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An approach based on the combined use of saturation transfer difference (STD), Tr‐NOESY and Inter‐ligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10 : 1 cis‐9), oleic acid (18 : 1 cis‐9), linoleic acid (18 : 2 cis‐9,12) and linolenic (18 : 3, cis‐9,12,15) free fatty acids (FFAs) with non‐labelled serum albumin (BSA/HSA). Significant negative inter‐ligand NOEs between the FFAs and the drugs ibuprofen and warfarin, through competition experiments, were observed. The inter‐ligand NOEs and docking calculations were interpreted in terms of competitive binding mode, the significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site (FA7), due to two potential distinctive anchoring polar groups of amino acids. This conformational flexibility is the reason that, the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous available X‐ray structural studies. α‐Linolenic acid competes favourably with warfarin at the binding site FA7. Isothermal titration calorimetry experiments of the preformed HSA/α‐linolenic acid complex upon titration with warfarin show a significant reduction in the binding constant of warfarin, in very good agreement with NMR and computational data. The combined use, therefore, of STD, Tr‐NOESY and INPHARMA NMR, ITC and docking calculations may find promising applications in the field of protein–lipid recognition research. [ABSTRACT FROM AUTHOR]

Fragment-based drug discovery is one of the most utilized approaches for the identification of novel weakly binding ligands, by efficiently covering a wide chemical space with rather few compounds and by allowing more diverse binding modes to be found. This approach has led to various clinical candidates and approved drugs. Halogen bonding, on the other hand, has gained traction in molecular design and lead optimization, but could offer additional benefits in early drug discovery. Screening halogen-enriched fragments (HEFLibs) could alleviate problems associated with the late introduction of such a highly geometry dependent interaction. Usually, the binding mode is then already dominated by other strong interactions. Due to the fewer competing interactions in fragments, the halogen bond should more often act as an anchor point for the binding mode. Previously, we proposed a fragment library with a focus on diverse binding modes that involve halogens for gaining initial affinity and selectivity. Herein, we demonstrate the applicability of these HEFLibs with a small set of diverse enzymes: the histone-lysine N-methyltransferase DOT1L, the indoleamine 2,3-dioxygenase 1 (IDO1), the AP2-associated protein kinase 1 (AAK1), and the calcium/calmodulin-dependent protein kinase type 1G (CAMK1G). We were able to identify various binding fragments via STD-NMR. Using ITC to verify these initial hits, we determined affinities for many of these fragments. The best binding fragments exhibit affinities in the one-digit micromolar range and ligand efficiencies up to 0.83 for AAK1. A small set of analogs was used to study structure-affinity relationships and hereby analyze the specific importance of each polar interaction. This data clearly suggests that the halogen bond is the most important interaction of fragment 9595 with AAK1.

Ethnopharmacological relevance: Two types of traditional Chinese formulas of botanical drugs are prescribed for treating perimenopausal syndrome (PMS), a disorder in middle-aged women during their transition to menopause. One is for treating PMS as kidney deficiency (KD) due to senescence and declining reproductive functions, and the other is for treating it as liver qi stagnation (LQS) in association with stress and anxiety. Despite the time-tested prescriptions, an objective attestation to the effectiveness of the traditional Chinese treatment of PMS is still to be established and the associated molecular mechanism is still to be investigated.Materials and methods: A model for PMS was generated from perimenopausal rats with chronic restraint stress (CRS). The effectiveness of traditional Chinese formulas of botanical drugs and a combination of two of the formulas was evaluated based on 1H NMR plasma metabolomic, as well as behavioral and physiological, indicators. To investigate whether the formulas contained ligands that could compensate for the declining level of estrogen, the primary cause of PMS, the ligand-based NMR technique of saturation transfer difference (STD) was employed to detect possible interacting molecules to estrogen receptors in the decoction.Results: Each prescription of the classical Chinese formula moderately attenuated the metabolomic state of the disease model. The best treatment strategy however was to combine two traditional Chinese formulas, each for a different etiology, to adjust the metabolomic state of the disease model to that of rats at a much younger age. In addition, this attenuation of the metabolomics of the disease model was by neither upregulating the estrogen level nor supplementing an estrogenic compound.Conclusion: Treatment of PMS with a traditional Chinese formula of botanical drugs targeting one of the two causes separately could ameliorate the disorder moderately. However, the best outcome was to treat the two causes simultaneously with a decoction that combined ingredients from two traditional prescriptions. The data also implicated a new paradigm for phytotherapy of PMS as the prescribed decoctions contained no interacting compound to modulate the activity of estrogen receptors, in contrast to the treatment strategy of hormone replacement therapy.

