Mechanisms of alpha-glycosidases

α-Retaining glycosidases are a class of hydrolytic enzymes that cleave α-glycosidic linkages with net retention of configuration at the anomeric center. The finer details of the mechanisms of two such enzymes, Golgi α-mannosidase II and human pancreatic α-amylase, were investigated in order to gain better understanding of this class of enzymes. α-Retaining mannosidases, like all other retaining glycosidases, employ a double displacement mechanism in which a transient glycosyl-enzyme intermediate is formed. In order to isolate or "trap" this key intermediate, two active site probes were synthesized for family 38 α-mannosidases. Both 5-fluoro-β-L-gulosyl fluoride and 2-deoxy-2-fluoro- α- D-mannosyl-fluoride were shown to be poor substrates for Golgi α-mannosidase II at room temperature. In both cases, by lowering the temperature, the lifetime of the intermediate increased so that the species was experimentally observable. Structural studies using X-ray crystallography showed that in both cases, the intermediate sugars adopted a ¹S₅ skew boat conformation, suggesting that the intermediate formed during the hydrolysis of the natural substrate also adopts this conformation. This suggests that intermediate distortion is an important part of the catalytic mechanism in α -glycosidases. α-Amylases are a family of α-retaining glycosidases involved in glucose polymer digestion. Several members of this family are activated in the presence of chloride ion. To investigate the mechanism of this phenomenon, a series of mutations were made to the residues constituting the chloride ion-binding site in human pancreatic a-amylase (HPA). Kinetic analysis of the resultant mutants showed that one of these residues, Arg337, in the absence of chloride, effectively inhibits the enzyme by modulating the pK[sub a] of the acid/base catalyst. The chloride ion activates HPA by relieving this inhibition through charge shielding. Upon mutation of this residue, the mutant HPA became a chloride-independent enzyme with full catalytic activity. Using a novel substrate, 2,4-dinitrophenyl α-maltotrioside, a new rapid screen was developed to identify potential HPA inhibitors. Using this screen, Dgluconohydroximino- l,5-lactam (GHIL) was determined to be a potential HPA inhibitor. Kinetic analysis of this compound showed that GHIL is a poor inhibitor of HPA with a K[sub i] value of 11 mM. However, in the presence of substrate, HPA will take this compound and modify it to create a more potent inhibitor in situ. This modification is most likely the result of a transglycosylation of the substrate to the inhibitor.Science, Faculty ofChemistry, Department ofGraduat

Feasibility and physiological relevance of designing highly potent aminopeptidase-sparing leukotriene A4 hydrolase inhibitors

Leukotriene A4 Hydrolase (LTA4H) is a bifunctional zinc metalloenzyme that comprises both epoxide hydrolase and aminopeptidase activity, exerted by two distinct but overlapping catalytic sites. The epoxide hydrolase function of the enzyme catalyzes the biosynthesis of the pro-inflammatory lipid mediator leukotriene B4 (LTB4). Recent literature suggests that the aminopeptidase function of LTA4H is responsible for degradation of the tripeptide Pro-Gly-Pro (PGP) for which neutrophil chemotactic activity has been postulated. It has been speculated that the design of epoxide hydrolase selective LTA4H inhibitors that spare the aminopeptidase pocket may therefore lead to more potent anti-inflammatory drugs. In this study, we conducted a high throughput screen (HTS) for LTA4H inhibitors and attempted to rationally design compounds that would spare the PGP degrading function. While we were able to identify compounds with preference for the epoxide hydrolase function, absolute selectivity was not achievable for highly potent compounds. In order to assess the relevance of designing aminopeptidase sparing LTA4H inhibitors, we studied the role of PGP in inducing inflammation in different settings in wild type and LTA4H deficient (LTA4H KO) animals but could not confirm its chemotactic potential. Attempts to design highly potent epoxide hydrolase selective LTA4H inhibitors, therefore seems to be neither feasible nor relevant

Evaluation of a liquid dispenser for assay development and enzymology in 1536 well format

