Exploration of the ligand binding site of the human 5‐HT<sub>4</sub> receptor by site‐directed mutagenesis and molecular modeling

Mialet, Jeanne; Dahmoune, Yamina; Lezoualc’h, Frank; Berque‐Bestel, Isabelle; Eftekhari, Pierre; Hoebeke, Johan; Sicsic, Sames; Langlois, Michel; Fischmeister, Rodolphe

doi:10.1038/sj.bjp.0703356

Cited by 52 publications

(62 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…One residue, Phe110 3.28 , is in TM3 and is predicted to be in the ancillary binding pocket (Teeter et al, 1994;Neve et al, 2003), and a second residue is in TM6 (His394 6.55 ). Five residues are in TM7, with three of them (Tyr409 7.35 , Thr413 7.39 , and Tyr417 7.43 ) predicted to be in the ancillary binding pocket (Table 1) D 2 -Y417W had substantially decreased affinity for most D 2 -selective antagonists, consistent with data from other receptors implicating residue 7.43 in ligand binding (Matsui et al, 1995;Cavalli et al, 1996;Roth et al, 1997;Mialet et al, 2000). Substituted benzamides (sulpiride, raclopride, tropapride, and YM-09151-02) were particularly sensitive to this mutation, with their binding reduced 60-to 200-fold.…”

Section: Resultssupporting

confidence: 74%

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

et al. 2005

View full text Add to dashboard Cite

To test the hypothesis that pharmacological differentiation between D 1 and D 2 dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D 2 receptor to the corresponding aligned residues in the D 1 receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D 2 receptors and 11 mutant D 1 receptors for D 1 -and D 2 -selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D 1 receptor mutant W99 3.28 F showed dramatically increased affinity for several D 2 -selective antagonists, particularly spiperone (225-fold), whereas the D 2 receptor mutant Y417 7.43 W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold).The binding of the,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V 5.39 caused modest but consistent and reciprocal changes in affinity of the receptors for D 1 and D 2 -selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D 1 /D 2 receptorselective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets. Dopamine modulates diverse biological functions, including movement, endocrine function, and memory formation, through activation of five distinct dopamine receptor subtypes that belong to the G protein-coupled receptor (GPCR) superfamily and are grouped into two subfamilies, D 1 -like dopamine receptors and D 2 -like dopamine receptors, based on their structure, pharmacology, and transduction pathways. The D 1 and D 2 receptors are the most abundant dopamine receptor subtypes and are most similar to the traditional pharmacologically defined D 1 and D 2 receptors (Kebabian and Calne, 1979). The D 1 receptor has a long carboxyl terminus and a short third intracellular loop, couples to the adenylate cyclase stimulatory G proteins G␣ s/olf , and stimulates cyclic AMP accumulation. In contrast, the D 2 receptor has a short carboxyl terminus and a long third intracellular loop, couples to the pertussis toxin-sensitive G proteins G␣ i/o , inhibits cyclic AMP accumulation, and also modulates a variety of G␤␥-regulated effectors such as calcium and potassium ion channels, mitogen-activated protein kinases, and phospholipases (Neve et al., 2004). D 1 and D 2 receptor-selective agonists and antagonists are current or potential therapeutic drugs for treatment o...

show abstract

Section: Resultssupporting

confidence: 74%

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

et al. 2005

View full text Add to dashboard Cite

show abstract

“…A binding pocket was easily identified on the extracellular side of the model, located between helices 3, 5, 6, and 7, in agreement with mutation data (45). Fig.…”