Ethnopharmacological relevance: Two types of traditional Chinese formulas of botanical drugs are prescribed for treating perimenopausal syndrome (PMS), a disorder in middle-aged women during their transition to menopause. One is for treating PMS as kidney deficiency (KD) due to senescence and declining reproductive functions, and the other is for treating it as liver qi stagnation (LQS) in association with stress and anxiety. Despite the time-tested prescriptions, an objective attestation to the effectiveness of the traditional Chinese treatment of PMS is still to be established and the associated molecular mechanism is still to be investigated. Materials and methods: A model for PMS was generated from perimenopausal rats with chronic restraint stress (CRS). The effectiveness of traditional Chinese formulas of botanical drugs and a combination of two of the formulas was evaluated based on 1H NMR plasma metabolomic, as well as behavioral and physiological, indicators. To investigate whether the formulas contained ligands that could compensate for the declining level of estrogen, the primary cause of PMS, the ligand-based NMR technique of saturation transfer difference (STD) was employed to detect possible interacting molecules to estrogen receptors in the decoction. Results: Each prescription of the classical Chinese formula moderately attenuated the metabolomic state of the disease model. The best treatment strategy however was to combine two traditional Chinese formulas, each for a different etiology, to adjust the metabolomic state of the disease model to that of rats at a much younger age. In addition, this attenuation of the metabolomics of the disease model was by neither upregulating the estrogen level nor supplementing an estrogenic compound. Conclusion: Treatment of PMS with a traditional Chinese formula of botanical drugs targeting one of the two causes separately could ameliorate the disorder moderately. However, the best outcome was to treat the two causes simultaneously with a decoction that combined ingredients from two traditional prescriptions. The data also implicated a new paradigm for phytotherapy of PMS as the prescribed decoctions contained no interacting compound to modulate the activity of estrogen receptors, in contrast to the treatment strategy of hormone replacement therapy. [ABSTRACT FROM AUTHOR]

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domainswapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development. [ABSTRACT FROM AUTHOR]

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein.

Since the exposure of organophosphate pesticides are known to cause severe health consequences, it is important to understand the molecular interaction of these pesticides metabolites with vital biomolecules, especially with the proteins. Here, considering bovine serum albumin (BSA) as a model protein, we have examined its interaction with two selected organophosphate metabolites, 3,5,6-trichloro-2-pyridinol (TCPy) and paraoxon methyl (PM). TCPy and PM are resultant metabolites of two most widely used organophosphate pesticides chlorpyrifos and parathion respectively. 1H NMR line broadening, selective spin-lattice relaxation rate measurements, saturation transfer difference (STD) NMR of both TCPy and PM were carried out in the presence and absence of BSA. The obtained values of the affinity index (A), binding constants (K a) and thermodynamic parameters indicated strong organophosphates-BSA interaction. Further, fluorescence quenching data on TCPy-BSA and PM-BSA interactions strongly supported the NMR results, besides providing the stoichiometry of these complexes. Molecular docking analysis unraveled viable, strong hydrogen bonds and electrostatic interactions in TCPy-BSA and PM-BSA complexes. This study also revealed substantial time-dependent changes in the 1H NMR intensity of PM in the presence of BSA, which suggests faster degradation of PM with increasing protein concentration during protein-metabolite interactions. The hydrolysis is attributed to the esterase-like action of BSA. The result provides key insights into the direct interaction of the organophosphate metabolites with a biologically important carrier protein, serum albumin. Unlabelled Image • Affinity constant of BSA-TCPy is highly greater than BSA-PM. • Thermodynamics parameters confirms electrostatic and hydrogen bonding between complexes. • Fluorescence quenching data shows static quenching for both metabolites. • Hydrolysis rate of PM was much faster than TCPy both in absence and presence of BSA. • PM will greatly diffuse to body tissues with accelerated excretion compared to TCPy. [ABSTRACT FROM AUTHOR]

