Imino- and Azasugar Protonation Inside Human Acid β-Glucosidase,the Enzyme that is Defective in Gaucher Disease

Gaucher disease is caused by mutations in human acid β-glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino- and azasugars such as 1-deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino- and azasugars bound in the active site having been resolved, the actual acid–base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1-deoxynojirimycin and isofagomine derivatives are protonated by human acid β-glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1-deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.     

Imino‐ and Azasugar Protonation Inside Human Acid β‐Glucosidase,the Enzyme that is Defective in Gaucher Disease

Gaucher disease is caused by mutations in human acid β‐glucosidase or glucocerebrosidase (GCase), the enzyme responsible for hydrolysis of glucosyl ceramide in the lysosomes. Imino‐ and azasugars such as 1‐deoxynojirimycin and isofagomine are strong inhibitors of the enzyme and are of interest in pharmacological chaperone therapy of the disease. Despite several crystal structures of the enzyme with the imino‐ and azasugars bound in the active site having been resolved, the actual acid–base chemistry of the binding is not known. In this study we show, using photoinduced electron transfer (PET), that 1‐deoxynojirimycin and isofagomine derivatives are protonated by human acid β‐glucosidase when bound, even if they are completely unprotonated outside the enzyme. While isofagomine derivative protonation to some degree was foreshadowed by earlier crystal structures, 1‐deoxynojirimycin derivatives were not believed to act as basic amines in the enzyme.     

Glycosidase inhibition: an assessment of the binding of 18 putative transition-state mimics

The inhibition of glycoside hydrolases, through transition-state mimicry, is important both as a probe of enzyme mechanism and in the continuing quest for new drugs, notably in the treatment of cancer, HIV, influenza, and diabetes. The high affinity with which these enzymes are known to bind the transition state provides a framework upon which to design potent inhibitors. Recent work [for example, Bülow, A. et al. J. Am. Chem. Soc. 2000, 122, 8567-8568; Zechel, D. L. et al. J. Am. Chem. Soc. 2003, 125, 14313-14323] has revealed quite confusing and counter-intuitive patterns of inhibition for a number of glycosidase inhibitors. Here we describe a synergistic approach for analysis of inhibitors with a single enzyme 'model system', the Thermotoga maritima family 1 beta-glucosidase, TmGH1. The pH dependence of enzyme activity and inhibition has been determined, structures of inhibitor complexes have been solved by X-ray crystallography, with data up to 1.65 A resolution, and isothermal titration calorimetry was used to establish the thermodynamic signature. This has allowed the characterization of 18 compounds, all putative transition-state mimics, in order to build an 'inhibition profile' that provides an insight into what governs binding. In contrast to our preconceptions, there is little correlation of inhibitor chemistry with the calorimetric dissection of thermodynamics. The ensemble of inhibitors shows strong enthalpy-entropy compensation, and the random distribution of similar inhibitors across the plot of DeltaH degrees a vs TDeltaS degrees a likely reflects the enormous contribution of solvation and desolvation effects on ligand binding.     

Aziridines as a structural motif to conformational restriction of azasugars

In order to investigate the hypothesis that the glycosidase inhibitor isofagomine was bound to alpha- or beta-glucosidase in a 1,4B conformation, a number of bicyclic aziridines that adopt the 1,4B or B1,4 conformations were synthesised and investigated. (1R)-2-endo,3-exo-2,3-Dihydroxy-4-endo-4-hydroxymethyl-6- azabicyclo[3.1.0]hexane (5) and its N-methyl and N-benzyl analogues and (1S)-2-exo-3-endo-2,3-dihydroxy-4- endo-4-hydroxymethyl-6-azabicyclo-[3.1.0]hexane (6) were synthesised. The aziridines 5 and 6 were found to be weak or not inhibitors of alpha-glucosidase, beta-glucosidase and alpha-fucosidase.     

Direct observation of the protonation state of an imino sugar glycosidase inhibitor upon binding

Glycosidases are some of the most ubiquitous enzyme in nature. Their biological significance, coupled to their enormous catalytic prowess derived from tight binding of the transition state, is reflected in their importance as therapeutic targets. Many glycosidase inhibitors are known. Imino sugars are often potent inhibitors, yet many facets of their mode of action, such as their degree, if any, of transition-state "mimicry" and their protonation state when bound to the target glycosidase remain unclear. Atomic resolution analysis of the endoglucanase, Cel5A, in complex with a cellobio-derived isofagomine in conjunction with the pH dependence of Ki and kcat/KM reveals that this compound binds as a protonated sugar. Surprisingly, both the enzymatic nucleophile and the acid/base are unprotonated in the complex.     