Section: Resultssupporting

confidence: 73%

G protein-coupled receptors:In silicodrug discovery in 3D

Becker¹,

Marantz²,

Shacham³

et al. 2004

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

164

158

View full text Add to dashboard Cite

The application of structure-based in silico methods to drug discovery is still considered a major challenge, especially when the x-ray structure of the target protein is unknown. Such is the case with human G protein-coupled receptors (GPCRs), one of the most important families of drug targets, where in the absence of x-ray structures, one has to rely on in silico 3D models. We report repeated success in using ab initio in silico GPCR models, generated by the PREDICT method, for blind in silico screening when applied to a set of five different GPCR drug targets. More than 100,000 compounds were typically screened in silico for each target, leading to a selection of <100 ''virtual hit'' compounds to be tested in the lab. In vitro binding assays of the selected compounds confirm high hit rates, of 12-21% (full dose-response curves, K i < 5 M). In most cases, the best hit was a novel compound (New Chemical Entity) in the 1-to 100-nM range, with very promising pharmacological properties, as measured by a variety of in vitro and in vivo assays. These assays validated the quality of the hits as lead compounds for drug discovery. The results demonstrate the usefulness and robustness of ab initio in silico 3D models and of in silico screening for GPCR drug discovery.modeling ͉ in silico screening ͉ structure-based G protein-coupled receptors (GPCRs) are membraneembedded proteins, responsible for communication between the cell and its environment (1). As a consequence, many major diseases, such as hypertension, cardiac dysfunction, depression, anxiety, obesity, inflammation, and pain, involve malfunction of these receptors (2), making them among the most important drug targets for pharmacological intervention (3-5). Thus, whereas GPCRs are only a small subset of the human genome, they are the targets for Ϸ50% of all recently launched drugs (6). As targets of paramount importance, it is expected that drug discovery for GPCRs would benefit from the introduction of computational methodologies (7), especially as these methods can be used in conjunction with such experimental methods as high-throughput screening (8, 9), NMR, and crystallography (10).Unfortunately, GPCRs, like other membrane-embedded proteins, have characteristics that make their 3D structure extremely difficult to determine experimentally. To date, the only GPCR for which a 3D structure was determined by x-ray crystallography is bovine rhodopsin (11), which is unique among GPCRs in that its ligand, retinal, is covalently bound and that it responds to light rather than to ligand binding. Hence, in the case of GPCRs, the limited availability of structural data has forced the computational design of ligands to heavily rely on ligand-based techniques. Indeed, for many GPCRs, the natural ligand can provide a good starting point, leading to useful pharmacophore models that can be used for identifying lead structures with novel scaffolds (6). These methods have been successfully applied for the discovery of peptide agonists to the somatostatin receptor (12) and for...

show abstract

“…The indole ring of 5-HT expands toward TMs 5 and 6 to hydrogen bond the key Ser5.43 and Asn6.55 side chains (Fig. 5A), as proposed by site-directed mutagenesis (Mialet et al, 2000), without forming a direct interaction with Trp6.48. This mode of binding explains the fact that T3.36A slightly influences both the binding affinity for 5-HT and R*-5-HT (E max and EC 50 values) and that W6.48A does not modify the binding affinity or E max value.…”

Section: Resultsmentioning

confidence: 99%

“…Computational models of the complexes between 5-HT 4 R and BIMU8 or zacopride. In these models, the protonated moiety of the ligands interact with Asp3.32; the 5-OH and NH moieties of 5-HT hydrogen bond the key Ser5.43 and Asn6.55 side chains (A) as proposed by site-directed mutagenesis (Mialet et al, 2000); the -C(CH 3 ) 2 and -Cl moieties of BIMU8 and (S)-zacopride, respectively, are located in a small hydrophobic cavity between TMs 3 to 5 (B and C) and the -NH 2 group of (S)-zacopride hydrogen bonds Ser5.43 (B). BIMU8 and (S)-zacopride form an intramolecular hydrogen bond between the -NH amide and a carbonyl or methoxy group, respectively.…”

Section: Discussionmentioning

confidence: 99%

Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Pellissier¹,

Sallander²,

Campillo³

et al. 2009

Mol Pharmacol

View full text Add to dashboard Cite

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R*. However, different structural active R* complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R* states of 5-hydroxytryptamine-4 (5-HT 4 ) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R*basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive gϪ to active gϩ and Trp6.48 from inactive gϩ to active t

show abstract

Exploration of the ligand binding site of the human 5‐HT₄ receptor by site‐directed mutagenesis and molecular modeling

Cited by 52 publications

References 30 publications

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

G protein-coupled receptors:In silicodrug discovery in 3D

Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Contact Info

Product

Resources

About

Exploration of the ligand binding site of the human 5‐HT4 receptor by site‐directed mutagenesis and molecular modeling

Cited by 52 publications

References 30 publications

Structural Determinants of Pharmacological Specificity Between D1and D2Dopamine Receptors

Structural Determinants of Pharmacological Specificity Between D1and D2Dopamine Receptors

G protein-coupled receptors:In silicodrug discovery in 3D

Conformational Toggle Switches Implicated in Basal Constitutive and Agonist-Induced Activated States of 5-Hydroxytryptamine-4 Receptors

Contact Info

Product

Resources

About

Exploration of the ligand binding site of the human 5‐HT₄ receptor by site‐directed mutagenesis and molecular modeling

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors

Structural Determinants of Pharmacological Specificity Between D₁and D₂Dopamine Receptors