Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments

Behling, Ronald W.; Yamane, T; Navon, Gil; Sammon, M.; Jelinski, Lynn W.

doi:10.1016/s0006-3495(88)83175-8

Cited by 23 publications

(21 citation statements)

References 31 publications

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“…The spectrum of hydroxamic acid I-1 with stromelysin shows a single set of broadened resonances and a slight change in the chemical shifts, indicative of a fast binding equilibrium between enzyme and inhibitor. Another very sensitive indication of ligand binding with the enzyme can be obtained from selective proton-relaxation measurements (Tisel) (22). Relaxation time measurements (Tisel) for inhibitor I-1 in both the free state and complexed to stromelysin are given in Table 1.…”

Section: Resultsmentioning

confidence: 99%

Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

Gonnella¹,

Bohacek²,

Zhang³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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The transferred nuclear Overhauser effect has been used to determine the biologically active conformations of two stromelysin inhibitors. Both inhibitors used in this study were hydroxamic acids generated via chemical synthesis. These structures, representing the conformation of each inhibitor bound to stromelysin, superimposed with excellent agreement.The study also provided information on the shape and orientation of the S2' and Si' pockets of the enzyme relative to thermolysin. Comparisons were made between stromelysin and thermolysin inhibitors to critically examine thermolysin as a template for stromelysin-inhibitor design. The enzyme-bound conformations of these stromelysin inhibitors were determined for use as a template in conformationally restricted drug design.Stromelysin 1 is a zinc metalloendoproteinase that is secreted by synoviocytes and articular chondrocytes in response to inflammatory mediators such as interleukin 1 (1). This enzyme is believed to cause the destruction of cartilage proteoglycans associated with osteo-and rheumatoid arthritis (2). Stromelysin has also been implicated in the acceleration of procollagenase activation, thereby enhancing collagenase-induced cartilage degradation and contributing to the progression of the arthritic disease state (3). Currently there are no therapies available to treat the cartilage degradation that occurs in these arthritic diseases. Because of the link of stromelysin to cartilage degradation, it is anticipated that the design of stromelysin inhibitors could result in the next major class of drugs for the treatment of arthritis.Stromelysin is secreted from cells as a 55-to 57-kDa monomeric proenzyme that, upon activation, loses 80 amino acids to yield a 45-kDa mature enzyme. Recently a Cterminally truncated form of the proenzyme was reported. This 28-kDa protein has been shown to activate identically to the wild type, yielding a 19-kDa protein with binding properties very similar to full-length stromelysin (4, 5). The threedimensional NMR structure of the inhibited catalytic domain of truncated human stromelysin 1 has been solved (6); however, to date the coordinates of these structures have not been released.As part of a rational drug-design strategy, it is important to obtain structural knowledge of the enzyme-inhibitor complex. In particular, it is necessary to know the shape of the binding pocket, as well as the conformation of the interacting inhibitor. Most NMR methods that allow complete structure elucidation of an enzyme-inhibitor complex require significant amounts of enzyme as well as isotopic labeling of the enzyme or inhibitor (7,8). The transferred nuclear Overhauser effect (TR-NOE), however, provides a different approach that focuses on the conformation of the inhibitor in the bound state. This experiment involves the generation of cross-relaxation effects between two protons in the bound state that are subsequently transferred to the free state via chemical exchange (9). The technique has an advantage over other NMR methods in t...

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Section: Resultsmentioning

confidence: 99%

Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

Gonnella¹,

Bohacek²,

Zhang³

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…The additional NMR peaks in this spectrum arise from the lipid (asolectin) in the vesicles and from mobile amino acid side chains on the protein. [The amount of asolectin was minimized by eluting the purified receptors from the affinity column without asolectin in the elution buffer (8).] The corresponding two-dimensional NOE spectrum of AcCho binding to the AcChoR is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The receptor was purified from the electroplax organs of T. californica as described (8). We have shown that under the conditions used in this study, AcCho binds to the AcChoR and that selective proton relaxation measurements are very sensitive to this binding.…”

Section: Methodsmentioning

confidence: 95%

See 1 more Smart Citation

Conformation of acetylcholine bound to the nicotinic acetylcholine receptor.

