The Nuclear Overhauser Effect in the Lead Identification Process Pharmacophore Models

Leone, Marilisa; Freeze, Hudson H.; Chan, Chui Sien; Pellecchia, Maurizio

doi:10.2174/157016306778108884

Cited by 21 publications

(17 citation statements)

References 77 publications

(150 reference statements)

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“…For the formulas of the compounds cf. Table 1 A number of protocols have been published that have applied NMR experiments to screen fragment libraries [19,22,23,[32][33][34][35][36][37][38][39]. To our knowledge, the combination of STD NMR and spin-lock filtered NMR experiments as a "readout system" for screening has not been reported before.…”

Section: Resultsmentioning

confidence: 99%

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

Rademacher¹,

Landström

Sindhuwinata³

et al. 2010

Glycoconj J

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A substantial body of work has been devoted to the design and synthesis of glycosyltransferase inhibitors. A major obstacle has always been the demanding chemistry. Therefore, only few potent and selective inhibitors are known to date. Glycosyltransferases possess two distinct binding sites, one for the donor substrate, and one for the acceptor substrate. In many cases binding to the donor site is well defined but data for acceptor binding is sparse. In particular, acceptor binding sites are often shallow, and in many cases the dimensions of the binding pocket are not well defined. One approach to glycosyltransferase inhibitors is to chemically link donor site and acceptor site ligands to generate high affinity binders. Here, we describe a novel approach to identify acceptor site ligands from a fragment library. We have chosen human blood group B galactosyltransferase (GTB) as a biologically important model target. The approach utilizes a combination of STD NMR, spin-lock filtered NMR experiments and surface plasmon resonance measurements. Following this route we have identified molecular fragments from a fragment library that bind to the acceptor site of GTB with affinities of the order of a natural acceptor substrate. Unlike natural substrates these fragments allow for straightforward chemical modifications and, therefore will serve as scaffolds for potent GTB inhibitors. In general, the approach described is applicable to any glycosyltransferase and may assist in the development of novel glycosyltransferase inhibitors.

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Section: Resultsmentioning

confidence: 99%

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

Rademacher¹,

Landström

Sindhuwinata³

et al. 2010

Glycoconj J

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“…This approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. The trNOE NMR technique is particularly useful in the identification of a moderate, ligand-binding protein as well as the determination of a bound conformation of the ligand through quantitative analysis of the intramolecular trNOEs (U46619-PGIS, [38][39][40]). The current study is the first proven experiment, which used advanced NMR techniques for the characterization of TP binding to the PGH 2 mimic (Figures 3-6), all of which has been made possible through the successful large-scale expression and purification of the full length and active TP protein (Figure 2).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the prostaglandin H2 mimic: Binding to the purified human thromboxane A2 receptor in solution

Ruan

Wijaya

Cervantes

et al. 2008

Archives of Biochemistry and Biophysics

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For decades, the binding of prostaglandin H 2 (PGH 2 ) to multiple target proteins of unrelated protein structures which mediate diverse biological functions has remained a real mystery in the field of eicosanoid biology. Here, we report that the structure of a PGH 2 mimic, U46619, bound to the purified human TP, was determined and compared with that of its conformation bound to the COXdownstream synthases, prostacyclin synthase (PGIS) and thromboxane A 2 synthase (TXAS). Active human TP protein, glycosylated and in full length, was expressed in Sf-9 cells using a baculovirus (BV) expression system and then purified to near homogeneity. The binding of U46619 to the purified receptor in a nonionic detergent-mimicked lipid environment was characterized by high-resolution NMR spectroscopy. The conformational change of U46619, upon binding to the active TP, was evidenced by the significant perturbation of the chemical shifts of its protons at H3 and H4 in a concentration-dependent manner. The detailed conformational changes and 3D structure of U46619 from the free form to the TP-bound form were further solved by 2D 1 H NMR experiments using a transferred NOE (trNOE) technique. The distances between the protons of H11 and H18, H11 and H19, H15 and H18, and H15 and H19 in U46619 were shorter following their binding to the TP in solution -down to within 5 Å, which were different than that of the U46619 bound to PGIS and U44069 (another PGH 2 mimic) bound to TXAS. These shorter distances led to further separation of the U46619 α and ω chains, forming a unique "rectangular" shape. This enabled the molecule to fit into the ligand-binding site pocket of a TP model, in which homology modeling was used for the transmembrane (TM) domain, and NMR structures were used for the extramembrane loops. The proton perturbations and 3D conformations in the TP-bound U46619 were different with that of the PGH 2 mimics bound to PGIS and TXAS. The studies indicated that PGH 2 can adopt multiple conformations in solution to satisfy the specific and unique shapes to fit the different binding pockets in the TP receptor and COX-downstream enzymes. The results also provided sufficient information for speculating the molecular basis of how PGH 2 binds to multiple target proteins even though unrelated in their protein sequences.

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“…Another method often used to identify second-site binders is based on the so-called inter-ligand NOE (ILOE experiments) (Li et al 1999, Leone et al 2006). Using this method, a library is screened in the presence of high concentrations of first-site binders and intermolecular ligand-ligand NOEs are detected in NOESY-type experiments.…”

Section: Generation Of Lead Compoundsmentioning

confidence: 99%

Fragment-based drug discovery using NMR spectroscopy

2013

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Nuclear magnetic resonance (NMR) spectroscopy has evolved into a powerful tool for fragment-based drug discovery over the last two decades. While NMR has been traditionally used to elucidate the three-dimensional structures and dynamics of biomacromolecules and their interactions, it can also be a very valuable tool for the reliable identification of small molecules that bind to proteins and for hit-to-lead optimization. Here, we describe the use of NMR spectroscopy as a method for fragment-based drug discovery and how to most effectively utilize this approach for discovering novel therapeutics based on our experience.

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The Nuclear Overhauser Effect in the Lead Identification Process Pharmacophore Models

Cited by 21 publications

References 77 publications

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

NMR-based exploration of the acceptor binding site of human blood group B galactosyltransferase with molecular fragments

Characterization of the prostaglandin H2 mimic: Binding to the purified human thromboxane A2 receptor in solution

Fragment-based drug discovery using NMR spectroscopy

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