WaterLOGSY is a sensitive ligand-observed NMR experiment for detection of interaction between a ligand and a protein and is now well-established as a screening technique for fragment-based lead discovery. Here we develop and assess a protocol to derive ligand epitope mapping from WaterLOGSY data and demonstrate its general applicability in studies of fragment-sized ligands binding to six different proteins (glycogen phosphorylase, protein peroxiredoxin 5, Bcl-xL, Mcl-1, HSP90, and human serum albumin). We compare the WaterLOGSY results to those obtained from the more widely used saturation transfer difference experiments and to the 3D structures of the complexes when available. In addition, we evaluate the impact of ligand labile protons on the WaterLOGSY data. Our results demonstrate that the WaterLOGSY experiment can be used as an additional confirmation of the binding mode of a ligand to a protein. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Solvent Paramagnetic Relaxation Enhancement has been introduced to study the detailed aggregation mode of CHAPS. • The core and surface of the micelle are formed mainly by the head, and tail of CHAPS, respectively. • The micelle in M2 state is mostly like to be a double layer structure with relatively tight but still messy aggregation Abstract CHAPS is a typical zwitterionic detergent widely used in protein purification. It has two peculiar aggregation states, whose aggregation patterns are still controversial and uncertain. In this paper, we analyzed the aggregation pattern of CHAPS by 1H solvent Paramagnetic Relaxation Enhancement (sPRE) experiment. It was found that the 1H sPREs of head groups of CHAPS are much smaller than those of the tail groups, and all of them become smaller simultaneously when the concentration of CHAPS is above CMC2. In addition, the sPREs of those protons in the steroid ring were found to be very similar to each other in both micellar states (M1 and M2). These results indicated that the micellar core of CHAPS is mainly formed by its steroid head which aggregate disorderly. On the other hand, a transition from single to double layer micelle is most likely to happen when the concentration of CHAPS is above CMC2. The micelle in M2 state is formed by the aggregation of the partially disordered primary ones as well. Furthermore, the STD (Saturation Transfer Difference) experiment showed that the apparent coherence transfer rates between water and those OH near protons at the steroid head are slower in M2 state, revealing that the CHAPS aggregate loosely in M1 state, but more tightly in M2 state, which may be driven by the increased hydrophobic interactions between the steroid groups of nearby CHAPS molecules, as well as the hydrogen bond between OH in the hydrophobic side and SO 3 −1, CO, or HN in the hydrophilic side of different molecules, which consequently extrude the water and stabilize the micelle. [ABSTRACT FROM AUTHOR]

The use of NMR as a tool to determine 3 dimensional protein solution structures, once a darling of the pharmaceutical industry, has largely given way to study of the interaction of prospective drugs with macromolecular targets. Many of these approaches involve ligand-centered studies, which have the advantage of speed and efficiency, but there are also many approaches that take directly from our learnings in macromolecular NMR and provide greater structural detail yet are still optimized for rapid turn-around of information. In the evolution of NMR in the pharmaceutical industry, the unique strengths of NMR to provide dynamic and atomic level information continue to be exploited to discover and design new drugs. Numerous methods have been developed over the past two decades that fall into the categories of fragment-based pre-lead discovery, ligand binding studies and qualitative structural screening. [ABSTRACT FROM AUTHOR]

