Trestatin C

Selectins play a major role in the inflammatory reaction by initiating neutrophil attachment to activated vascular endothelium. Some heparin preparations can interact with L- and P-selectin; however, the determinants required for inhibiting selectin-mediated cell adhesion have not yet been characterized. We now report that carboxyl-reduced and sulfated heparin (prepared by chemical modifications of porcine intestinal mucosal heparin leading to the replacement of carboxylates by O-sulfate groups) and trestatin A sulfate (obtained by sulfation of trestatin A, a non-uronic pseudo-nonasaccharide extracted from Streptomyces dimorphogenes) exhibit strong anti-P-selectin and anti-L-selectin activity while lacking antithrombin-mediated anticoagulant activity. In vitro experiments revealed that both compounds inhibited P-selectin- and L-selectin-mediated cell adhesion under laminar flow conditions. Moreover, carboxyl-reduced and sulfated heparin and trestatin A sulfate were also active in vivo, as assessed by experiments showing 1) that microinfusion of trestatin A sulfate reduced by 96% leukocyte rolling along rat mesenteric postcapillary venules and 2) that both compounds inhibited (by 58-81%) neutrophil migration into thioglycollate-inflamed peritoneum of BALB/c mice. These results indicate that nonanticoagulant sulfated saccharides targeted at P-selectin and L-selectin may have therapeutic potential in inflammatory disorders.

Crystal structures of human pancreatic alpha-amylase (HPA) in complex with naturally occurring inhibitors have been solved. The tetrasaccharide acarbose and a pseudo-pentasaccharide of the trestatin family produced identical continuous electron densities corresponding to a pentasaccharide species, spanning the -3 to +2 subsites of the enzyme, presumably resulting from transglycosylation. Binding of the acarviosine core linked to a glucose residue at subsites -1 to +2 appears to be a critical part of the interaction process between alpha-amylases and trestatin-derived inhibitors. Two crystal forms, obtained at different values of pH, for the complex of HPA with the protein inhibitor from Phaseolus vulgaris (alpha-amylase inhibitor) have been solved. The flexible loop typical of the mammalian alpha-amylases was shown to exist in two different conformations, suggesting that loop closure is pH-sensitive. Structural information is provided for the important inhibitor residue, Arg-74, which has not been observed previously in structural analyses.

Acarbose is a well-known microbial specialized metabolite used clinically to treat type 2 diabetes. This natural pseudo-oligosaccharide (PsOS) shows potent inhibitory activity toward various glycosyl hydrolases, including α-glucosidases and α-amylases. While acarbose and other PsOSs are produced by many different bacteria, their ecological or biological role in microbial communities is still an open question. Here, we show that several PsOS-producing actinobacteria, i.e., Actinoplanes sp. SE50/110 (acarbose producer), Streptomyces glaucescens GLA.O (acarbose producer), and Streptomyces dimorphogenes ATCC 31484 (trestatin producer), can grow in the presence of acarbose, while the growth of the non-PsOS-producing organism Streptomyces coelicolor M1152 was suppressed when starch is the main source of energy. Further investigations using recombinant α-amylases from S. coelicolor M1152 and the PsOS-producing actinobacteria revealed that the S. coelicolor α-amylase was inhibited by acarbose, whereas those from the PsOS-producing bacteria were not inhibited by acarbose. Bioinformatic and protein modeling studies suggested that a point mutation in the α-amylases of the PsOS-producing actinobacteria is responsible for the resistance of those enzymes toward acarbose. Converting the acarbose-resistant α-amylase AcbE to its A304H variant diminished its acarbose-resistance property. Taken together, the results suggest that acarbose is used by the producing bacteria as a competitive exclusion agent to suppress the growth of other microorganisms in their natural environment, while the producing organisms equip themselves with α-amylase variants that are resistant to acarbose.