Exploring the subtleties of drug-receptor interactions: the case of matrix metalloproteinases

By solving high-resolution crystal structures of a large number (14 in this case) of adducts of matrix metalloproteinase 12 (MMP12) with strong, nanomolar, inhibitors all derived from a single ligand scaffold, it is shown that the energetics of the ligand-protein interactions can be accounted for directly from the structures to a level of detail that allows us to rationalize for the differential binding affinity between pairs of closely related ligands. In each case, variations in binding affinities can be traced back to slight improvements or worsening of specific interactions with the protein of one or more ligand atoms. Isothermal calorimetry measurements show that the binding of this class of MMP inhibitors is largely enthalpy driven, but a favorable entropic contribution is always present. The binding enthalpy of acetohydroxamic acid (AHA), the prototype zinc-binding group in MMP drug discovery, has been also accurately measured. In principle, this research permits the planning of either improved inhibitors, or inhibitors with improved selectivity for one or another MMP. The present analysis is applicable to any drug target for which structural information on adducts with a series of homologous ligands can be obtained, while structural information obtained from in silico docking is probably not accurate enough for this type of study.     

Van der Waals interactions dominate ligand-protein association in a protein binding site occluded from solvent water

In the present study we examine the enthalpy of binding of 2-methoxy-3-isobutylpyrazine (IBMP) to the mouse major urinary protein (MUP), using a combination of isothermal titration calorimetry (ITC), NMR, X-ray crystallography, all-atom molecular dynamics simulations, and site-directed mutagenesis. Global thermodynamics data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than a classical entropy-driven signature that might be expected given that the binding pocket of MUP-1 is very hydrophobic. The only ligand-protein hydrogen bond is formed between the side-chain hydroxyl of Tyr120 and the ring nitrogen of the ligand in the wild-type protein. ITC measurements on the binding of IBMP to the Y120F mutant demonstrate a reduced enthalpy of binding, but nonetheless binding is still enthalpy dominated. A combination of solvent isotopic substitution ITC measurements and all-atom molecular dynamics simulations with explicit inclusion of solvent water suggests that solvation is not a major contributor to the overall binding enthalpy. Moreover, hydrogen/deuterium exchange measurements suggest that there is no significant contribution to the enthalpy of binding derived from "tightening" of the protein structure. Data are consistent with binding thermodynamics dominated by favorable dispersion interactions, arising from the inequality of solvent-solute dispersion interactions before complexation versus solute-solute dispersion interactions after complexation, by virtue of poor solvation of the binding pocket.     

Thermodynamic aspects of dicarboxylate recognition by simple artificial receptors.

Recognition of dicarboxylates by bis-functional hydrogen-bonding receptors displays divergent thermodynamics in different solvent systems. NMR titration and isothermal titration calorimetry indicated that neutral bis-urea and bis-thiourea receptors form exothermic complexes with dicarboxylates in DMSO, with a near zero entropic contribution to binding. The increased binding strength of bis-guanidinium receptors precluded quantitative measurement of binding constants in DMSO, but titration calorimetry offered a qualitative picture of the association. Formation of these 1:1 complexes was also exothermic, but additional endothermic events occurred at both lower and higher host-guest ratios. These events indicated multiple binding equilibria but did not always occur at a discrete 2:1 or 1:2 host-guest molar ratio, suggesting higher aggregates. With increasing amounts of methanol as solvent, bis-guanidinium receptors form more endothermic complexes with dicarboxylates, with a favorable entropy of association. This switch from association driven by enthalpy to one driven by entropy may reflect a change from complexation involving the formation of hydrogen bonds to that promoted by solvent liberation from binding sites.     

