Complete differentiation between enkephalinase and angiotensin-converting enzyme inhibition by retro-thiorphan.

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Abstract
Thiorphan, N-[(R,S)-3-mercapto-2-benzylpropanoyl]glycine is a highly potent inhibitor (Ki [inhibition constant] = 3.5 nM) of enkephalinase, a metalloendopeptidase cleaving the Gly-Phe bond (positions 3 and 4) of enkephalins in brain tissue. In accordance with this property, thiorphan displays antinociceptive activity after systemic administration. Thiorphan also inhibits to a lesser extent (Ki = 140 nM) the widely distributed angiotensin-converting enzyme, a carboxydipeptidase implicated in blood pressure regulation. In view of an eventual clinical use of enkephalinase inhibitors, fully specific compounds were developed. Such derivatives were obtained taking into account that N-methylation of the ultimate amide bond of dipeptides strongly decreases enkephalinase affinity without affecting angiotensin-converting enzyme recognition, whereas retro-inversion of the amide bond leads to the inverse effect. The retro-inverso dipeptide (R)-H2N-CH(CH2.vphi.)-NHCO-CH2-CO2H exhibits an inhibitory potency on enkephalinase (IC50 .simeq. 12 .mu.M) close to that of the natural dipeptide L-Phe-Gly (IC50 .simeq. 3 .mu.M). This result shows the topological analogy between the crucial components involved in enkephalinase recognition both in active dipeptides and structurally related retro-inverso isomers. Taking into account these observations, retro-thiorphan, (R,S)-HS-CH2-CH-(CH2.vphi.)-NHCO-CH2-COOH, was prepared. As compared to thiorphan, the retro isomer is 50% as potent (Ki = 6 nM) on enkephalinase but displays a drastic loss of potency on angiotensin-converting enzyme (IC50 > 10,000 nM). This specificity was interpreted as a consequence of differences in the stereochemical constraints involving enzyme-inhibitor H-bonding. This hypothesis is supported by reported crystallographic studies on related enzymes such as thermolysin and carboxypeptidase A. As expected, retro-thiorphan exhibits about the same analgesic potency as thiorphan on the hot plate and writhing tests in mice. The topological concept of retro-inverso isomers could be extended to other enkephalinase inhibitors, allowing the design of potent and highly selective compounds occurring as new classes of analgesic and psychoactive agents.

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