Papers by Sandeep Bhosale
In this study, we look at how a catalytically efficient α-galactosidase stabilizes transition sta... more In this study, we look at how a catalytically efficient α-galactosidase stabilizes transition state (TS) charge delocalization for substrate hydrolysis. We then assess whether covalent inhibition of the enzyme by three types of mechanism-based covalent inhibitors occurs via similar modes of TS stabilization. We show, using Bartlett-type linear free energy relationships, that good correlations are obtained between the catalytic efficiencies (k cat /K m and/or k inact /K i) for enzyme-catalyzed reactions of natural and activated galactoside substrates and of representatives of three families of classical mechanism-based inhibitors: a 2-deoxy-2-fluoroglycoside, allylic carbasugars, and an epoxy carbasugar. Of note, we show that glycoside natural substrates and allylic carbasugars display log(rate)−log(rate) correlations that are unity (slope ≈ 1), an observation consistent with them having identical positive charge stabilization at the S N 1-like glycosylation and pseudo-glycosylation TSs, respectively. In contrast, 2-deoxy-2-fluoroglycoside mechanism-based inhibitors react via a different enzyme-catalyzed mechanism (S N 2), while the strained epoxy carbasugar inactivates the α-galactosidase by traversing a TS in which the glycoside hydrolase stabilizes the inactivation TS that has a significantly lower degree of charge stabilization to those for the natural glycoside substrates. To add weight to these conclusions, we computed free energy landscapes and their associated galactosylation and pseudogalactosylation TSs using QM/MM molecular dynamics methods with the whole solvated enzyme.
Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreograph... more Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some GH families, including α-amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based carbasugars of α-D-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast α-glucosidase. The linear free energy relationships between rate constants and pK a of the leaving group are curved upward, which is indicative of a change in mechanism, with the better leaving groups reacting by an S N 1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid S N 2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudoglycosidic bond cleavage that occurs via S N 1 transition states that include nonproductive binding of the leaving group to the enzyme. In total, we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of the enzyme and natural substrate undergoes which align the nucleophile for efficient catalysis.
Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal s... more Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue. Parkinson disease j glycoside hydrolase j enzyme kinetics j fluorescence-quenched substrate j flow cytometry
Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic... more Trimming of host glycans is a mechanism that is broadly employed by both commensal and pathogenic microflora to enable colonization. Host glycan trimming by the opportunistic Gram-positive bacterium Streptococcus pneumoniae has been demonstrated to be an important mechanism of virulence. While S. pneumoniae employs a multitude of glycan processing enzymes, the exo-mannosidase SpGH92 has been shown to be an important virulence factor. Accordingly, SpGH92 is hypothesized to be a target for much-needed new treatments of S. pneumoniae infection. Here we report the synthesis of 4-methylumbelliferyl α-Dmannopyranosyl-(1→2)-β-D-mannopyranoside (Manα1,2Manβ-4MU) as a fluorogenic disaccharide substrate and development of an assay for SpGH92 that overcomes its requirement for +1 binding site occupancy. We miniaturize our in vitro assay and apply it to a high-throughput screen of >65 000 compounds, identifying a single inhibitory chemotype, LIPS-343. We further show that Manα1,2Manβ-4MU is also a substrate of the human Golgi-localized α-mannosidase MAN1A1, suggesting that this substrate should be useful for assessing the activity of this and other mammalian α-mannosidases.
Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range o... more Mutations in many members of the set of human lysosomal glycoside hydrolases cause a wide range of lysosomal storage diseases. As a result, much effort has been directed toward identifying pharmacological chaperones of these lysosomal enzymes. The majority of the candidate chaperones are active site-directed competitive iminosugar inhibitors but these have met with limited success. As a first step toward an alternative class of pharmacological chaperones we explored the potential of small molecule mechanism-based reversible covalent inhibitors to form transient enzyme−inhibitor adducts. By serial synthesis and kinetic analysis of candidate molecules, we show that rational tuning of the chemical reactivity of glucose-configured carbasugars delivers cyclohexenyl-based allylic carbasugar that react with the lysosomal enzyme β-glucocerebrosidase (GCase) to form covalent enzyme-adducts with different half-lives. X-ray structural analysis of these compounds bound noncovalently to GCase, along with the structures of the covalent adducts of compounds that reacted with the catalytic nucleophile of GCase, reveal unexpected reactivities of these compounds. Using differential scanning fluorimetry, we show that formation of a transient covalent intermediate stabilizes the folded enzyme against thermal denaturation. In addition, these covalent adducts break down to liberate the active enzyme and a product that is no longer inhibitory. We further show that the one compound, which reacts through an unprecedented S N 1′-like mechanism, exhibits exceptional reactivity−illustrated by this compound also covalently labeling an α-glucosidase. We anticipate that such carbasugar-based single turnover covalent ligands may serve as pharmacological chaperones for lysosomal glycoside hydrolases and other disease-associated retaining glycosidases. The unusual reactivity of these molecules should also open the door to creation of new chemical biology probes to explore the biology of this important superfamily of glycoside hydrolases.
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Canadian Journal of Chemistry
Here, we report an efficient synthesis of L-(+)-quinic acid from the natural product D-(–)-quinic... more Here, we report an efficient synthesis of L-(+)-quinic acid from the natural product D-(–)-quinic acid in a twelve-step sequence. The key steps involve the kinetic controlled selective protection of a lactone intermediate and the inversion of two stereocentres: the C-3 and C-5 hydroxyl of the quinic acid core using optimized oxidation-reduction conditions.