等温滴定量热法和荧光滴定法研究十二烷基硫酸钠与纤维素酶的结合

用等温滴定量热法和荧光滴定法研究了阴离子型去垢剂十二烷基硫酸钠（SDS ）与绿色木霉纤维素酶相互作用的热力学，SDS结合纤维素酶的亲和力较弱，为较 小的放热反应，并伴随着一定程度的熵增，为焓和熵共同驱动的反应，而且存在着 显著的焓-熵补偿作用。该结合过程的摩尔恒压热容为较大的负值（-186 J·mol~ (-1)·K~(-1)），这表明疏水相互作用是形成复合物的主要作用力，SDS的加入使 纤维素酶的内源荧光发生猝灭，同时导致该蛋白荧光光谱最大发射峰位的红移和酶 活力的部分丧失，这表明SDS与纤维素酶的相互作用既包含结合反应也包含SDS诱导 该蛋白部分去折叠的过程。     

N?O Bond as a Glycosidic‐Bond Surrogate: Synthetic Studies Toward Polyhydroxylated N‐Alkoxypiperidines

A series of novel polyhydroxylated N‐alkoxypiperidines has been synthesized by ring‐closing double reductive amination (DRA) of highly functionalized 1,5‐dialdehydes with various hydroxylamines. The required saccharide‐based dialdehydes were prepared efficiently from sodium cyclopentadienylide in seven steps. A two‐step protocol has been developed for the DRA; it led, after deprotection, to isofagomine, 3‐deoxyisofagomine, and numerous other N‐alkoxy analogues. The barrier to inversion in these polyhydroxylated N‐alkoxypiperidine derivatives was found by variable‐temperature NMR methods to be approximately 15 kcal mol^?1. With the exception of N‐hydroxyisofagomine itself, none of the compounds prepared showed significant inhibitory activity against sweet almond β‐glucosidase.     

人血清白蛋白与季铵盐双子表面活性剂的相互作用

在298.15 K下, 应用等温滴定量热法研究了人血清白蛋白(HSA)与两种季铵盐双子表面活性剂[(C_nN)₂Cl₂, n＝12, 14]在缓冲溶液(pH＝7.0)中相互作用的热力学性质. 实验结果表明, HSA对这两种表面活性剂有两类结合位点, 分别为结合时需要吸收热量的强结合位点和可放出热量的弱结合位点. 两种表面活性剂对应的第一类结合——强结合为熵驱动过程, 且该结合位点对应的结合位点数、结合常数和热力学参数差别不大. 至于第二类结合——弱结合位点, 由于 (C₁₄N)₂Cl₂ 疏水链过长, 只有部分进入HSA的疏水空腔内, 因此相应的的结合位点数和放热量减小, 而熵变增加, 为焓和熵共同驱动的反应. 圆二色研究表明(C_nN)₂Cl₂的加入使HSA的二级结构发生变化, 这说明(C_nN)₂Cl₂与HSA的相互作用既包含结合反应也包含(C_nN)₂Cl₂诱导该蛋白部分结构改变的过程.     

Dissection of aminoglycoside-enzyme interactions: a calorimetric and NMR study of neomycin B binding to the aminoglycoside phosphotransferase(3')-IIIa

In this work, for the first time, we report pKa values of the amino functions in a target-bound aminoglycoside antibiotic, which permitted dissection of the thermodynamic properties of an enzyme-aminoglycoside complex. Uniformly enriched 15N-neomycin was isolated from cultures of Streptomyces fradiae and used to study its binding to the aminoglycoside phosphotransferase(3')-IIIa (APH) by 15N NMR spectroscopy. 15N NMR studies showed that binding of neomycin to APH causes upshifts of approximately 1 pKa unit for the amines N2' and N2' ' while N6' experienced a 0.3 pKa unit shift. The pKa of N6' ' remained unaltered, and resonances of N1 and N3 showed significant broadening upon binding to the enzyme. The binding-linked protonation and pH dependence of the association constant (Kb) for the enzyme-aminoglycoside complex was determined by isothermal titration calorimetry. The enthalpy of binding became more favorable (negative) with increasing pH. At high pH, binding-linked protonation was attributable mostly to the amino functions of neomycin; however, at neutral pH, functional groups of the enzyme, possibly remote from the active site, also underwent protonation/deprotonation upon formation of the binary enzyme-neomycin complex. The Kb for the enzyme-neomycin complex showed a complicated dependence on pH, indicating that multiple interactions may affect the affinity of the ligand to the enzyme and altered conditions, such as pH, may favor one or another. This work highlights the importance of determining thermodynamic parameters of aminoglycoside-target interactions under different conditions before making attributions to specific sites and their effects on these global parameters.     

