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.