ABSTRACT
Cytosolic carboxypeptidases (CCPs) comprise a unique subfamily of M14 carboxypeptidases and are erasers of the reversible protein posttranslational modification- polyglutamylation. Potent inhibitors for CCPs may serve as leading compounds targeting imbalanced polyglutamylation. However, no efficient CCP inhibitor has yet been reported. Here, we showed that 2-phosphonomethylpentanedioic acid (2-PMPA), a potent inhibitor of the distant M28 family member glutamate carboxypeptidase II (GCPII), rather than the typical M14 inhibitor 2-benzylsuccinic acid, could efficiently inhibit CCP activities. 2-PMPA inhibited the recombinant Nna1 (a.k.a. CCP1) for hydrolyzing a synthetic peptide in a mixed manner, with Ki and Ki' being 0.11 µM and 0.24 µM respectively. It inhibited Nna1 for deglutamylating tubulin, the best-known polyglutamylated protein, with an IC50 of 0.21 mM. Homology modeling predicted that the R-form of 2-PMPA is more favorable to bind Nna1, unlike that GCPII prefers to S-form. This work for the first time identified a potent inhibitor for CCP family.
Subject(s)
Glutamate Carboxypeptidase II/antagonists & inhibitors , Organophosphorus Compounds/pharmacology , Protease Inhibitors/pharmacology , Carboxypeptidases/antagonists & inhibitors , Carboxypeptidases/genetics , Carboxypeptidases/metabolism , Cytosol/enzymology , Drug Evaluation, Preclinical/methods , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Glutamate Carboxypeptidase II/chemistry , Glutamate Carboxypeptidase II/metabolism , Glutarates/pharmacology , Kinetics , Molecular Docking Simulation , Organophosphorus Compounds/chemistry , Protease Inhibitors/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Serine-Type D-Ala-D-Ala Carboxypeptidase/genetics , Serine-Type D-Ala-D-Ala Carboxypeptidase/metabolism , Succinic Acid/pharmacologyABSTRACT
Here we evaluate the potential for local administration of a small molecule FOLH1/GCPII inhibitor 2-phosphonomethyl pentanedioic acid (2-PMPA) as a novel treatment for inflammatory bowel disease (IBD). We found that FOLH1/GCPII enzyme activity was increased in the colorectal tissues of mice with TNBS-induced colitis, and confirmed that 2-PMPA inhibited FOLH1/GCPII enzyme activity ex vivo. In order to maximize local enema delivery of 2-PMPA, we studied the effect of vehicle tonicity on the absorption of 2-PMPA in the colon. Local administration of 2-PMPA in a hypotonic enema vehicle resulted in increased colorectal tissue absorption at 30min compared to 2-PMPA administered in an isotonic enema vehicle. Furthermore, local delivery of 2-PMPA in hypotonic enema vehicle resulted in prolonged drug concentrations for at least 24h with minimal systemic exposure. Finally, daily treatment with the hypotonic 2-PMPA enema ameliorated macroscopic and microscopic symptoms of IBD in the TNBS-induced colitis mouse model, indicating the potential of FOLH1/GCPII inhibitors for the local treatment of IBD.