As a natural dietary polyphenol, 3,4,5-tri- O -caffeoylquinic acid (3,4,5-triCQA) exhibits numerous stronger pharmacological activities than that of its analogues. Studies on interaction between 3,4,5-triCQA and protein are very helpful for understanding the mechanism of these enhanced biological functions. In this study, 1 H saturation transfer difference NMR ( 1 H STD-NMR) combined with multi-spectroscopy were used to probe the interaction of 3,4,5-triCQA with human serum albumin (HSA). Both qualitative and quantitative 1 H STD-NMR indicated that 3,4,5-triCQA can specifically bind to HSA at the favored Sudlow's site II with caffeoyl groups as the main recognizable moiety. Fluorescence emission spectra showed that Stern-Volmer quenching constant ( K SV ) decreases from 10.132 × 10 4 M − 1 to 9.711 × 10 4 M − 1 with temperature raise, indicating that 3,4,5-triCQA quenches HSA fluorescence through a static mechanism. Binding constant ( K b = 5.557 × 10 5 M − 1 ) and the number of binding sites ( n ≈ 1) at 298 K suggested that 3,4,5-triCQA only occupies one site in HSA with high affinity. Enthalpy (Δ H = − 28.802 kJ/mol) and entropy (Δ S = 12.429 J/mol/K) change proved the dominant role of electrostatic interaction in binding process. Multi-spectroscopic analysis also confirmed that the protein secondary structure and hydrophobicity were significantly affected. Molecular docking further verified the NMR and spectroscopic results. Overall, 3,4,5-triCQA exhibited a strong albumin affinity owing to the plural caffeoyl groups, which lead to the enhanced pharmacological activities. This study clarified the molecular mechanism of 3,4,5-triCQA in binding to HSA, and the findings are beneficial for the research on polyphenol-like drugs and antioxidants in foods or cosmetics. [ABSTRACT FROM AUTHOR]

Innovative approaches in nanotechnology can provide drug delivery systems with a high potential in different fields. To avoid trial and error assays as a main driving force governing new designs and, furthermore, to develop successful nanosystem optimization strategies, it is of the greatest importance to develop specific characterisation techniques beyond conventional determinations of size, zeta potential and morphology. However, the application of techniques able to determine some key characteristics, such as nanostructure (i.e., solid structure vs vesicular), and the way in which the reorganization of components takes place on these structures has been scarcely explored. The present work has been devoted to provide some insights about the potential offered by some NMR techniques to those scientists working on nanotechnological approaches. For this purpose, we selected our nanosystems based on sorbitan monooleate as a case study. We used 1 H NMR methods, including a recently proposed method relying in the well-known Saturation Transfer Difference (STD) experiment for the observation of ‘invisible signals’ in large aggregates (Invisible State STD or ISSTD). Overall, these techniques revealed the presence in these nanosystems of a gradient of flexibility from an internal rigid core towards a more flexible region located on their surface, as well as the absence of water content in both regions. Such structure, corresponding to a solid nanostructure rather than a vesicular one, can explain some of the interesting properties previously observed for these innovative nanosystems, such as their high stability, and allows us to refer to these nanosystems with the term “Solid Sorbitan esters Nanoparticles” (SSN). On the basis of the valuable information provided by the mentioned characterisation techniques, it is our understanding that they could facilitate the future design of new drug delivery nanosystems as well as the improvement of existing ones and/or the development of new applications for classical drug delivery concepts. [ABSTRACT FROM AUTHOR]

The fragment FOL7185 (compound 17 ) was found to be a hit against IspD and IspE enzymes isolated from bacteria, and a series of analogs containing the pyrazolopyrimidine core were synthesized. The majority of these compounds inhibited the growth of Burkholderia thailandensis ( Bt ) and Pseudomonas aeruginosa ( Pa ) in the Kirby–Bauer disk diffusion susceptibility test. Compound 29 shows inhibitory activity at 0.1 mM (32.2 μg/mL), which is comparable to the control compound kanamycin (48.5 μg/mL). Compound 29 also shows inhibitory activity at 0.5 mM against kanamycin resistant P. aeruginosa . Saturation transfer difference NMR (STD-NMR) screening of these compounds against Bt IspD and Bt IspE indicated that most of these compounds significantly interact with Bt IspE, suggesting that the compounds may inhibit the growth of Bt by disrupting isoprenoid biosynthesis. Ligand epitope mapping of compound 29 with Bt IspE indicated that hydrogens on 2,4-dichlorophenyl group have higher proximity to the surface of the enzyme than hydrogens on the pyrazolopyrimidine ring. [ABSTRACT FROM AUTHOR]

