1143 JPP 2004, 56: 1143–1153 ß 2004 The Authors Received March 30, 2004 Accepted June 15, 2004 DOI 10.1211/0022357044139 ISSN 0022-3573 Cyclic Peptide Research Unit, Department of Pharmacy, University of Port Elizabeth, Box 1600, Port Elizabeth 6000, South Africa K. McCleland, P. J. Milne, F. R. Lucieto Department of Biochemistry and Microbiology, University of Port Elizabeth, Box 1600, Port Elizabeth 6000, South Africa C. Frost, S. C. Brauns, M. Van De Venter School of Physiology, Nutrition and Consumer Sciences, North-West University, Potchefstroom Campus, Private Bag X6001, Potchefstroom, 2520, South Africa J. Du Plessis, K. Dyason Correspondence: P. J. Milne, Department of Pharmacy, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth, 6001, South Africa. E-mail: pmapjm@upe.ac.za Acknowledgement and funding: Thank you to Prof. P. J. Milne, Dr C. Frost, Mr C. Brauns, Mr F. R. Lucietto, Mr G. Kilian, Mrs A. Erasmus and Dr K. Dyason for all their contributions and assistance. The financial assistance of the National Research Foundation (NRF) towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the authors and are not necessarily attributable to the NRF. An investigation into the biological activity of the selected histidine-containing diketopiperazines cyclo(His-Phe) and cyclo(His-Tyr) K. McCleland, P. J. Milne, F. R. Lucieto, C. Frost, S. C. Brauns, M. Van De Venter, J. Du Plessis and K. Dyason Abstract Although cyclic diketopiperazines have been known since the beginning of the century, only now have they attracted considerable interest with respect to their biological activity. The aim of this study was to determine if the diketopiperazines cyclo(L-histidyl-L-phenylalanyl) (cyclo(His-Phe)) and cyclo(L-histidyl-L-tyrosyl) (cyclo(His-Tyr)) have significant biological activity relevant to the treatment of cardiovascular-related disease states, cancer and infectious diseases. Haematological studies were performed, including thrombin substrate binding, blood clotting time, platelet adhesion, platelet aggregation and fibrinolysis assays. A cytotoxicity screening utilizing a tetrazolium-based assay on the cell lines HeLa, WHCO3, and MCF-7 was performed. The whole-cell patch-clamp technique was used to investigate ion-channel activity in ventricular myocytes of rats, and isolated rat heart studies were performed to investigate the cardiac effects involving heart rate and coronary flow rate. Cyclo(His-Tyr) produced a significant prolongation of blood clotting time, slowing of clot lysis and inhibition of ADP-induced platelet adhesion and aggregation (P < 0.05). Cyclo(His-Phe) showed significant (P < 0.05) anti-tumour activity, causing greatest reduction of cell viability in cervical carcinoma cells. Preliminary results from patch-clamp studies indicate that both diketopiperazines caused blocking of sodium and calcium ion channels, but opening of inward rectifying potassium ion channels. In the rat isolated heart studies, cyclo(His-Phe) caused a gradual reduction in heart rate (P ¼ 0.0027) and a decrease in coronary flow rate (P ¼ 0.0017). Cyclo(His-Tyr) significantly increased the heart rate (P ¼ 0.0016) but did not cause any significant change of coronary flow rate (P > 0.05). Cyclo(His-Tyr) showed notable (P < 0.05) antibacterial activity and both diketopiperazines showed excellent antifungal activity (P < 0.05). These observations reveal diketopiperazines to be ideal lead compounds for the rational design of an agent capable of preventing metastasis, inhibiting tumour growth, and as potential chemotherapeutic, antiarrhythmic and antihypertensive agents, as well as potential antibacterial and antifungal agents. Introduction In recent years there has been a growing awareness of the diversity and biological roles played by many of over one-hundred diketopiperazines found in nature. Many deriva- tives have antiviral (e.g., the gliotoxins and sporidesmins), phytotoxic (e.g., cyclo(Pro- Tyr)) and antibiotic (e.g., bicyclomycin) properties, whereas simple members like cyclo(His-Pro), cyclo(Pro-Leu), cyclo(Asp-Pro), cyclo(Pro-Val) and cyclo(Pro-Phe) show various biological activities (Prasad 1995). For this reason, many of these diketo- piperazines are now regarded as important metabolic intermediates rather than as protein artifacts. A host of other diketopiperazines, or cyclic dipeptides, are reportedly endogenous in different living organisms or exist as either by-products of manufacturing processes or simply degradation products of larger proteins and peptides. Although there is no doubt that many of these cyclic dipeptides may possess interesting and possibly economically beneficial biological activity, evaluations on a small scale have only begun in 1995 (Prasad 1995). It has been shown that these agents can be used as templates from which peptidomi- metic analogues with enhanced activity can be designed (Henczi & Weaver 1995). The