Characterisation of the Carboxypeptidase G2 Catalytic Site and Design of New Inhibitors for Cancer Therapy

Abstract

The enzyme carboxypeptidase G2 (CPG2) is used in antibody-directed enzyme prodrug therapy (ADEPT) to catalyse the formation of an active drug from an inert prodrug. Free CPG2 in the bloodstream must be inhibited before administration of the prodrug in order to avoid a systemic reaction in the patient. Although a few small-molecule CPG2 inhibitors have been reported, none has been taken forward thus far. This lack of progress is due in part to a lack of structural understanding of the CPG2 active site as well as the absence of small molecules that can block the active site whilst targeting the complex for clearance. The work described here aimed to address both areas. We report the structural/functional impact of extensive point mutation across the putative CPG2 catalytic site and adjacent regions for the first time, revealing that residues outside the catalytic region (K208A, S210A and T357A) are crucial to enzyme activity. We also describe novel molecules that inhibit CPG2 whilst maintaining the accessibility of galactosylated moieties aimed at targeting the enzyme for clearance. This work acts as a platform for the future development of high-affinity CPG2 inhibitors that occupy new chemical space and will advance the safe application of ADEPT in cancer treatment.

Sphingomonas sp. KT-1 PahZ2 Structure Reveals a Role for Conformational Dynamics in Peptide Bond Hydrolysis

“…The overall positioning of PAA 5 in the PahZ2 KT-1 binding site is similar to contacts made between the structural homologues DapE and carboxypeptidase G2 with their substrates where intermolecular interactions occur between substrate and both dimerization/catalytic domains. 38,86,87 We note condition-dependent differences that exist for PahZ2 KT-1 with respect to the orientation of PAA 5 in the binding site not previously reported for structural homologues. Figure 7A reveals that incubation of Zn 2+ /PAA 5 -bound PahZ2 KT-1 in the absence of salt promotes an open conformation that does not position the catalytic zinc ions adjacent to bound polypeptide substrate.…”

“…Inspection of the equilibrated PahZ2 KT‑1 :PAA 5 complex reveals that substrate binds at the hinge between dimerization and catalytic domains such that contacts are made across both domains independent of incubation conditions (Figure A,B). The overall positioning of PAA 5 in the PahZ2 KT‑1 binding site is similar to contacts made between the structural homologues DapE and carboxypeptidase G2 with their substrates where intermolecular interactions occur between substrate and both dimerization/catalytic domains. ,, …”

Considerations on the Rational Design of Covalently Conjugated Cell-Penetrating Peptides (CPPs) for Intracellular Delivery of Proteins: A Guide to CPP Selection Using Glucarpidase as the Model Cargo Molecule

“…CPG2 also known as glucarpidase (Voraxaz ® ) is an FDA-approved bacterial enzyme breaking methotrexate (MTX) down into two noncytotoxic metabolites [21,22,23]. Due to its capacity for hydrolyzing a wide range of folate analogs, CPG2 is also able to convert non-toxic glutamated nitrogen mustard prodrugs into cytotoxic substances; hence, it is proposed as an intriguing choice for the antibody/gene-directed enzyme prodrug therapy modalities known as ADEPT and GDEPT, respectively [24,25,26]. In glucarpidase therapy, the enzyme only reduces the circulating MTX levels and has no access to the drug inside the cells.…”