Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice.

Pancreatic islet ß-cells secrete the hormones insulin and amylin, and defective ß-cell function plays a central role in the pathogenesis of type-2 diabetes (T2D). Human amylin (hA, also termed hIAPP) misfolds and forms amyloid aggregates whereas orthologous mouse amylin does neither. Furthermore, hA elicits apoptosis in cultured ß-cells and ß-cell death in ex-vivo islets. In addition, hA-transgenic mice that selectively express hA in their ß-cells, manifest ß-cell apoptosis and progressive islet damage that leads to diabetes closely resembling that in patients with T2D. Aggregation of hA is thus linked to the causation of diabetes. We employed time-dependent thioflavin-T spectroscopy and ion-mobility mass spectrometry to screen potential suppressors of hA misfolding for anti-diabetic activity. We identified the dietary flavonol rutin as an inhibitor of hA-misfolding and measured its anti-diabetic efficacy in hA-transgenic mice. In vitro, rutin bound hA, suppressed misfolding, disaggregated oligomers and reverted hA-conformation towards the physiological. In hA-transgenic mice, measurements of glucose, fluid-intake, and body-weight showed that rutin-treatment slowed diabetes-progression by lowering of rates of elevation in blood glucose (P = 0.030), retarding deterioration from symptomatic diabetes to death (P = 0.014) and stabilizing body-weight (P < 0.0001). In conclusion, rutin treatment suppressed hA-aggregation in vitro and doubled the lifespan of diabetic mice (P = 0.011) by a median of 69 days compared with vehicle-treated control-diabetic hA-transgenic mice.

Development of a technique to determine bicyclomycin-rho binding and stoichiometry by isothermal titration calorimetry and mass spectrometry.

Bicyclomycin (1) is the only natural product inhibitor of the transcription termination factor rho. Rho is a hexameric helicase that terminates nascent RNA transcripts utilizing ATP hydrolysis and is an essential protein for many bacteria. The paucity of information concerning the 1-rho interaction stems from the weak binding affinity of 1. We report a novel technique using imine formation with rho to enhance the affinity of a bicyclomycin analogue and determine the binding stoichiometry by isothermal titration calorimetry (ITC) and mass spectrometry (MS). Our designed bicyclomycin ligand, 5a-(3-formyl-phenylsulfanyl)-dihydrobicyclomycin (2) (apparent I(50) = 4 muM), inhibits rho an order of magnitude more efficiently than 1 (I(50) = 60 muM). MS shows that 2 selectively forms an imine with K181 in rho. We found that despite the heterogeneity of ATP binding (three tight and three weak) imposed on the rho hexamer, the nearby bicyclomycin binding pocket is not affected, and both 1 and 2 bind with equal affinity to all six subunits.