Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme

Abstract

Carboxypeptidase G2 (CPG2) is an Food and Drug Administration (FDA)-approved enzyme drug used to treat methotrexate (MTX) toxicity in cancer patients receiving MTX treatment. It has also been used in directed enzyme-prodrug chemotherapy, but this strategy has been hampered by off-site activation of the prodrug by the circulating enzyme. The development of a tumor protease activatable CPG2, which could be achieved using a circular permutation of CPG2 fused to an inactivating 'prodomain', would aid in these applications. We report the development of a protease accessibility-based screen to identify candidate sites for circular permutation in proximity of the CPG2 active site. The resulting six circular permutants showed similar expression, structure, thermal stability, and, in four cases, activity levels compared to the wild-type enzyme. We rationalize these results based on structural models of the permutants obtained using the Rosetta software. We developed a cell growth-based selection system, and demonstrated that when fused to periplasm-directing signal peptides, one of our circular permutants confers MTX resistance in Escherichia coli with equal efficiency as the wild-type enzyme. As the permutants have similar properties to wild-type CPG2, these enzymes are promising starting points for the development of autoinhibited, protease-activatable zymogen forms of CPG2 for use in therapeutic contexts.

Massively parallel, computationally-guided design of a pro-enzyme

“…We determined that this variant maintains the overall tertiary structure of the enzyme (Figure S2), indicating that the circular permutation does not perturb the structure or function of CPG2, as expected from our previous results. 24 We could not locate density for the appended helical pro-domain in the structure, a finding that is consistent with the weak inhibition observed for this design.…”

“…We previously designed active circular permutations of CPG2 (CPG2 CP-N89 ) with termini proximate to the active site, creating the possibility of attaching a terminal pro-domain that can access and effectively inhibit the active site. 24 The CPG2 CP-N89 variant (in which the polypeptide chain is re-organized such that the N-terminal residue is wild-type residue 89) served as the starting point for our design efforts. As CPG2 is a zinc metallo-enzyme, we reasoned that using a zinc-coordinating sidechain, such as glutamate, to guide pro-domain placement would result in enhanced auto-inhibition by rendering the catalytic site inaccessible while the pro-domain is bound (Figure 2).…”

“…We previously reported that periplasmically-expressed CPG2 is able to protect E. coli CAG12184 cells from methotrexate toxicity. 24,26–28 We employed this screen to discriminate between inhibiting and non-inhibiting pro-CPG2 designs from the set of 7469 computationally-generated designs. In summary, cells expressing designs with poorly inhibiting pro-domains (i.e.…”

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

“…Glucarpidase, like other peptide and protein-based biotherapeutics, is prone to proteolytic degradation. Although its stability in the blood is high enough to eliminate high concentrations of blood MTX, this might not be enough for successful application in ADEPT [75]. Therefore, several strategies have been applied to increase the resistance of glucarpidase to proteolytic enzymes, such as PEGylation, fusion with human serum albumin [76,77], and circular permutations [75].…”

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

“…showed that R324A substitution resulted in a mutant CPG2 with very low activity towards MTX, thereby confirming that the S1′ pocket participates in substrate binding/recognition. Surprisingly, this is the only residue in the proposed MTX binding region that has been studied experimentally by site‐directed mutagenesis in vivo or in vitro (although Yachnin and co‐workers recently reported the creation of CPG2 permutants at sites adjacent to this region). The residues in the putative S1 and S1′ pockets appear to be poorly conserved in our sequence alignments of other family members, as shown in Figure S1.…”