Assessment of the Roles of Serines 5.43(239) and 5.46(242) for Binding and Potency of Agonist Ligands at the Human Serotonin 5-HT2A Receptor

Abstract

We assessed the relative importance of two serine residues located near the top of transmembrane helix 5 of the human 5-HT 2A receptor, comparing the wild type with S5.43(239)A or S5.46(242)A mutations. Using the ergoline lysergic acid diethylamide (LSD), and a series of substituted tryptamine and phenethylamine 5-HT 2A receptor agonists, we found that Ser5.43(239) is more critical for agonist binding and function than Ser5.46(242). Ser5.43(239) seems to engage oxygen substituents at either the 4-or 5-position of tryptamine ligands and the 5-position of phenylalkylamine ligands. Even when a direct binding interaction cannot occur, our data suggest that Ser5.43(239) is still important for receptor activation. Polar ring-substituted tryptamine ligands also seem to engage Ser5.46(242), but tryptamines lacking such a substituent may adopt an alternate binding orientation that does not engage this residue. Our results are consistent with the role of Ser5.43(239) as a hydrogen bond donor, whereas Ser5.46(242) seems to serve as a hydrogen bond acceptor. These results are consistent with the functional topography and utility of our in silico-activated homology model of the h5-HT 2A receptor. In addition, being more distal from the absolutely conserved Pro5.50, a strong interaction with Ser5.43(239) may be more effective in straightening the kink in helix 5, a feature that is possibly common to all type A GPCRs that have polar residues at position 5.43.The serotonin 2A (5-HT 2A ) receptor is a member of the monoamine family A type GPCRs. It seems to play an essential role in cognition, memory, and consciousness (Nichols, 2004). Although no crystal structure exists for this or any of the monoamine GPCRs, advances in X-ray crystallography have provided structures for the dark-adapted inverse agonist form of bovine rhodopsin (Palczewski et al., 2000;Li et al., 2004), creating opportunities to develop homology models of general GPCR structure. Given sufficient sequence similarity/identity, the accuracy and reliability of comparative homology models is generally believed to be superior to de novo models (Baker and Sali, 2001) As an example of this approach, the in silico-activated homology model of the h5-HT 2A receptor developed in our laboratory has been used to predict several ligand-receptor interactions. Our results thus far have provided qualitative support for the model and have identified directions for further investigation of the structure-activity relationships of agonist ligands (Parrish et al., 2005;Braden et al., 2006; McLean et al., 2006a,b). Although such homology receptor models must be viewed with caution, they can often be validated by receptor mutagenesis experiments, along with complementary changes in ligand structure.To refine our homology model and provide empirical support for the receptor functional topography it defines, we extended results derived from previous mutagenesis studies Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10....

Molecular Pharmacology and Ligand Docking Studies Reveal a Single Amino Acid Difference between Mouse and Human Serotonin 5-HT2A Receptors That Impacts Behavioral Translation of Novel 4-Phenyl-2-dimethylaminotetralin Ligands

“…Affinity and Function of (1) 3 H]ketanserin at m5-HT2A A5.46S and h5-HT2A S5.46A receptors were 1.2 6 0.3 and 1.6 6 0.4 nM, respectively, closely matching many reported literature values (Almaula et al, 1996a;Braden and Nichols, 2007) and not statistically different from 5-HT2A WT receptors (P . 0.05).…”

“…Thus, the interaction of the (1)-6-OH-7-Cl-PAT 6-OH moiety with S5.46 was crucial for its binding, whereas this amino acid hardly impacted binding of (2)-6-OH-7-Cl-PAT. This observation is unusual compared with most other 5-HT2A-targeting compounds reported (Almaula et al, 1996a;Braden and Nichols, 2007;Shan et al, 2012), including DOI. For example, of more than 20 compounds tested, only the ergot derivatives, e.g., ergonovine, showed major losses in affinity at the human S5.46A 5-HT2A receptor (Almaula et al, 1996a;Braden and Nichols, 2007).…”

Psychedelics

“…Although some of the compounds studied by Martí-Solano et al (2015) did have a high bias for PI signaling over AA release, the basis for their structural conclusions must be considered suspect because of significant flaws in their molecular modeling. For example, Braden and Nichols (2007) showed that TM5 serine 239 (5.43) in the 5-HT 2A receptor was critical for high affinity and potency of 4-or 5-oxygenated tryptamines, potentially serving as a hydrogen bond donor to the ligand, but Martí-Solano et al (2015) essentially ignore its role in their molecular dynamics simulations. In addition, they (incorrectly) state that "S5.43 is able to establish indirect interactions with different serotonergic agonists," citing Psychedelics Braden and Nichols (2007).…”

Structure–activity relationships of serotonin 5‐HT2A agonists

“…Although indoles engage Ser242 within the human 5-HT 2A receptor, the dimethoxy-substituted phenethylamines do not. 80 The bicyclic indole nucleus of serotonin also is larger than the phenyl ring of the phenethylamines, and it seems possible that the hydrophobic 4-substituent of the phenethylamines acts as a sort of wedge or spacer to fill more fully the binding cavity that has evolved to accommodate the relatively larger indole nucleus of serotonin.…”

“…Construction of a homology model of the 5-HT 2A receptor from this in silico-activated rhodopsin template, and virtual docking with various 5-HT 2A agonist ligands, allowed the formulation of a number of hypothesis about the functional topography of the ligand binding site, which were subsequently validated by site-directed mutagenesis and ligand testing. 80,106 In addition, virtual docking studies allowed the design of new agonist ligands as well as the prediction of their more active enantiomers. 104,105 More recently, Isberg et al 79 have developed an in silico-activated model of the 5-HT 2A receptor starting with the published crystal structure of the β-2-adrenergic receptor.…”