CXCR4 is a GPCR involved in leukocyte trafficking. Small molecule antagonists of the receptor may treat inflammatory disease, cancer and HIV. Here we probe the binding of a tetrahydroisoquinoline-based antagonist (TIQ-10) to CXCR4 using saturation transfer double-difference (STDD) NMR. STDD spectra were acquired using extracts from Chinese Hamster Ovary cells expressing membrane-embedded CXCR4. The experiments demonstrate competitive binding between TIQ-10 and established antagonists and provide the TIQ-10 – CXCR4 binding epitope. Molecular modeling of TIQ-10 into the binding pocket provides a pose consistent with STDD-derived interactions. This study paves the way for future investigations of GPCR-ligand interactions in a biological milieu for use in chemical biology, biochemistry, structural biology, and rational drug design. [ABSTRACT FROM AUTHOR]

Natural organic matter (NOM) plays a critical role in regulating the transport and the fate of organic contaminants in the environment. NMR spectroscopy is a powerful technique for the investigation of the sorption and binding mechanisms between NOM and pollutants, as well as their mutual chemical transformations. Despite NMR relatively low sensibility but due to its wide versatility to investigating samples in the liquid, gel, and solid phases, NMR application to environmental NOM-pollutants relations enables the achievement of specific and complementary molecular information. This report is a brief outline of the potentialities of the different NMR techniques and pulse sequences to elucidate the interactions between NOM and organic pollutants, with and without their labeling with nuclei that enhance NMR sensitivity. Copyright © 2015 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

UDP-galactopyranose mutase (UGM) is an enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall, and is essential for the growth and survival of the organism. A micromolar inhibitor developed by tetrafluorination of the UGM substrate has been previously studied by saturation transfer difference (STD) NMR spectroscopy. To elucidate the bioactive conformation of the inhibitor bound to UGM, we employ molecular dynamics (MD) simulations to construct a structural model. The MD model is subsequently validated by a good fit between experimental and theoretical STD effects, the latter calculated by a complete relaxation and conformational exchange matrix (CORCEMA) analysis. This structural model is used to explain the relative binding affinities of the inhibitor and the parent substrate. [ABSTRACT FROM AUTHOR]

Abstract: Fluorinated, arylsulfone-based inhibitors of Matrix Metalloproteinases (MMP) have been used, in the [18F]-radiolabelled version, as radiotracers targeted to MMP-2/9 for Positron Emission Tomography (PET). Although they showed acceptable tumour uptake, specificity was rather low. To get further insights into the reason of low specificity, the binding interaction of these compounds with Human Serum Albumin (HSA) has been investigated. 19F NMR spectroscopy showed that all compounds considered partition between multiple HSA binding sites, being characterized by either slow-exchange kinetics (with K a in the order of 105 M−1) and fast-exchange kinetics (with K a in the order of 104 M-1). For 2-(2-(4′-(2-fluoroethoxy)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1a) and 2-(2-(4′-(2-fluoroacetamido)biphenyl-4-ylsulfonyl)phenyl)acetic acid (1c), these slow and fast-exchanging binding sites could be mapped to Sudlow's site I and II, respectively. It is shown that high affinity albumin binding constitutes a theoretical limitation for the specificity achievable by MMP-inhibitors as MMP-targeted PET tracers in cancer imaging, because albumin accumulating aspecifically in tumours lowers the binding potential of radiotracers. [Copyright &y& Elsevier]

Highlights: [•] Applications of modern NMR screening techniques to the pharmaceutical target Chk1. [•] The ligand GF109203X was found to be the most potent inhibitor. [•] Epitope mapping studies by NMR were performed. [•] Specific NMR experiments were used to characterize the bound conformation of GF109203X. [•] A 3D model for the bound form of GF109203X in solution was proposed. [ABSTRACT FROM AUTHOR]

