Trapping and Characterization of the Reaction Intermediate in Cyclodextrin Glycosyltransferase by Use of Activated Substrates and a Mutant Enzyme

Cyclodextrin glycosyltransferases (CGTases) catalyze the degradation of starch into linear or cyclic oligosaccharides via a glycosyl transfer reaction occurring with retention of anomeric configuration. They are also shown to catalyze the coupling of maltooligosaccharyl fluorides. Reaction is thought to proceed via a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. This intermediate can be trapped by use of 4-deoxymaltotriosyl α-fluoride (4DG3αF). This substrate contains a good leaving group, fluoride, thus facilitating formation of the intermediate, but cannot undergo the transglycosylation step since the nucleophilic hydroxyl group at the 4-position is missing. When 4DG3αF was reacted with wild-type CGTase (Bacillus circulans 251), it was found to be a slow substrate (kcat = 2 s-1) compared with the parent glycosyl fluoride, maltotriosyl R-fluoride (kcat = 275 s-1). Unfortunately, a competing hydrolysis reaction reduces the lifetime of the intermediate precluding its trapping and identification. However, when 4DG3αF was used in the presence of the presumed acid/base catalyst mutant Glu257Gln, the intermediate could be trapped and analyzed because the first step remained fast while the second step was further slowed (kcat = 0.6 s-1). Two glycosylated peptides were identified in a proteolytic digest of the inhibited enzyme by means of neutral loss tandem mass spectrometry. Edman sequencing of these labeled peptides allowed identification of Asp229 as the catalytic nucleophile and provided evidence for a covalent intermediate in CGTase. Asp229 is found to be conserved in all members of the family 13 glycosyl transferases.

Reassessment of Acarbose as a Transition State Analogue Inhibitor of Cyclodextrin Glycosyltransferase

The binding of several different active site mutants of Bacillus circulans cyclodextrin glycosyltransferase to the inhibitor acarbose has been investigated through measurement of Ki values. The mutations represent several key amino acid positions, most of which are believed to play important roles in governing the product specificity of cyclodextrin glycosyltransferase. Michaelis-Menten parameters for the substrates α-maltotriosyl fluoride (αG3F) and α-glucosyl fluoride (αGF) with each mutant have been determined by following the enzyme-catalyzed release of fluoride with an ion-selective fluoride electrode. In both cases, reasonable correlations are observed in logarithmic plots relating the Ki value for acarbose with each mutant and both kcat/Km and Km for the hydrolysis of either substrate by the corresponding mutants. This indicates that acarbose, as an inhibitor, is mimicking aspects of both the ground state and the transition state. A better correlation is observed for αGF (r = 0.98) than αG3F (r = 0.90), which can be explained in terms of the modes of binding of these substrates and acarbose. Re-refinement of the previously determined crystal structure of wild-type CGTase complexed with acarbose reveals a binding mode consistent with the transition state analogue character of this inhibitor.

Structures of Lactate Dehydrogenase A (LDHA) In Apo, Ternary an Inhibitor-Bound Forms

Lactate dehydrogenase (LDH) is an essential metabolic enzyme that catalyzes the interconversion of pyruvate and lactate using NADH/NAD + as a co-substrate. Many cancer cells exhibit a glycolytic phenotype known as the Warburg effect, in which elevated LDH levels enhance the conversion of glucose to lactate, making LDH an attractive therapeutic target for oncology. Two known inhibitors of the human muscle LDH isoform, LDHA, designated 1 and 2 , were selected, and their IC 50 values were determined to be 14.4 ± 3.77 and 2.20 ± 0.15 µ M , respectively. The X-ray crystal structures of LDHA in complex with each inhibitor were determined; both inhibitors bind to a site overlapping with the NADH-binding site. Further, an apo LDHA crystal structure solved in a new space group is reported, as well as a complex with both NADH and the substrate analogue oxalate bound in seven of the eight molecules and an oxalate only bound in the eighth molecule in the asymmetric unit. In this latter structure, a kanamycin molecule is located in the inhibitor-binding site, thereby blocking NADH binding. These structures provide insights into LDHA enzyme mechanism and inhibition and a framework for structure-assisted drug design that may contribute to new cancer therapie

Synthesis of 4-methylumbelliferyl α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside and development of a coupled fluorescent assay for GH125 exo-α-1,6-mannosidases

