Biophysical study into the structure and substrate binding properties of peptido-mimetic ligands to carboxypeptidase G2

Doctoral thesis English OPEN
Jeyaharan, Dhadchayini
  • Subject: QD | QP

The antibody directed enzyme pro-drug therapy (ADEPT) is a new anticancer treatment, where pre-clinical experiments concluded that an intermediate step involving the inhibition of carboxypeptidase G2 (CPG2) from the circulatory system prior to pro-drug administration is crucial to prevent systemic toxicity (Bagshawe et al. 1991). The research described in this thesis sought to better understand the mode of binding of these inhibitors to CPG2 using solution state nuclear magnetic resonance (NMR) spectroscopy.\ud \ud A high-yield expression of active and soluble mature CPG2 (in the absence of the leader peptide) in E. coli suitable for NMR studies and co-crystallisation screening is reported. We have used this method to routinely produce milligrams quantities of 1H/13C/15N isotopically-labelled protein suitable for NMR studies. The second aim of this thesis is interactions; interactions between CPG2 and selected inhibitors provided by our industrial partner Mologic Ltd. (Bedford, UK). Different structural parts of the inhibitors were identified by NMR to directly interact with CPG2: the naphthalene and the glutamate groups. Chemical shift perturbations studies show different patterns for CP06 and CP67 inhibitors suggesting that they have different binding mechanisms. Site-directed mutagenesis of residues in P1 pocket of CPG2 reveal no activity against methotrexate (MTX), suggesting that they are key players in substrate recognition, while H285A and E200A mutant proteins display similar activity to wild type CPG2 protein.\ud \ud Although the NMR data described here for CPG2 were incomplete and thus did not yield resonance assignment, we show attempts at a "divide-and-conquer" approach. The CPG2CAT construct shows great promise for downstream NMR studies as it has favourable solution properties and retains key properties of the parent protein, namely enzymatic activity and the ability to self associate.
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