Objectives The main function of influenza neuraminidase (NA) involves enzymatic cleavage of sialic acid from the surface of host cells resulting in the release of the newly produced virions from infected cells, as well as aiding the movement of virions through sialylated mucus present in the respiratory tract. However, there has previously been little information on the binding affinity of different forms of sialylated glycan with NA. Our objectives were then to investigate both sialic acid binding and cleavage of neuraminidase at an atomic resolution level. Design Nuclear magnetic resonance (NMR) spectroscopy was used to investigate pH and temperature effects on binding and cleavage as well as to interrogate the selectivity of human-like or avian-like receptors for influenza neuraminidase N1 derived from a range of different influenza virus strains including human seasonal H1N1, H1N1pdm09 and avian H5N1. Results We demonstrated that an acidic pH and physiological temperature are required for efficient NA enzymatic activity; however a change in the pH had a minimum effect on the NA-sialic acid binding affinity. Our data comparing α-2,3- and α-2,6-sialyllactose indicated that the variation in neuraminidase activity on different ligands correlated with a change in binding affinity. Epitope mapping of the sialylglycans interacting with NAs from different viral origin showed different binding profiles suggesting that different binding conformations were adopted. Conclusions The data presented in this study demonstrated that physicochemical conditions (pH in particular) could affect the NA enzymatic activity with minor effect on ligand binding. NA cleavage specificity seemed to be associated with a difference in binding affinity to different ligands, suggesting a relationship between the two events. These findings have implications regarding the replication cycle of influenza infection in the host where different sialidase activities would influence penetration through the respiratory mucin barrier and the release of the newly generated virus from the infected cells. [ABSTRACT FROM AUTHOR]

Physiological processes are mainly controlled by intermolecular recognition mechanisms involving protein-protein and protein-ligand (low molecular weight molecules) interactions. One of the most important tools for probing these interactions is high-field solution nuclear magnetic resonance (NMR) through protein-observed and ligand-observed experiments, where the protein receptor or the organic compounds are selectively detected. NMR binding experiments rely on comparison of NMR parameters of the free and bound states of the molecules. Ligand-observed methods are not limited by the protein molecular size and therefore have great applicability for analysing protein-ligand interactions. The use of these NMR techniques has considerably expanded in recent years, both in chemical biology and in drug discovery. We review here three major ligand-observed NMR methods that depend on the nuclear Overhauser effect-transferred nuclear Overhauser effect spectroscopy, saturation transfer difference spectroscopy and water-ligand interactions observed via gradient spectroscopy experiments-with the aim of reporting recent developments and applications for the characterization of protein-ligand complexes, including affinity measurements and structural determination. [ABSTRACT FROM AUTHOR]

1H solution NMR spectroscopy is used synergistically with 3D crystallographic structures to map experimentally significant hydrophobic interactions upon substrate binding in solution under thermodynamic equilibrium. Using saturation transfer difference spectroscopy ( STD NMR), a comparison is made between wild-type xylanase XT6 and its acid/base catalytic mutant E159Q - a non-active, single-heteroatom alteration that has been previously utilized to measure binding thermodynamics across a series of xylooligosaccharide-xylanase complexes [Zolotnitsky et al. (2004) Proc Natl Acad Sci USA 101, 11275-11280). In this study, performing STD NMR of one substrate screens binding interactions to two proteins, avoiding many disadvantages inherent to the technique and clearly revealing subtle changes in binding induced upon mutation of the catalytic Glu. To visualize and compare the binding epitopes of xylobiose-xylanase complexes, a ' SASSY' plot (saturation difference transfer spectroscopy) is used. Two extraordinarily strong, but previously unrecognized, non-covalent interactions with H2-5 of xylobiose were observed in the wild-type enzyme but not in the E159Q mutant. Based on the crystal structure, these interactions were assigned to tryptophan residues at the −1 subsite. The mutant selectively binds only the β-xylobiose anomer. The 1H solution NMR spectrum of a xylotriose-E159Q complex displays non-uniform broadening of the NMR signals. Differential broadening provides a unique subsite assignment tool based on structural knowledge of face-to-face stacking with a conserved tyrosine residue at the +1 subsite. The results obtained herein by substrate-observed NMR spectroscopy are discussed further in terms of methodological contributions and mechanistic understanding of substrate-binding adjustments upon a charge change in the E159Q construct. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) methods at the Institute of Molecular Pharmacy (IMP) provide crucial information in several fundamental themes in drug discovery. Assignments of complex carbohydrates are obtained to a high degree of completion and accuracy, thereby accelerating their identity. Conformational analysis of ligands in their unbound state reveals to what extent the steric considerations, essential for minimizing entropy losses upon binding, are optimized. Small libraries of compounds are screened for their binding to receptors. Promising ligands are analyzed in the presence of their receptor to determine the geometric arrangements in the bound state, as well as the functional groups responsible for the observed binding.