Behling

Yamane²,

Navon³

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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We report here the biologically active conformation ofacetylcholine when bound to the high-affinity state of the receptor from Torpedo cakfornica. The acetylcholine conformation was determined in the free and bound states by proton NMR two-dimensional nuclear Overhauser effects. In agreement with x-ray crystallographic data, acetylcholine in solution has an extended conformation with an average distance between the acetyl methyl and choline methyl protons of =5 A. When bound to the acetylcholine receptor, acetylcholine adopts a conformation where the acetyl methyl group is dose (3.3 A) to the methyl groups of the choline moiety. This bent conformation places the oxygens adjacent to one another and allows the methyl groups to form an uninterrupted hydrophobic surface over the rest of the acetylcholine molecule. The significant difference between the fre-and bound-state conformations implies that structure-activity studies based solely on molecular modeling strategies must be approached with caution. To understand the interactions of biologically interesting molecules, it is necessary to know the shape ofthe interaction sites as well as the conformations of the interacting molecules. We report here the biologically active conformation of acetylcholine (AcCho) when bound to the high-affinity (desensitized) state ofthe acetylcholine receptor (AcChoR) from Torpedo californica. The exact location ofthe AcCho binding site on the a-subunits of the AcChoR is still unknown, although it has recently been localized to residues 158-216 by several groups using a variety oftechniques (1-4). In contrast to the uncertainty in the AcCho binding site, crystal structures are available for AcCho and other agonists that also bind to the AcChoR (5-7). However, we show here that the conformation of AcCho in its receptor-bound state is distinctly different from its conformation in solution and in the crystalline solid state. MATERIALS AND METHODSAll experiments used purified AcChoR in asolectin vesicles. The receptor was purified from the electroplax organs of T. californica as described (8). We have shown that under the conditions used in this study, AcCho binds to the AcChoR and that selective proton relaxation measurements are very sensitive to this binding. [Under these conditions the ratio of Kd (acetylcholine)/Kd (nicotine) is 0.14.] Conditions (see text) have been determined to distinguish between the contributions from site-specific binding ofAcCho to the AcChoR and contributions from nonspecific interactions of AcCho both with the lipid and with other areas of the receptor (8).All proton NMR spectra were acquired on a 360-MHz Bruker AM series spectrometer with an Aspect 3000 computer. The two-dimensional nuclear Overhauser effect (NOE) data sets were obtained in the phase-sensitive mode (9) and were processed with Dennis Hare's Fourier transform NMR program. The temperature in all experiments was held at 250C to within PC with the Bruker variable temperature unit by passing heated nitrogen gas through the probe.The conf...

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“…One of the first methodologies for the use of selective spin-lattice relaxation times on the binding of ligands to biomolecular targets was the study of agonist binding to the acetylcholine receptor of Torpedo californica [61]. In that work, considering that the concentration of the target in relation to the ligand ([L]>>[T]), it was used the equation 1/T 1obs -1/T 1free = f/(T 1 S bound +t bound ), where f is the fraction of the bound ligand and t bound is the lifetime of the ligand-target complex [62].…”

Section: Equationmentioning

confidence: 99%

Spin-Lattice Relaxation Time in Drug Discovery and Design

Figueroa‐Villar

Tinoco²

2009

CTMC

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NMR is one of the most powerful techniques for ligand-biomolecule interaction studies and drug screening and design. There are several methods that are strongly used, including chemical shift perturbation (CSP), saturation transfer difference (STD) and diffusion coefficients. However, one of the most useful and easy to apply NMR parameters in medicinal chemistry studies is the spin-lattice relaxation data, which can be employed to investigate the strength and topology of intermolecular interactions, such as drug-drug, drug-protein, drug-DNA, drug-micelle (or vesicle) and biomolecule-biomolecule interactions. This review deals with the newest applications of T(1) in different studies of interest for drug design and evaluation.

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Measuring relative acetylcholine receptor agonist binding by selective proton nuclear magnetic resonance relaxation experiments

Cited by 23 publications

References 31 publications

Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

Bioactive conformation of stromelysin inhibitors determined by transferred nuclear Overhauser effects.

Conformation of acetylcholine bound to the nicotinic acetylcholine receptor.

Spin-Lattice Relaxation Time in Drug Discovery and Design

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