Revealing the Mechanism for Covalent Inhibition of Glycoside Hydrolases by Carbasugars at an Atomic Level

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect. Nature

The worldwide prevalence of chronic hepatitis C virus (HCV) infection is estimated to be approaching 200 million people We designed a mechanistically unbiased approach based on chemical genetics to identify chemical starting points for interfering with HCV replication. Our differentiating strategy centred on the identification of compounds functionally distinct from those acting on the traditional targets of antiviral research in this field, the NS3 protease and the NS5B RNA-dependent RNA polymerase 10 . BMS-858 formed the basis of an extensive series of chemical refinements that focused on improving antiviral potency, broadening inhibitory activity to encompass the HCV 1a genotype, and optimizing for oral bioavailability and sustained pharmacokinetic properties. After defining symmetry as an important contributor to antiviral activity 10 , a discovery that preceded the disclosure of structural information (see below), we subsequently identified BMS-79005

A de novo nucleoside synthesis and late-stage heterobenzylic fluorination strategy

Nucleoside analogues constitute almost half of today’s major anticancer and antiviral therapeutics. Despite this, synthetic routes to these valuable molecules have typically relied on carbohydrate starting materials, which can significantly impair efforts in medicinal chemistry. Moreover, nucleoside scaffolds with increased complexity (e.g., C2’ or C4’ substitution) often require lengthy syntheses (up to 18 steps). Toward a goal of streamlining nucleoside synthesis, we have developed a one-pot proline-catalyzed α-fluorination/aldol reaction that generates enantiomerically enriched fluorohydrins that can serve as versatile building blocks for the construction of nucleoside analogues. Most importantly, this process enables access to variously functionalized nucleoside analogues in only 3 steps from commercial starting materials. The development of this process and practical application in rapidly accessing C2’- and C4’- modified nucleoside analogues, locked nucleic acids (LNAs), and iminonucleosides should inspire future efforts in drug design. Similar challenges also obstruct the synthesis of carbohydrate analogues (CAs), another important class of molecules to drug discovery efforts. To streamline CA synthesis, we developed several new proline-catalyzed α-functionalization/aldol reactions for constructing stereochemically rich and densely functionalized aldol adducts. In only 2 steps, these aldol adducts were then readily converted into a structurally diverse collection of CAs including iminosugars, annulated furanoses, bicyclic nucleosides, and fluorinated carbacycles. Incorporation of a fluorine atom can have several profound effects on a drug’s physiochemical properties – including metabolic stability, membrane permeability, and potency. However, the introduction of fluorine into the heterobenzylic position of drug molecules has remained an unsolved synthetic challenge. Towards this goal, we describe the first unified platform for the late-stage mono- and difluorination and trifluoromethylthiolation at heterobenzylic positions. This technology should become a dynamic tool for drug-lead diversification

Synthesis of Heterobenzylic Fluorides

Fluorination at heterobenzylic positions can have a significant impact on basicity, lipophilicity, and metabolism of drug leads. As a consequence, the development of new methods to access heterobenzylic fluorides has particular relevance to medicinal chemistry. This Short Review provides a survey of common methods used to synthesize heterobenzylic fluorides and includes fluoride displacement reactions of previously functionalized molecules (e.g., deoxyfluorination and halide exchange) and electrophilic fluorination of resonance stabilized heterobenzylic anions. In addition, recent advances in the direct fluorination of heterobenzylic C(sp3)-H bonds and monofluoromethylation of heterocyclic C(sp2)-H bonds are presented

Application of Sequential Proline Catalyzed α-Chlorination and Aldol Reactions in the Total Synthesis of 1-Deoxygalactonojirimycin

A short enantioselective total synthesis of the natural product drug 1-deoxygalactonojirimycin (migalastat) has been achieved that does not rely on chiral pool starting materials. Instead, this synthesis exploits a one-pot proline-catalyzed Îą-chlorination and aldol reaction of a commercially available aldehyde to assemble the entire carbon skeleton in a single step. The key role played by a nitrogen protecting group in the final epoxide opening reaction is highlighted as is the amenability to access structural analogues using this route.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Branched-chain and dendritic lipids for nanoparticles

