Michael Szelke

A transition-state analogue inhibitor of human renin (H.261): test in vitro and a comparison with captopril in the anaesthetized baboon.

H.261, a new transition state inhibitor of human renin with an IC50 of 6.9 X 10(-10) M, was given by intravenous infusion to six anaesthetized baboons. The inhibitor was infused first at 0.1 mumol/kg/h for 15 min, then at 1.0 mumol/kg/h for a further 15 min. After a recovery period of 2 h in which the animals received 5% dextrose, they were infused with captopril, 25 mumol/kg/h for 15 min. At both rates of infusion H.261 markedly and significantly reduced the enzymatic action of renin in plasma, the blood concentration of angiotensin I, the plasma concentration of angiotensin II and mean arterial pressure. All changes reverted towards or to control values in the subsequent control period. Captopril also lowered plasma angiotensin II concentration and mean arterial pressure markedly and significantly but, as expected for an inhibitor of the angiotensin I-converting enzyme, plasma active renin concentration and blood angiotensin I concentration increased. The changes of angiotensin II and arterial pressure were similar with captopril and H.261.

Synthetic inhibitors of human tissue kallikrein

We have developed a series of novel, potent low molecular weight (4-600 Da) inhibitors of TK which were stable to cleavage by the enzyme and showed a high degree of selectivity for TK over several other serine proteases. Compound 7 (CH-2856) was found to reduce eosinophilia in a model of allergic inflammation. The effects observed in vivo provide further evidence for the involvement of TK and kinins in the pathophysiology of allergic diseases such as asthma.

MECHANISM OF IRRITANT-INDUCED COUGH : STUDIES WITH A KININ ANTAGONIST AND A KALLIKREIN INHIBITOR

It has been suggested that bradykinin may play a role in stimulating cough in at least one pathological condition in humans. We have employed an animal model to investigate the possible role of this peptide in irritant-induced cough. The kinin antagonist Hoe 140 and codeine both produced dose-related inhibition of cough responses to inhalation of citric acid or bradykinin aerosols by conscious guinea pigs. The selective tissue kallikrein inhibitor CH694 inhibited cough caused by citric acid but not by bradykinin. Indomethacin pretreatment attenuated the responses to both stimuli as did phosphoramidon. It is concluded that cough produced by citric acid inhalation may be mediated, at least in part, by generation of kinins; secondary to this, a release of prostanoids also appears to participate in the response.

Design and synthesis of thrombin inhibitors

As part of our programme directed at the development of enzyme inhibitors based on transition-state mimics, we discovered in the early 1980s that P3-P3′ fragments of human fibrinogen Aα, containing the ketomethylene isostere Arg-Ψ-[COCH2]Gly at P1-P1′, were potent inhibitors of thrombin. Such low-molecular-weight inhibitors are expected to be clinically useful as anticoagultant drugs. In our more recent investigations, the P1-P1′ moiety has been replaced with various arginine or lysine ketones. The resulting compounds showed the following order of thrombin inhibitory potency: α-ketoesters > fluoroketones >alkoxymethylketones > difluoro-β-ketoamides >β-ketoesters >alkyl ketones. In contrast to all other lysine/arginine pairs studied previously, the inhibitor based on a lysine α-ketoester proved superior to the corresponding arginine analogue. A possible explanation for this finding is discussed. All the highly electrophilic ketones (e.g., fluoroketones) were found to exhibit slow-binding kinetics with thrombin, which is likely to be a disadvantage in clinical use. Alkoxymethyl ketones were devoid of such behaviour and have been developed further to yield nanomolar inhibitors of low molecular weight and good selectivity for thrombin. One of these ketones was found to compare favourably with known thrombin inhibitors in anticoagulant assays. The synthesis of various types of inhibitor mentioned above is described, together with structure-activity correlations for inhibition of thrombin.

Novel cholecystokinin receptor ligands: Oxopiperazines derived from Boc-CCK-4

Abstract Replacement of the C-terminal dipeptide amide of Boc-CCK-4 (1) with oxopiperazines yields a series of weak CCK-receptor ligands. Further modifications result in more potent and receptor subtype selective compounds.

Selective inhibitors of plasma kallikrein

Based on a tetrapeptide fragment [Pro387-Ser390] of HK we have developed a series of potent low molecular weight (5-600 Da) inhibitors of PK which are stable to the enzyme. These inhibitors show good selectivity for PK versus tissue kallikrein, thrombin and plasmin. Such inhibitors will help define the role of PK and kinins in human physiology and pathophysiology. They may also find clinical use in the treatment of diseases where kinins are important mediators.

Thrombin Inhibitors Based on Ketone Derivatives of Arginine and Lysine

Much attention is currently focused on inhibitors of thrombin as potential anticoagulants. We have previously reported thrombin inhibitors based on fragments of fibrinogen containing a ketomethylene isostere at P1-P1. We now expand on these early findings by reporting on tripeptide based inhibitors of thrombin containing arginine or lysine ketones at the C-terminus. A large variety of such ketones have been studied and compared in their ability to increase the thrombin time in human plasma. In the case of arginine or lysine ketones the order of activity (i.e. decreasing IC50 TT) was: alkyl ketones < beta-ketoesters < difluoro beta-ketoamides < alkyloxymethyl ketones < fluoroketones. Lysine analogues were generally found to be ca. ten-fold less active than their arginine counterparts. However, in the case of alpha-ketoesters the lysine derivatives were superior to all the types of arginine ketones studied (including the arginine alpha-keto ester derived thrombin inhibitor). A mechanistic explanation of the relative inactivity of the arginine alpha-keto ester derivative is proposed. All the highly electrophilic ketones were found to be slow-binding with thrombin.