A Strategy for Neuraminidase Inhibitors Using Mechanism‐Based Labeling Information

A potent inhibitor for Vibrio cholerae neuraminidase (VCNA) was developed by using a novel two‐step strategy, a target amino acid validation using mechanism‐based labeling information, and a potent inhibitor search using a focused library. The labeling information suggested the hidden dynamics of a loop structure of VCNA, which can be a potential target of the novel inhibitor. A focused library composed of 187 compounds was prepared from a 9‐azide derivative of 2,3‐dehydro‐N‐acetylneuraminic acid (DANA) to interrupt the function of the loop of the labeled residues. Inhibitor 3c showed potent inhibition properties and was the strongest inhibitor with FANA, a N‐trifluoroacetyl derivative of DANA. Validation studies of the inhibitor with a detergent and a Lineweaver–Burk plot suggested that the 9‐substitution group would interact hydrophobically with the target loop moiety, adding a noncompetitive inhibition property to the DANA skeleton. This information enabled us to design compound 4 having the combined structure of 3c and FANA. Compound 4 showed the most potent inhibition (K_i=73 nM , mixed inhibition) of VCNA with high selectivity among the tested viral, bacterial, and mammal neuraminidases.     

Recent Advances in Biocatalytic Promiscuity: Hydrolase‐Catalyzed Reactions for Nonconventional Transformations

Enzymes have emerged in recent decades as ideal catalysts for synthetic transformations under mild reaction conditions. Their capacity to accelerate a myriad of biotransformations with high levels of selectivity and broad substrate specificity including excellent atom economy has led to a current full recognition. The six classes of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases) possess outstanding abilities to perform specific modifications in target molecules. Nevertheless, in the last fifteen years, novel examples have appeared related to nonconventional processes catalyzed by various classes of biocatalysts. Amongst these, hydrolases have received special attention since they display remarkable activities in initially unexpected reactions such as carbon–carbon and carbon–heteroatom bond‐formation reactions, oxidative processes and novel hydrolytic transformations. In this review, the main findings in this area will be disclosed, highlighting the catalytic properties of hydrolases not only to catalyze single processes but also multicomponent and tandem nonconventional reactions.     

19F NMR and DFT Analysis Reveal Structural and Electronic Transition State Features for RhoA‐Catalyzed GTP Hydrolysis

Molecular details for RhoA/GAP catalysis of the hydrolysis of GTP to GDP are poorly understood. We use ¹⁹F NMR chemical shifts in the MgF₃^? transition state analogue (TSA) complex as a spectroscopic reporter to indicate electron distribution for the γ‐PO₃^? oxygens in the corresponding TS, implying that oxygen coordinated to Mg has the greatest electron density. This was validated by QM calculations giving a picture of the electronic properties of the transition state (TS) for nucleophilic attack of water on the γ‐PO₃^? group based on the structure of a RhoA/GAP‐GDP‐MgF₃^? TSA complex. The TS model displays a network of 20 hydrogen bonds, including the GAP Arg85′ side chain, but neither phosphate torsional strain nor general base catalysis is evident. The nucleophilic water occupies a reactive location different from that in multiple ground state complexes, arising from reorientation of the Gln‐63 carboxamide by Arg85′ to preclude direct hydrogen bonding from water to the target γ‐PO₃^? group.     

2‐Phenylquinoline–Sugar Hybrids as Photoswitchable α‐Glucosidase Inhibitors

Purpose‐designed 2‐phenylquinoline (PQ)‐sugar hybrids 1 and 2 were synthesized and evaluated for their photodegradation activities against an α‐glucosidase target. The results indicated that PQ‐mannose hybrid 2 selectively and effectively photodegraded α‐glucosidase and significantly inhibited its enzymatic activity upon irradiation with long‐wavelength UV light in the absence of any additives under neutral and aqueous conditions. Furthermore, 2 selectively and effectively inhibited α‐glucosidase activity only with photo‐irradiation even in complex cell lysate.     

Accelerating Enzymatic Catalysis Using Vortex Fluidics

Enzymes catalyze chemical transformations with outstanding stereo‐ and regio‐specificities, but many enzymes are limited by their long reaction times. A general method to accelerate enzymes using pressure waves contained within thin films is described. Each enzyme responds best to specific frequencies of pressure waves, and an acceleration landscape for each protein is reported. A vortex fluidic device introduces pressure waves that drive increased rate constants (k_cat) and enzymatic efficiency (k_cat/K_m). Four enzymes displayed an average seven‐fold acceleration, with deoxyribose‐5‐phosphate aldolase (DERA) achieving an average 15‐fold enhancement using this approach. In solving a common problem in enzyme catalysis, a powerful, generalizable tool for enzyme acceleration has been uncovered. This research provides new insights into previously uncontrolled factors affecting enzyme function.     

Mutations in distant residues moderately increase the enantioselectivity of Pseudomonas fluorescens esterase towards methyl 3bromo-2-methylpropanoate and ethyl 3phenylbutyrate

Directed evolution combined with saturation mutagenesis identified six different point mutations that each moderately increases the enantioselectivity of an esterase from Pseudomonas fluorescens (PFE) towards either of two chiral synthons. Directed evolution identified a Thr230Ile mutation that increased the enantioselectivity from 12 to 19 towards methyl (S)-3-bromo-2-methylpropanoate. Saturation mutagenesis at Thr230 identified another mutant, Thr230Pro, with higher-than-wild-type enantioselectivity (E=17). Previous directed evolution identified mutants Asp158Asn and Leu181Gln that increased the enantioselectivity from 3.5 to 5.8 and 6.6, respectively, towards ethyl (R)-3-phenylbutyrate. In this work, saturation mutagenesis identified other mutations that further increase the enantioselectivity to 12 (Asp158Leu) and 10 (Leu181Ser). A homology model of PFE indicates that all mutations lie outside the active site, 12-14 A from the substrate and suggests how the distant mutations might indirectly change the substrate-binding site. Since proteins contain many more residues far from the active site than close to the active site, random mutagenesis is strongly biased in favor of distant mutations. Directed evolution rarely screens all mutations, so it usually finds the distant mutations because they are more common, but probably not the most effective.     

Rational Enzyme Design without Structural Knowledge: A Sequence-Based Approach for Efficient Generation of Transglycosylases

Glycobiology is dogged by the relative scarcity of synthetic, defined oligosaccharides. Enzyme-catalysed glycosylation using glycoside hydrolases is feasible but is hampered by the innate hydrolytic activity of these enzymes. Protein engineering is useful to remedy this, but it usually requires prior structural knowledge of the target enzyme, and/or relies on extensive, time-consuming screening and analysis. Here, a straightforward strategy that involves rational rapid in silico analysis of protein sequences is described. The method pinpoints 6–12 single-mutant candidates to improve transglycosylation yields. Requiring very little prior knowledge of the target enzyme other than its sequence, the method is generic and procures catalysts for the formation of glycosidic bonds involving various d /l -, α/β-pyranosides or furanosides, and exo or endo action. Moreover, mutations validated in one enzyme can be transposed to others, even distantly related enzymes.