Arginyl-histidine

The in vitro and in vivo activities of a mixture of six oleane triterpene saponins, recovered from the methanolic extract of the leaves of the Vietnamese plant Maesa balansae (PX-6518), were evaluated against drug-sensitive visceral Leishmania strains. The in vitro 50% inhibitory concentration (IC(50)) against intracellular Leishmania infantum amastigotes was 0.04 micro g/ml. The cytotoxic concentrations causing 50% cell death (CC(50)s) were about 1 micro g/ml in murine macrophage host cells and >32 micro g/ml in human fibroblasts (MRC-5 cell line). Evaluation in the Leishmania donovani BALB/c mouse model indicated that a single subcutaneous administration of 0.4 mg/kg at 1 day after infection reduced liver amastigote burdens by about 95% in all treated animals. If treatment was delayed until 14 days after infection, a dose of 1.6 mg/kg of body weight was required to maintain the same level of activity. Single 250-mg/kg doses of sodium stibogluconate (Pentostam) 1 and 14 days after infection produced comparable efficacies. A single dose of PX-6518 at 2.5 mg/kg administered 5 days before infection was still 100% effective in preventing liver infection, suggesting a particularly long residual action. Spleen and bone marrow could not be cleared by PX-6518 nor sodium stibogluconate. PX-6518 did not show activity after oral dosing at up to 200 mg/kg for 5 days. This study concludes that triterpenoid saponins from M. balansae show promising in vitro and in vivo antileishmanial potential and can be considered as new lead structures in the search for novel antileishmanial drugs.

The antimicrobial peptide poly(arginyl-histidine) is secreted by the ergot fungus Verticillium kibiense. We previously showed that poly(arginyl-histidine) from the fungus inhibits the growth of certain microorganisms more effectively than that chemically synthesized from the L-form of arginine and histidine, implying some substantial differences between the fungal and synthetic peptides. To elucidate what causes such differences, we here investigated the structural features of the fungal peptides. The acid hydrolysates of the fungal peptide contained d-histidine. When synthetic poly(L-arginyl-D-histidine) mimicking the fungal peptide was added to the culture of Salmonella typhimurium together with poly(L-arginyl-L-histidine), poly(L-arginyl-D-histidine) was not easily degraded during the incubation compared with poly(L-arginyl-L-histidine). We concluded that the d-form of histidine residues in the fungal peptide prolongs the life of the peptide leading to the enhancement of antimicrobial activity.

We have recently developed a convenient method of screening a broad range of microorganisms that produce epsilon-poly-L-lysine (M. Nishikawa and K. Ogawa, Appl. Environ. Microbiol. 68:3575-3581, 2002). Using this method, we found an ergot fungus that secretes a charged polypeptide other than epsilon-poly-L-lysine. It was identified as a new species on the basis of its 28S rRNA sequence and was named Verticillium kibiense (formerly Epichloe kibiensis). Peptide sequencing and mass spectrometry revealed that the polypeptide is a linear peptide composed of repeated units of arginyl-histidine. The numbers of repeated units were in most cases five and in some cases four or six. This peptide showed activity against a broad range of bacteria and fungi but lost its activity under conditions of high ionic strength. Zinc and copper ions specifically changed the circular dichroism spectra of the peptide and restored the antimicrobial activity from abrogation under high ionic conditions, although these ions had no reinforcing effect on antimicrobial activity when they were added to solutions at a low ionic strength. The peptide labeled with fluorescein was able to permeate the cell membranes of target microbes, but its ability to permeate cell membranes decreased under conditions of high ionic strength. This decreased ability was partially recovered specifically by the addition of zinc and copper ions. These results indicate that poly(arginyl-histidine) is a cationic polypeptide characterized by specific metal binding and resistance to salts.