The throughput of a biomimetic lipid-mediated assay used to investigate the effects of inhibitors on the kinetics of β-hematin formation has been optimized through the use of 24-well microplates. The rate constant for β-hematin formation mediated by monopalmitoyl-rac-glycerol was reduced from 0.17 ± 0.04 min−1 previously measured in Falcon tubes to 0.019 ± 0.002 min−1 in the optimized assay. While this necessitated longer incubation times, transferring aliquots from multiple 24-well plates to a single 96-well plate for final absorbance measurements actually improved the overall turnaround time per inhibitor. This assay has been applied to investigate the effects of four clinically-relevant antimalarial drugs (chloroquine, amodiaquine, quinidine and quinine) as well as several short-chain 4-aminoquinoline derivatives and non-quinoline (benzamide) compounds on the kinetics of β-hematin formation. The adsorption strength of these inhibitors to crystalline β-hematin (Kads) was quantified using a theoretical kinetic model that is based on the Avrami equation and the Langmuir isotherm. Statistically-significant linear correlations between lipid-mediated β-hematin inhibitory activity and Kads values for quinoline (r2 = 0.76, P-value = 0.0046) and non-quinoline compounds (r2 = 0.99, P-stat = 0.0006), as well as between parasite inhibitory activity (D10) and Kads values for quinoline antimalarial drugs and short-chain chloroquine derivatives (r2 = 0.64, P-value = 0.0098) provides a strong indication that drug action involves adsorption to the surface of β-hematin crystals. Independent support in this regard is provided by experiments that spectrophotometrically monitor the direct adsorption of antimalarial drugs to pre-formed β-hematin.

We report here on the preparation of an ovel a-thioglycoligase that can be used for the fast and efficient synthesis of a-N-acetylglucosaminebased glycosides. Using the a-N-acetyl-glucosaminidase from Clostridium perfringens of family GH89 (according to the Carbohydrate Active Enzymes classification) as starting point,w ep reparedm utants in the acid/base residue glutamic acid 483 that were found to have different synthetic efficiencies (maximal yields > 80% after 24 h) in the presenceo fa n activated donor ands uitable acceptors.T he synthetic potential of the Glu483 alanine mutant as an a-thioglycoligase -o nly the third biocatalyst with this stereospecificity reported to date,a nd the first selective for the N-acetylglucosamine moiety-w as demonstrated by producing for the first time N-acetyl-a-dglucosaminyl azide and N-acetylglucosamine a-thioglycosides in high yields.T os howcase the application of such compounds,w es how that they offer the wild-type CpGH89 protection from thermalu nfolding, demonstrating their potentiala sp harmacological chaperones for the treatment of mucopolysaccharidosis IIIB (Sanfilippo syndrome).

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Background Pelargonium sidoides is an important traditional medicine in South Africa with a well-defined history of both traditional and documented use of an aqueous-ethanolic formulation of the roots of P. sidoides (EPs 7630), which is successfully employed for the treatment of respiratory tract infections. There is also historical evidence of use in the treatment of tuberculosis. The aim of this study was to develop a platform of Mycobacterium tuberculosis (Mtb) kinase enzymes that may be used for the identification of therapeutically relevant ethnobotanical extracts that will allow drug target identification, as well as the subsequent isolation of the active compounds. Results Mtb kinases, Nucleoside diphosphokinase, Homoserine kinase, Acetate kinase, Glycerol kinase, Thiamine monophosphate kinase, Ribokinase, Aspartokinase and Shikimate kinase were cloned, produced in Escherichia coli and characterized. HPLC-based assays were used to determine the enzyme activities and subsequently the inhibitory potentials of varying concentrations of a P. sidoides extract against the produced enzymes. The enzyme activity assays indicated that these enzymes were active at low ATP concentrations. The 50% inhibitory concentration (IC50) of an aqueous root extract of P. sidoides against the kinases indicated SK has an IC50 of 1.2 μg/ml and GK 1.4 μg/ml. These enzyme targets were further assessed for compound identification from the P. sidoides literature. Conclusion This study suggests P. sidoides is potentially a source of anti-tubercular compounds and the Mtb kinase platform has significant potential as a tool for the subsequent screening of P. sidoides extracts and plant extracts in general, for compound identification and elaboration by selected extract target inhibitor profiling.