While developments in liquid dispensers have made the use of 1536 well plates for high throughput screen (HTS) standard, there is still a gap in dispenser technology for performing matrix experiments with several components. Experiments such as those performed during assay development and enzymological studies are therefore still performed by manual pipetting in lower density plates. We have evaluated a new dispenser, the Certus liquid dispenser (Gyger Fluidics GmbH, Switzerland), that is capable of flexible dispensing in 1536 well format, with a dead volume of less than 200 µl. Taking advantage of the precision of the dispenser for volumes down to 50 nl, we have create concentration gradients on plates by dispensing different volumes of reagent and then backfilling with buffer. Using this method and the flexibility of the dispenser software, we have performed several multidimensional experiments varying 2 – 3 components, including an assay development for an HTS, a mode of inhibition study, and a co-factor optimization, where we determined 32 KM values. Overall, the flexibility of plate layout for multiple components, the accuracy to dispense volumes ranging 2 log orders, and minimal reagent usage, makes this dispenser enabling for complex biochemical experiments

Advancements in assay technologies and strategies to enable drug discovery

ABSTRACT: Assays drive drug discovery from the exploratory phases to the clinical testing of drug candidates. As such, numerous assay technologies and methodologies have arisen to support drug discovery efforts. Robust identification and characterization of tractable chemical matter requires biochemical, biophysical, and cellular approaches and often bene-fits from high-throughput methods. To increase throughput, efforts have been made to provide assays in miniaturized volumes which can be arrayed in microtiter plates to support the testing of as many as 100,000 samples/day. Alongside these efforts has been the growth of microtiter plate-free formats with encoded libraries that can support the screening billions of compounds, a hunt for new drug modalities, as well as the emphasis on more disease relevant formats using complex cell models of disease states. This review will focus on recent developments in high-throughput assay technolo-gies applied to identify starting points for drug discovery. We also provide recommendations on strategies for implement-ing various assay types to select high quality leads for drug development

N-linked glycosylation of folded proteins by the bacterial oligosaccharyltransferase

N-linked protein glycosylation is found in all domains of life. In eukaryotes, it is the most abundant protein modification of secretory and membrane proteins, and the process is coupled to protein translocation and folding. We found that in bacteria, N-glycosylation can occur independently of the protein translocation machinery. In an in vitro assay, bacterial oligosaccharyltransferase glycosylated a folded endogenous substrate protein with high efficiency and folded bovine ribonuclease A with low efficiency. Unfolding the eukaryotic substrate greatly increased glycosylation. We propose that in the bacterial system, glycosylation sites are located in flexible parts of folded proteins, whereas the eukaryotic cotranslational glycosylation evolved to a mechanism presenting the substrate in a flexible form before folding

Evaluation of 5H-Thiazolo[3,2-α]pyrimidin-5-ones as Potential GluN2A PET Tracers

We describe here our efforts to develop a PET tracer for imaging GluN2A-containing NMDA receptors, based on a 5H-thiazolo[3,2-α]pyrimidin-5-one scaffold. The metabolic stability and overall properties could be optimized satisfactorily, although binding affinities remained a limiting factor for in vivo imaging. We nevertheless identified 7-(((2-fluoroethyl)(3-fluorophenyl)amino)-methyl)-3-(2-(hydroxymethyl)cyclopropyl)-2-methyl-5H-thiazolo-[3,2-α]pyrimidin-5-one ([18 F]7b) as a radioligand providing good-quality images in autoradiographic studies, as well as a tritiated derivative, 2-(7-(((2-fluoroethyl)(4-fluorophenyl)amino)methyl)-2-methyl-5-oxo-5H-thiazolo[3,2-α]pyrimidin-3-yl)cyclopropane-1-carbonitrile ([3 H2 ]15b), which was used for the successful development of a radioligand binding assay. These are valuable new tools for the study of GluN2A-containing NMDA receptors, and for the optimization of allosteric modulators binding to the pharmacophore located at the dimer interface of the GluN1-GluN2A ligand-binding domain. Keywords: NMDA; defluorination; imaging agent; radiochemistry; receptor