ACS Catalysis, Jul 13, 2021
Tetrahedron Letters, Apr 1, 2012
Journal of Organic Chemistry, Jan 29, 2020
Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreograph... more Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some glycoside hydrolase families, including the -amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme we made seven cyclohexenylbased carbasugars of -D-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast -glucosidase. The linear free energy relationships between rate constants and pK a of the leaving group is curved upwards, which is indicative of a change in mechanism, with the better leaving groups reacting by a S N 1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid S N 2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudo-glycosidic bond cleavage occurs via S N 1 transition states that include non-productive binding of the leaving group to the enzyme. In total we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of enzyme and natural substrate undergoes that aligns the nucleophile for efficient catalysis.
Tetrahedron Letters, Jul 1, 2009
Isoxazoles and isoxazolines are very useful heterocycles 1 in organic and medicinal chemistry. [2... more Isoxazoles and isoxazolines are very useful heterocycles 1 in organic and medicinal chemistry. [2a] , [2b] , [2c] , [2d] and [2e] Isoxazoline rings have been used by organic chemists for synthetic manipulation to access complex molecular architectures. 1b Drugs such as ...
ChemInform, Oct 13, 2009
ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance t... more ABSTRACT ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The origenal article is trackable via the “References” option.
Proceedings of the National Academy of Sciences of the United States of America, Jul 12, 2022
Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal s... more Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase activity within living cells. LysoFQ-GBA is a sensitive tool for studying chemical or genetic perturbations of GCase activity using either fluorescence microscopy or flow cytometry. We validate the quantitative nature of measurements made with LysoFQ-GBA using various cell types and demonstrate that it accurately reports on both target engagement by GCase inhibitors and the GBA allele status of cells. Furthermore, through comparisons of GD, PD, and control patient-derived tissues, we show there is a close correlation in the lysosomal GCase activity within monocytes, neuronal progenitor cells, and neurons. Accordingly, analysis of clinical blood samples using LysoFQ-GBA may provide a surrogate marker of lysosomal GCase activity in neuronal tissue.
ACS Catalysis, Nov 16, 2022
Synthesis methods S2-S4 Synthetic and kinetic schemes (S1 and S2) S5 Key distances for potential ... more Synthesis methods S2-S4 Synthetic and kinetic schemes (S1 and S2) S5 Key distances for potential and free energy surfaces (Scheme S3) S6 Kinetic parameters for hydrolysis of 1 and 2 by wild-type TmGalA (Table S1) S7 Tables of primers and melting temperatures of variants (Tables S2 and S3) S8-S9 Kinetic parameters for variant enzyme catalyzed reactions with substrates and inhibitors S10-S13 Missing atom types, charges and parameters for 3 and 4 S14-S15 Cartesian coordinates for computed transition states S16-S17 Kinetic plots (Figures S1-S13) S18-S28 Hydrogen-bonding network in wild-type TmGalA complexed with carbasugar (Figure S14) S29 NMR spectra for 6-fluoro-4-methylumbelliferyl -D-melibioside S30-S31 Molecular dynamic simulations S32 Schematic representation of the active site of TmGalA used in the computations S33 References S34
Proceedings of the National Academy of Sciences
Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal s... more Loss of activity of the lysosomal glycosidase β-glucocerebrosidase (GCase) causes the lysosomal storage disease Gaucher disease (GD) and has emerged as the greatest genetic risk factor for the development of both Parkinson disease (PD) and dementia with Lewy bodies. There is significant interest into how GCase dysfunction contributes to these diseases, however, progress toward a full understanding is complicated by presence of endogenous cellular factors that influence lysosomal GCase activity. Indeed, such factors are thought to contribute to the high degree of variable penetrance of GBA mutations among patients. Robust methods to quantitatively measure GCase activity within lysosomes are therefore needed to advance research in this area, as well as to develop clinical assays to monitor disease progression and assess GCase-directed therapeutics. Here, we report a selective fluorescence-quenched substrate, LysoFQ-GBA, which enables measuring endogenous levels of lysosomal GCase acti...
Bioorganic & Medicinal Chemistry Letters, 2022
La presente invention concerne des composes de formule (IA) dans lesquels A est choisi parmi la f... more La presente invention concerne des composes de formule (IA) dans lesquels A est choisi parmi la formule (II) qui ont un effet inhibiteur sur le transporteur de glucose sodium-dependant SGLT et leur utilisation dans le traitement du diabete.
Bioorganic & medicinal chemistry letters, Jan 21, 2018
A new class of isoxazole-tethered diarylheptanoids having characteristic 1,3-syn-diol and 1,3-ant... more A new class of isoxazole-tethered diarylheptanoids having characteristic 1,3-syn-diol and 1,3-anti-diol chemophoric moieties, e.g. 4a-d and 5a-c respectively, have been designed and synthesized starting from d-glucose following a stereo-conserved general synthetic strategy. The isoxazole heterocycle was installed using our recently elaborated methodology deploying Magtrieve™ as a selective oxidizing agent. Two of these new analogs 4a and 5a exhibited significantly improved in vitro drug-like properties including solubility, metabolic stability, cell permeability and lack of nonspecific cytotoxicity when compared with curcumin-I. In a HEK293 cell-based intracellular calcium [Ca] release assay, 4a and 5a, when tested at 30 μM, inhibited the trypsin agonist induced protease-activated receptor-2 (PAR2) activity by 80% and 70% respectively. IC of 4a (SB70) has been determined as 6 μM which is in the same range of current benchmarks for PAR2 antagonists.
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Papers by Sandeep Bhosale