Discovery of Mechanism-Based Inactivators for Human Pancreatic Carboxypeptidase A from a Focused Synthetic Library

Abstract

Metallocarboxypeptidases (MCPs) are involved in many biological processes such as fibrinolysis or inflammation, development, Alzheimer's disease, and various types of cancer. We describe the synthesis and kinetic characterization of a focused library of 22 thiirane- and oxirane-based potential mechanism-based inhibitors, which led to discovery of an inhibitor for the human pro-carboxypeptidase A1. Our structural analyses show that the thiirane-based small-molecule inhibitor penetrates the barrier of the pro-domain to bind within the active site. This binding leads to a chemical reaction that covalently modifies the catalytic Glu270. These results highlight the importance of combined structural, biophysical, and biochemical evaluation of inhibitors in design strategies for the development of spectroscopically nonsilent probes as effective beacons for in vitro, in cellulo, and/or in vivo localization in clinical and industrial applications.

Keywords: Carboxypeptidase A; X-ray crystallography; mechanism-based inactivators; thiiranes.

Scheme 1. Recognition of a Polypeptide Substrate within the MCPs Active Site (Top Panel; Boxed), Mimicked by That of a Thiirane- or Oxirane-Based Inhibitors

The metal-ion coordination chemistry is necessary for the onset of covalent modification.

Figure 1

Library of synthetic oxiranes and thiiranes. Each compound was synthesized as a racemic mixture.

Scheme 2. General Synthetic Scheme for the Preparation of Compound Libraries

Figure 2

(A) Stereoimage of human proCPA1 in complex with compound 11. The backbone of the pro and the catalytic domains are shown in gray and blue, respectively. The inhibitor is shown in capped sticks. (B) 2_Fobs-F_calc electron-density map showing the hCPA1 active site covalently bound inhibitor 11, calculated by deleting the Glu270 side chain and inhibitor coordinates, is contoured at 1.5σ level. The inhibitor and residues important for binding (labeled) are shown in capped sticks (green and yellow, respectively). The catalytic zinc ion is in magenta, while the sulfur is in yellow. Oxygen and nitrogen are colored red and blue, respectively. A continuous electron density is clearly observed along the bond linking Glu270 side chain and the inhibitor. (C) Schematic representation of the binding of inhibitor 11.

Figure 3

Molecular modeling of the hCPA1–fluorine inhibitor complex structure. The inhibitor and residues important for binding are shown in capped sticks, in green and yellow, respectively. The catalytic zinc ion is in magenta, the sulfur is in yellow, and the fluorine is in light blue. Oxygens and nitrogens are in red and blue, respectively. The model has been constructed with PyMOL using PDB file 5OM9.