Abstract: In this study we explore the relationship between solution pH and the distribution of the binding interactions at different domains of a dissolved humic acid (HA) for three xenobiotics: pentafluoroaniline (PFA), pentafluorophenol (PFP), and hexafluorobenzene (HFB). The components of HA where xenobiotic interactions occur are identified using the 1H{19F} Reverse Heteronuclear Saturation Transfer Difference (RHSTD) Nuclear Magnetic Resonance (NMR) spectroscopy experiment. At low pH, PFA and PFP interact preferentially with aromatic components of HA. Increasing pH reduces this preference. Conversely, HFB interacts with all components of HA equally, across the entire pH range. The possible roles of both aromatic-specific interactions and conformational changes of HA behind these observations are explored. It is shown that T-oriented π–π interactions at π-electron accepting HA structures are slightly stronger for PFA and PFP than for HFB. Using DOSY NMR it is shown that the pH-dependence of the interactions is correlated with changes in the conformation of the carbohydrate components of HA rather than with the aromatic components. It is argued that the observed preference for aromatic HA is caused by restricted access to the non-aromatic components of HA at low pH. These HA components form tightly bound hydrophobic domains due to strong inter- and intra-molecular hydrogen bonds. At high pH, these structures open up, making them more available for interactions with polar compounds. [Copyright &y& Elsevier]

Abstract: Zein is a protein containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids. NMR techniques were used to detect binding interactions and measure affinity between zein and three different drugs: tetracycline, amoxicillin and indomethacin. The release study of zein microparticle formulations containing any of these drugs was confronted with the affinity results, showing a remarkable correlation. The feasible methodology employed, focused in the functionality of the protein–drug interaction, can be very promising for the rational design of appropriate drug vehicles for drug delivery. [Copyright &y& Elsevier]

Abstract: This work shows that high resolution magic angle spinning (HR-MAS) can be used to elucidate ligand–receptor binding phenomena for mass-limited samples in liquid solution. With respect to conventional 5mm liquid-state NMR probe heads, HR-MAS allows for a considerable reduction (about one order of magnitude) in sample volume requirement, which represents a considerable advantage when working with biological samples present in low amount. More specifically, using a 20-μL active volume HR-MAS probe head, NMR experiments typically used to describe binding events (e.g. Saturation Transfer Difference, WaterLOGSY, Diffusion Ordered Spectroscopy) were successfully recorded. In fact, contrary to other NMR experiments (e.g. TOCSY), which require special care when performed on liquid samples by HR-MAS, no interferences due to fast sample spinning were observed here. Collectively, the results indicate that HR-MAS allows for a significant mass sensitivity enhancement (a 3-fold increase in signal-to-noise ratio per unit of measuring time and mass was obtained here), hereby improving correspondingly the detection limit of the previously mentioned NMR experiments. More importantly, because MAS removes magnetic susceptibility broadenings in the sample, a good spectral resolution can be easily achieved in HR-MAS even for slightly heterogeneous samples, such as those arising from incomplete purification. [Copyright &y& Elsevier]

Solution-state nuclear magnetic resonance (NMR) is a versatile tool for the study of binding interactions between small molecules and macromolecular targets. We applied ligand-based NMR techniques to the study of human thymidylate synthase (hTS) using known nanomolar inhibitors and a library of small molecule fragments. Screening by NMR led to the rapid identification of ligand pairs that bind in proximal sites within the cofactor-binding pocket of hTS. Screening hits were used as search criteria within commercially available sources, and a subset of catalog analogs were tested for potency by in vitro assay and binding affinity by quantitative saturation transfer difference (STD)-NMR titration. Two compounds identified by this approach possess low micromolar affinity and potency, as well as excellent binding efficiency against hTS. Relative binding orientations for both leads were modeled using AutoDock, and the most likely bound conformations were validated using experimentally derived STD-NMR binding epitope data. These ligands represent novel starting points for fragment-based drug design of non-canonical TS inhibitors, and their binding epitopes highlight important and previously unexploited interactions with conserved residues in the cofactor-binding site. [ABSTRACT FROM AUTHOR]

