Expression and Characterization of Human Pancreatic Preprocarboxypeptidase A1 and Preprocarboxypeptidase A2

Laethem, Ronald M.; Blumenkopf, Todd A.; Cory, Michael; Elwell, Lynn P.; Moxham, Christopher M.; Ray, Paul H.; Walton, Leslie; Smith, Gary K.

doi:10.1006/abbi.1996.0310

Cited by 28 publications

(23 citation statements)

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“…Our initial target to generate a human enzyme with nonhuman specificity was human pancreatic carboxypeptidase A, recently cloned and expressed in our laboratory (31). Pancreatic carboxypeptidase A is a zinc-containing exopeptidase released into the small intestine from the pancreas as a zymogen (32,33).…”

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

“…Pancreatic carboxypeptidase A is a zinc-containing exopeptidase released into the small intestine from the pancreas as a zymogen (32,33). The pancreatic CPA has two further subclasses, CPA1 and CPA2, in both humans (31,34,35) and rats (36,37). While the amino acid sequences of CPA1 and CPA2 active sites are similar and both enzymes prefer aromatic C-terminal amino acids, CPA2 enzyme prefers bulkier aromatic C-terminal amino acids (31,37).…”

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

“…The pancreatic CPA has two further subclasses, CPA1 and CPA2, in both humans (31,34,35) and rats (36,37). While the amino acid sequences of CPA1 and CPA2 active sites are similar and both enzymes prefer aromatic C-terminal amino acids, CPA2 enzyme prefers bulkier aromatic C-terminal amino acids (31,37). This was shown for the rat enzyme with di-and tripeptide substrates and for the human with amino acid prodrugs of MTX (31,37).…”

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

“…While the amino acid sequences of CPA1 and CPA2 active sites are similar and both enzymes prefer aromatic C-terminal amino acids, CPA2 enzyme prefers bulkier aromatic C-terminal amino acids (31,37). This was shown for the rat enzyme with di-and tripeptide substrates and for the human with amino acid prodrugs of MTX (31,37). High resolution crystal structures for bCPA have been determined (32).…”

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

“…High resolution crystal structures for bCPA have been determined (32). Of the nine active site residues within 4.5 Å of the bound substrate, only three vary among bovine CPA (32), rat CPA1 (36), rCPA2 (37), hCPA1 (31,34), and hCPA2 (31,35). These changes, at residues 253, 254, and 268, describe a larger binding pocket for CPA2 than CPA1 and provide a rationale for the substrate specificities.…”

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

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Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate

Smith

Banks

Blumenkopf³

et al. 1997

Journal of Biological Chemistry

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Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stable in vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine-and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human carboxypeptidases A. Two of these analogs are MTX-␣-3-cyclobutylphenylalanine and MTX-␣-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best sub-

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Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

Section: From Glaxowellcome Inc Research Triangle Park North Carolmentioning

confidence: 99%

See 3 more Smart Citations

Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate

Smith

Banks

Blumenkopf³

et al. 1997

Journal of Biological Chemistry

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Enzymes involved in the bioconversion of ester-based prodrugs

Liederer

Borchardt

2006

Journal of Pharmaceutical Sciences

330

238

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Enzymes and Prodrugs Used for ADEPT

Knox

1999

Enzyme-Prodrug Strategies for Cancer Therapy

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Expression and Characterization of Human Pancreatic Preprocarboxypeptidase A1 and Preprocarboxypeptidase A2

Cited by 28 publications

References 0 publications

Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate

Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate

Enzymes involved in the bioconversion of ester-based prodrugs

Enzymes and Prodrugs Used for ADEPT

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