Certain bacterial pathogens possess a repertoire of carbohydrate processing enzymes that process host N-linked glycans and many of these enzymes are required for full virulence of harmful human pathogens such as Clostridium perfringens and Streptococcus pneumoniae. One bacterial carbohydrate processing enzyme that has been studied is the pneumococcal virulence factor SpGH125 from S. pneumoniae and its homologue, CpGH125, from C. perfringens. These exo-α-1,6-mannosidases from glycoside hydrolase family 125 show poor activity toward aryl α-mannopyranosides. To circumvent this problem, we describe a convenient synthesis of the fluorogenic disaccharide substrate 4-methylumbelliferone α-d-mannopyranosyl-(1→6)-β-d-mannopyranoside. We show this substrate can be used in a coupled fluorescent assay by using β-mannosidases from either Cellulomonas fimi or Helix pomatia as the coupling enzyme. We find that this disaccharide substrate is processed much more efficiently than aryl α-mannopyranosides by CpGH125, most likely because inclusion of the second mannose residue makes this substrate more like the natural host glycan substrates of this enzyme, which enables it to bind better. Using this sensitive coupled assay, the detailed characterization of these metal-independent exo-α-mannosidases GH125 enzymes should be possible, as should screening chemical libraries for inhibitors of these virulence factors

The molecular pharmacology of AMD11070: An orally bioavailable CXCR4 HIV entry inhibitor

In order to enter and infect human cells HIV must bind to CD4 in addition to either the CXCR4 or the CCR5 chemokine receptor. AMD11070 was the first orally available small molecule antagonist of CXCR4 to enter the clinic. Herein we report the molecular pharmacology of AMD11070 which is a potent inhibitor of X4 HIV-1 replication and the gp120/CXCR4 interaction. Using the CCRF-CEM T cell line that endogenously expresses CXCR4 we have demonstrated that AMD11070 is an antagonist of SDF-1α ligand binding (IC(50)=12.5±1.3nM), inhibits SDF-1 mediated calcium flux (IC(50)=9.0±2.0nM) and SDF-1α mediated activation of the CXCR4 receptor as measured by a Eu-GTP binding assay (IC(50)=39.8±2.5nM) or a [(35)S]-GTPγS binding assay (IC(50)=19.0±4.1nM), and inhibits SDF-1α stimulated chemotaxis (IC(50)=19.0±4.0nM). AMD11070 does not inhibit calcium flux of cells expressing CXCR3, CCR1, CCR2b, CCR4, CCR5 or CCR7, or ligand binding to CXCR7 and BLT(1), demonstrating selectivity for CXCR4. In addition AMD11070 is able to inhibit the SDF-1β isoform interactions with CXCR4; and N-terminal truncated variants of CXCR4 with equal potency to wild type receptor. Further mechanistic studies indicate that AMD11070 is an allosteric inhibitor of CXCR4.status: publishe

Mitigating hERG Inhibition: Design of Orally Bioavailable CCR5 Antagonists as Potent Inhibitors of R5 HIV-1 Replication

A series of CCR5 antagonists representing the thiophene-3-yl-methyl ureas were designed that met the pharmacological criteria for HIV-1 inhibition and mitigated a human ether-a-go-go related gene (hERG) inhibition liability. Reducing lipophilicity was the main design criteria used to identify compounds that did not inhibit the hERG channel, but subtle structural modifications were also important. Interestingly, within this series, compounds with low hERG inhibition prolonged the action potential duration (APD) in dog Purkinje fibers, suggesting a mixed effect on cardiac ion channels

Design of Substituted Imidazolidinylpiperidinylbenzoic Acids as Chemokine Receptor 5 Antagonists: Potent Inhibitors of R5 HIV‑1 Replication

The redesign of the previously reported thiophene-3-yl-methyl urea series, as a result of potential cardiotoxicity, was successfully accomplished, resulting in the identification of a novel potent series of CCR5 antagonists containing the imidazolidinylpiperidinyl scaffold. The main redesign criteria were to reduce the number of rotatable bonds and to maintain an acceptable lipophilicity to mitigate hERG inhibition. The structure–activity relationship (SAR) that was developed was used to identify compounds with the best pharmacological profile to inhibit HIV-1. As a result, five advanced compounds, <b>6d</b>, <b>6e</b>, <b>6i</b>, <b>6h</b>, and <b>6k</b>, were further evaluated for receptor selectivity, antiviral activity against CCR5 using (R5) HIV-1 clinical isolates, and in vitro and in vivo safety. On the basis of these results, <b>6d</b> and <b>6h</b> were selected for further development