trp-AMC in the lower graph are typical for all compounds with the sequence Glp-Asn-Pro-Xaa, thus far tested. The goodness of fit (r²) for both non-linear and linear regression analysis plots was >0.95. All V_max values fell within the confidence intervals of the intercept obtained from linear regression analysis, and the slope of a plot of V_max versus inhibitor concentration (not shown) was not found to deviate significantly from 0, supporting the interpretation that Glp-Asn-Pro-Tyr-Trp-Trp-AMC is acting as a classical competitive inhibitor of PPII.

Figure 2. Effects of (a) Glp-Asn-Pro-AMC and (b) Glp-Asn-Pro-Tyr-Trp-Trp-AMC on the release of TRH from rat brain hypothalamic slices

TRH release was measured under basal and depolarizing conditions in the presence of vehicle (saline or DMSO) or (a) Glp-Asn-Pro-AMC (0.1 mM in saline) or (b) Glp-Asn-Pro-Tyr-Trp-Trp-AMC (0.1 mM in DMSO). Results are means±S.E.M. (n=6). *P<0.05; **P<0.01 compared with corresponding basal; ###P<0.001 compared with corresponding conditions in the absence of inhibitor (Student's t test).

Figure 3. Predicted conserved binding mode for Glp-Asn-Pro inhibitory motif (green), compared with that obtained for TRH (yellow)

The Figure shows the active-site zinc (pink) and key active-site residues of human PPII (labelled in black). Broken lines highlight possible hydrogen-bond interactions that arise in inhibitory peptides that contain a P₁′ asparagine residue. Such interactions are not indicated for the substrate, TRH, and suggest a possible mechanism of action for the inhibitor series shown in the present study.