Lipid nanoparticles (LNPs) for drug-delivery applications are largely derived from natural lipids. Synthetic lipids, particularly those incorporating branched hydrocarbons and hyper-branched hydrocarbon architectures may afford enhanced lipophilicity with enhanced fluidity and thereby lead to LNP stabilization. Hydrocarbon anchors based on serinol diesters were prepared from linear Cn (n = 14, 16, 18) and branched (n = 16) acids with Boc-protected serinol. These diesters were further dimerized on an iminodiacetamide backbone to provide eight branched-chain and dendritic lipid anchors. Derivatization of these core structures provided eight PEG-lipids and seven thiopurine linked lipid-drug conjugates. LNPs were prepared by microfluidic mixing from mixed lipids in ethanol diluted into aqueous media. The lipid-drug conjugates incorporated 5 mol% of a phosphocholine and 5 mol% of a commercial PEG-lipid to form LNPs with a thiopurine drug loading of 15 wt%. The PEG-lipids prepared were formulated at 1.5 mol% as a surface stabilizer to LNPs containing dsDNA lipoplexes. The stability of the LNPs was assessed under different storage conditions through monitoring of particle size. For both LNPs from lipid-thiopurine conjugates and the PEG-lipid systems there is strong preliminary evidence that hydrocarbon branching results in LNP stabilization. Four of the lipid-drug conjugate formulations were stable to cell culture conditions (10% serum, 37째C) and the toxicity of these LNPs was assessed in two cell lines relative to the free thiopurines in the medium. The observed toxicity is consistent with cellular uptake of the LNPs and reductive release of the cargo thiopurine within the cell.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

A county-level cross-sectional analysis of positive deviance to assess multiple population health outcomes in Indiana

Objective: To test a positive deviance method to identify counties that are performing better than statistical expectations on a set of population health indicators. Design: Quantitative, cross-sectional county-level secondary analysis of risk variables and outcomes in Indiana. Data are analysed using multiple linear regression to identify counties performing better or worse than expected given traditional risk indicators, with a focus on ‘positive deviants’ or counties performing better than expected. Participants: Counties in Indiana (n=92) constitute the unit of analysis. Main outcome measures: Per cent adult obesity, per cent fair/poor health, low birth weight per cent, per cent with diabetes, years of potential life lost, colorectal cancer incidence rate and circulatory disease mortality rate. Results: County performance that outperforms expectations is for the most part outcome specific. But there are a few counties that performed particularly well across most measures. Conclusions: The positive deviance approach provides a means for state and local public health departments to identify places that show better health outcomes despite demographic, social, economic or behavioural disadvantage. These places may serve as case studies or models for subsequent investigations to uncover best practices in the face of adversity and generalise effective approaches to other areas

Probing the substrate specificity of Trypanosoma brucei GlcNAc-PI de-N-acetylase with synthetic substrate analogues

A series of synthetic analogues of 1-d-(2-amino-2-deoxy-α-d-glucopyranosyl)-myo-inositol 1-(1,2-di-O-hexadecanoyl-sn-glycerol 3-phosphate), consisting of 7 variants of either the d-myo-inositol, d-GlcpN or the phospholipid components, were prepared and tested as substrates and inhibitors of GlcNAc-PI de-N-acetylase, a genetically validated drug target enzyme responsible for the second step in the glycosylphosphatidylinositol (GPI) biosynthetic pathway of Trypanosoma brucei. The d-myo-inositol in the physiological substrate was successfully replaced by cyclohexanediol and is still a substrate for T. brucei GlcNAc-PI de-N-acetylase. However, this compound became sensitive to the stereochemistry of the glycoside linkage (the β-anomer was neither substrate or inhibitor) and the structure of the lipid moiety (the hexadecyl derivatives were inhibitors). Chemistry was successfully developed to replace the phosphate with a sulphonamide, but the compound was neither a substrate or an inhibitor, confirming the importance of the phosphate for molecular recognition. We also replaced the glucosamine by an acyclic analogue, but this also was inactive, both as a substrate and inhibitor. These findings add significantly to our understanding of substrate and inhibitor binding to the GlcNAc-PI de-N-acetylase enzyme and will have a bearing on the design of future inhibitors