Spectroscopic and Isothermal Titration Calorimetry Studies of Binding Interactions Between Carbon Nanodots and Serum Albumins

Carbon nanodots (C-dots) have attracted great attention as a new class of luminescent nanomaterials. In order to better understand the basic behavior of C-dots in biological systems, the binding characteristics of C-dots with bovine serum albumin (BSA) and human serum albumin (HSA) were investigated using spectroscopic approaches and isothermal titration calorimetry at pH 7.4. We found that the intrinsic fluorescence of BSA and HSA was quenched by the C-dots with a dynamic quenching mode. It was proved that the C-dots had little influence on the conformation of BSA and HSA by their UV–vis and circular dichroism spectra. Some important thermodynamic parameters were calculated, and the positive values of ΔH° and ΔS° indicate that the binding process was endothermic, and that the interaction was driven by favorable entropy and unfavorable enthalpy. It also showed that the hydrophobic force played a major role in the binding process.     

环糊精与新型表面活性剂的主客体相互作用

在298.15 K下用微量热法结合核磁共振法研究了α-环糊精, β-环糊精与十二烷基多氧乙烯磺酸钠C12EnS(n=1, 3)在水溶液中的包结作用. 实验结果表明, β-环糊精与客体的包合是焓、熵共驱的过程, α-环糊精与客体的包合则是焓驱动过程. β-环糊精与两种客体包合的化学计量比随客体中氧乙烯链的不同而不同, 而α-环糊精与两种客体包合的化学计量比则无差别. 1H NMR数据表明, C12EnS的存在使两种环糊精上各质子的化学位移向高场移动, 从微观上证明了包结作用的发生.     

Synthesis and aggregation behavior of pluronic F87/poly(acrylic acid) block copolymer in the presence of doxorubicin

Poly(acrylic acid) (PAA) was polymerized on both termini of Pluronic F87 copolymer using the atom transfer radical polymerization technique to produce a novel block copolymer, PAA-b-F87-b-PAA (F87PAA). The loading of a cationic anticancer drug, doxorubicin (DOX), to F87PAA at different pH values was investigated using isothermal titration calorimetry (ITC), laser light scattering techniques, and UV-vis spectroscopy. At pH of 4.3-7.1, the ITC profile exhibited a significant exothermic peak, which indicated that the drug loading is an enthalpically driven process. At a pH of 4.3, the enthalpy maximum was significantly reduced in the presence of 2 M urea, indicating the existence of hydrogen bonds between the DOX and F87PAA copolymer. At a pH of 7.1, the fraction of bound DOX was close to the stoichiometric proportion of 1:1 to the molar concentration of carboxyl groups in the copolymer, where the drug loading is governed by electrostatic and stacking interactions. The TEM image of the complex indicated the formation of large compound micelles induced by the binding of DOX to the PAA segments.     

Thermodynamic characterization of the binding of tetrahydropterins to phenylalanine hydroxylase

Phenylalanine hydroxylase (PAH) is the key enzyme in the catabolism of L-Phe. The natural cofactor of PAH, 6R-tetrahydrobiopterin (BH4), negatively regulates the enzyme activity in addition to being an essential cosubstrate for catalysis. The analogue 6-methyltetrahydropterin (6M-PH4) is effective in catalysis but does not regulate PAH. Here, the thermodynamics of binding of BH4 and 6M-PH4 to human PAH have been studied by isothermal titration calorimetry. At neutral pH and 25 degrees C, BH4 binds to PAH with higher affinity (Kd = 0.75 +/- 0.18 microM) than 6M-PH4 (Kd = 16.5 +/- 2.7 microM). While BH4 binding is a strongly exothermic process (DeltaH = -11.8 +/- 0.4 kcal/mol) accompanied by an entropic penalty (-TDeltaS = 3.4 +/- 0.4 kcal/mol), 6M-PH4 binding is both enthalpically (DeltaH = -3.3 +/- 0.3 kcal/mol) and entropically (-TDeltaS = -3.2 kcal/mol) driven. No significant changes in binding affinity were observed in the 5-35 degrees C temperature range for both pterins at neutral pH, but the enthalpic contribution increased with temperature rendering a heat capacity change (DeltaCp) of -357 +/- 26 cal/mol/K for BH4 and -63 +/- 12 cal/mol/K for 6M-PH4. Protons do not seem to be taken up or released upon pterin binding. Structure-based energetics calculations applied on the molecular dynamics simulated structures of the complexes suggest that in the case of BH4 binding, the conformational rearrangement of the N-terminal tail of PAH contribute with favorable enthalpic and unfavorable entropic contributions to the intrinsic thermodynamic parameters of binding. The entropic penalty is most probably associated to the reduction of conformational flexibility at the protein level and disappears for the L-Phe activated enzyme. The calculated energetic parameters aid to elucidate the molecular mechanism for cofactor recognition and the regulation of PAH by the dihydroxypropyl side chain of BH4.     