Abstract: We demonstrate here that pentagalloyl glucose (PGG), a main component of gallotannins, was an effective inhibitor of HSA and it exerted similar inhibitory potency to Aleppo tannin used in this study. The inhibition of HSA by PGG was found to be non-competitive and inhibitory constants of K EI =2.6 µM and K ESI =3.9 µM were determined from Lineweaver–Burk secondary plots. PGG as a model compound for gallotannins was selected to study the inhibitory mechanism and to characterize the interaction of HSA with this type of molecules. Surface plasmon resonance (SPR) binding experiments confirmed the direct interaction of HSA and PGG, and it also established similar binding of Aleppo tannin to HSA. Saturation transfer difference (STD) experiment by NMR clearly demonstrated the aromatic rings of PGG may be involved in the interaction suggesting a possible stacking with the aromatic side chains of HSA. The role of aromatic amino acids of HSA in PGG binding was reinforced by kinetic studies with the W58L and Y151M mutants of HSA: the replacement of the active site aromatic amino acids with aliphatic ones decreased the PGG inhibition dramatically, which justified the importance of these residues in the interaction. [Copyright &y& Elsevier]

Abstract: Lipopolysaccharide (LPS), the major constituent of the outer membrane of Gram-negative bacteria, is the very first site of interactions with the antimicrobial peptides. In this work, we have determined a solution conformation of melittin, a well-known membrane active amphiphilic peptide from honey bee venom, by transferred nuclear Overhauser effect (Tr-NOE) spectroscopy in its bound state with lipopolysaccharide. The LPS bound conformation of melittin is characterized by a helical structure restricted only to the C-terminus region (residues A15-R24) of the molecule. Saturation transfer difference (STD) NMR studies reveal that several C-terminal residues of melittin including Trp19 are in close proximity with LPS. Isothermal titration calorimetry (ITC) data demonstrates that melittin binding to LPS or lipid A is an endothermic process. The interaction between melittin and lipid A is further characterized by an equilibrium association constant (K a) of 2.85×106 M−1 and a stoichiometry of 0.80, melittin/lipid A. The estimated free energy of binding (ΔG 0), −8.8 kcal mol−1, obtained from ITC experiments correlates well with a partial helical structure of melittin in complex with LPS. Moreover, a synthetic peptide fragment, residues L13-Q26 or mel-C, derived from the C-terminus of melittin has been found to contain comparable outer membrane permeabilizing activity against Escherichia coli cells. Intrinsic tryptophan fluorescence experiments of melittin and mel-C demonstrate very similar emission maxima and quenching in presence of LPS micelles. The Red Edge Excitation Shift (REES) studies of tryptophan residue indicate that both peptides are located in very similar environment in complex with LPS. Collectively, these results suggest that a helical conformation of melittin, at its C-terminus, could be an important element in recognition of LPS in the outer membrane. [Copyright &y& Elsevier]

The esterolytic catalytic antibody (catAb) has the positive charged region interacting with the carbonyl group of the ester substrate. To examine how such a region interacts with the substrate, we compared the catAb with the non-catalytic antibody (non-catAb) for interaction with the non-cleavable amide substrate (a mimic of the ester substrate) and the two end products. Surface plasmon resonance (SPR) analysis revealed that the amide substrate gave the equivalent Kd values for the two antibodies, whereas both the on-rate and off-rate of the catAb were five-times lower than those of the non-catAb. In agreement with SPR analysis, saturation transfer difference (STD) NMR spectroscopy detected the STD signals only between the catAb and one of the product, suggesting the slower exchange rates of the amide substrate in the catAb as compared with the mixing times, whereas it was not the case with the non-catAb. Transferred nuclear Overhauser effect NMR spectroscopy showed the negative signals for only between the non-catAb and the amide substrate or the product, again suggesting the lower off-rates of the catAb as compared with the mixing times. The decreased interaction rates should be the primary consequence of the positively charged region in the combining site in the catAb. [ABSTRACT FROM PUBLISHER]