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Characterization of the deamidase enzyme responsible for the metabolism of the anticancer peptide: H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2.

Abstract：

:H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2, a broad spectrum neuropeptide growth factor antagonist (antagonist G), is soon to enter a phase I clinical trial for the treatment of small-cell lung cancer (SCLC). The pre-clinical pharmacology of this peptide has revealed that its metabolism proceeds from the C-terminus via deamidation. In this study the class of enzyme responsible for the degradation of antagonist G has been characterized. Tissue distribution studies on the enzyme have shown it to be very widespread with high specific activity being detected in the spleen, kidney, H69 SCLC xenograft and liver (12.64, 9.58, 8.00 and 6.94 nmols G/mg protein/hr, respectively). HPLC gel filtration indicated that the G-deamidase enzyme had an apparent molecular mass of 81 kDa. The sub-cellular distribution of the enzyme using differential centrifugation indicates that it is largely soluble with > 85% of the activity located in the cytosolic fraction. The distribution of activity towards antagonist G closely resembles that of esterase and acid carboxypeptidase activity, two activities, along with deamidase activity, known to be possessed by serine carboxypeptidases. Studies using a range of protease inhibitors showed clear inhibition of metabolism by phenylmethylsulphonylfluoride and benzyloxycarbonylphenylalanine chloromethylketone, indicating that the enzyme is a chymotrypsin-like serine carboxypeptidase. This knowledge of the enzyme will be invaluable in the further development of antagonist G and similar compounds. Moreover, the widespread distribution of this enzyme together with its broad specificity for C-terminal group suggests that it should be given serious consideration when designing C-terminally modified peptide drugs.

journal_name

Biochem Pharmacol

journal_title

Biochemical pharmacology

authors

Jones DA,Cummings J,Langdon SP,MacLellan A,Smyth JF

doi

10.1016/0006-2952(95)00191-2

subject

Has Abstract

pub_date

1995-08-25 00:00:00

pages

585-90

issue

eissn

0006-2952

issn

1873-2968

pii

0006-2952(95)00191-2

journal_volume

pub_type

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Characterization of the deamidase enzyme responsible for the metabolism of the anticancer peptide: H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH2.