Studies on the binding of paeonol and two of its isomers to human serum albumin by using microcalorimetry and circular dichroism

Interactions of paeonol and two of its isomers with human serum albumin (HSA) in buffer solutions (pH 7.0) have been investigated by calorimetry and circular dichroism. Heats of the interactions have been determined with isothermal titration microcalorimetry at 298.15 K. Data process has been based on the supposition that there are several independent classes of binding sites on each HSA molecule for molecules of each one of the drugs. The results obtained by using this supposition combined with Langmuir adsorption model show that there are two classes of such binding sites. The binding constant, changes of enthalpy, entropy, and Gibbs free energy are obtained, which show that the two classes of binding are mainly driven by enthalpy except that the first-class binding of Ace is predominantly driven by entropy. On the same class of binding site, the negative value of binding enthalpy decreases in the order of Pae, Hma, and Ace. The difference of thermodynamic data is caused by the different locations of substituent groups on aromatic benzene ring of guest molecules. Circular dichroism (CD) spectra show that the three isomers change the secondary structure of HSA. These results indicate that the interaction includes contributions of the binding and the partial change of molecular structure of HSA induced by the three isomers.     

Synthesis and α-amylase inhibitory activity of glucose-deoxynojirimycin conjugates

Inhibitors of α-amylase have attracted attention for their putative effects against diabetes mellitus. Although numerous studies have explored natural small molecule inhibitors, acarbose is currently the only compound with sufficient inhibitory potency and drug-like characteristics to be considered as a potential therapeutic agent. We have synthesized conjugates of the potent glucosidase inhibitor, 1-deoxynojirimycin, and glucose, with the aim of enhancing inhibitory activity against α-amylase. This synthetic conjugate showed increased inhibition of α-amylase compared to 1-deoxynojirimycin alone, suggesting that similar modifications of existing glucosidase inhibitors may yield more potent α-amylase inhibitors.     

Highlights in oleuropein aglycone structure

Oleuropein hydrolysis products, obtained both by endogenous beta-glucosidase and almond commercial one, were investigated by classic methodologies and by 1HNMR experiments. We found that both presence and relative amount of the different compounds in the reaction mixture are stricly related to the used enzyme.     

Development of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives as selective inhibitors of glucosylceramide metabolism in man

In this article, we present a straightforward synthesis of adamantan-1-yl-methoxy-functionalized 1-deoxynojirimycin derivatives. The used synthetic routes are flexible and can be used to create a wide variety of lipophilic mono- and difunctionalized 1-deoxynojirimycin derivatives. The compounds reported here are lipophilic iminosugar based on lead compound 4, a potent inhibitor of the three enzymes involved in the metabolism of the glycosphingolipid glucosylceramide. Iminosugar-based inhibitors of glucosylceramide synthase, one of these three enzymes, have attracted increasing interest over the past decade due to the crucial role of this enzyme in glycosphingolipid biosynthesis. Combined with the fact that an increasing number of pathological processes are being linked to excessive glycosphingolipid levels, glucosylceramide synthase becomes a very attractive therapeutic and research target. Our results presented here demonstrate that relocating the lipophilic moiety from the nitrogen atom to other positions on the 1-deoxynojirimycin ring system does not lead to a more potent or selective inhibitor of glucosylceramide synthase. The beta-aza-C-glycoside analogue (17) retained the best inhibitory potency for glucosylceramide synthase and is a more potent inhibitor than the therapeutic agent N-butyl-1-deoxynojirimycin (3), marketed as treatment for Gaucher disease under the commercial name Zavesca.