Purification and Properties of Carboxypeptidase G1

McCullough, Jerry L.; Ba, Chabner; Bertino, Joseph R.

doi:10.1016/s0021-9258(19)45873-0

Cited by 135 publications

(25 citation statements)

References 21 publications

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“…Carboxypeptidase G enzymes have been isolated from a number of pseudomonads [9][10][11][12] and a Flavobacterium sp. [13], and all of them have very similar substrate specificities.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy

et al. 1997

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We find that the CPG2 catalytic domain has structural homology with other zinc-dependent exopeptidases, both those with a single zinc ion and those with a pair of zinc ions in the active site. The closest structural homology is with the aminopeptidase from Aeromonas proteolytica, where the similarity includes superposable zinc ligands but does not extend to the rest of the active-site residues, consistent with the different substrate specificities. The mechanism of peptide cleavage is likely to be very similar in these two enzymes and may involve the bridging hydroxyl ion ligand acting as a primary nucleophile.

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“…Carboxypeptidase G enzymes have been isolated from a number of pseudomonads [9][10][11][12] and a Flavobacterium sp. [13], and all of them have very similar substrate specificities.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy

et al. 1997

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“…As previously described, CPG2 catalytic activity was assessed employing MTX. 19 Briefly, purified PEGylated CPG2 fusion protein (50 µg/mL) was added to a mixture of MTX with its buffer (0.1 M Tris-HCl pH 7.3 containing 0.2 mM ZnSO4 and 5 µL of MTX “0.45 mM”) and incubated at 37 ο C. The decrease in absorbance was measured every 10 min using NANODROP 1000 spectrophotometer (Thermo Scientific). Different MTX concentrations were used to determine the catalytic activity of the PEGylated CPG2 fusion proteins.…”

Section: Methodsmentioning

confidence: 99%

Production of Long-Acting CNGRC–CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment

Al-Mansoori

Qahtani

Elsinga

et al. 2021

Technol Cancer Res Treat

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Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC–carboxypeptidase G2 (CPG2) and CNGRC–CPG2–CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC–CPG2, and CNGRC–CPG2–CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug “ZD2767P” cytotoxic effect in association with PEG CPG2–CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC–CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

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“…This provides an alternative nonrenal route of methotrexate elimination by converting methotrexate to its inactive metabolites, DAMPA and glutamate (Figure 1). 1,5,8-13 This is particularly important for patients who already have impaired renal function.…”

Section: Glucarpidasementioning

confidence: 99%