1. 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, K Dyason J Pharm Pharmacol. 2004 Sep;56(9):1143-53. doi: 10.1211/0022357044139.
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 = 0.0027) and a decrease in coronary flow rate (P = 0.0017). Cyclo(His-Tyr) significantly increased the heart rate (P = 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.
2. Enzymatic synthesis of dehydro cyclo(His-Phe)s, analogs of the potent cell cycle inhibitor, dehydrophenylahistin, and their inhibitory activities toward cell division
Hiroshi Kanzaki, Satohiro Yanagisawa, Teruhiko Nitoda Biosci Biotechnol Biochem. 2004 Nov;68(11):2341-5. doi: 10.1271/bbb.68.2341.
Cyclo(His-Phe) was effectively converted to its dehydro derivatives by the enzyme of Streptomyces albulus KO-23, an albonoursin-producing actinomycete. Two types of dehydro derivatives were isolated from the reaction mixture and identified as cyclo(DeltaHis-DeltaPhe) and cyclo(His-DeltaPhe). This is the first report on cyclo(His-DeltaPhe) and the enzymatic preparation of both compounds. Cyclo(DeltaHis-DeltaPhe), a tetradehydro cyclic dipeptide, exhibited a minimum inhibitory concentration of 0.78 mumol/ml inhibitory activity toward the first cleavage of sea urchin embryos, in contrast to cyclo(His-DeltaPhe) that had no activity. The finding that the isoprenylated derivative of cyclo(DeltaHis-DeltaPhe), dehydrophyenylahistin, had 2,000 times higher activity than cyclo(DeltaHis-DeltaPhe) indicates that an isoprenyl group attached to an imidazole ring of the compound was essential for the inhibitory activity.
3. Active site remodelling of a cyclodipeptide synthase redefines substrate scope
Emmajay Sutherland, Christopher John Harding, Clarissa Melo Czekster Commun Chem. 2022 Aug 25;5:101. doi: 10.1038/s42004-022-00715-2.
Cyclodipeptide synthases (CDPSs) generate a wide range of cyclic dipeptides using aminoacylated tRNAs as substrates. Histidine-containing cyclic dipeptides have important biological activities as anticancer and neuroprotective molecules. Out of the 120 experimentally validated CDPS members, only two are known to accept histidine as a substrate yielding cyclo(His-Phe) and cyclo(His-Pro) as products. It is not fully understood how CDPSs select their substrates, and we must rely on bioprospecting to find new enzymes and novel bioactive cyclic dipeptides. Here, we developed an in vitro system to generate an extensive library of molecules using canonical and non-canonical amino acids as substrates, expanding the chemical space of histidine-containing cyclic dipeptide analogues. To investigate substrate selection we determined the structure of a cyclo(His-Pro)-producing CDPS. Three consecutive generations harbouring single, double and triple residue substitutions elucidated the histidine selection mechanism. Moreover, substrate selection was redefined, yielding enzyme variants that became capable of utilising phenylalanine and leucine. Our work successfully engineered a CDPS to yield different products, paving the way to direct the promiscuity of these enzymes to produce molecules of our choosing.
