Homology Model Versus X-ray Structure in Receptor-based Drug Design: A Retrospective Analysis with the Dopamine D3 Receptor

Abstract

Structure-based design methods commonly used in medicinal chemistry rely on a three-dimensional representation of the receptor. However, few crystal structures are solved in comparison with the huge number of pharmaceutical targets. This often renders homology models the only information available. It is particularly true for G protein-coupled receptors (GPCRs), one of the most important targets for approved medicines and current drug discovery projects. However, very few studies have tested their validity in comparison with corresponding crystal structures, especially in a lead optimization perspective. The recent solving of dopamine D3 receptor crystal structure allowed us to assess our historical homology model. We performed a statistical analysis, by docking our in-house lead optimization library of 1500 molecules. We demonstrate here that the refined homology model suits at least as well as the X-ray structure. It is concluded that when the crystal structure of a given GPCR is not available, homology modeling can be an excellent surrogate to support drug discovery efforts.KEYWORDS: Dopamine D3 receptor, G protein-coupled receptors, docking, homology model, lead optimization S tructure-based design (SBD) uses the information brought by a receptor three-dimensional (3D) model to help the discovery and optimization of ligands. The protein 3D representation gives insights into the shape and electrostatics of the binding site, as well as the nature of its constitutive amino acids. Indeed, it gives insights into the possible interactions that ligands could form. SBD is widely used in medicinal chemistry at different stages of the drug discovery process. In the earliest, SBD can be instrumental in selecting new hits from a large library, thus reducing the size of the compounds to be experimentally tested (hit finding). Many examples in the literature report the success of virtual screening studies.1,2 SBD has also proved very useful in the following step of lead optimization, notably as a tool to understand and rationalize the structure-activity relationships. 3This approach commonly starts from a reliable crystal structure of the target receptor. However, in comparison with the huge number of proteins of pharmaceutical interest, the number of available crystal structures is limited. This is particularly true for G protein-coupled receptors (GPCRs), the most important class of drug targets whose ligands represent about 50% of the currently marketed drugs. 4 To compensate for the scarcity of crystal structures, molecular models of target proteins are often built based on a close structural neighbor (comparative/homology modeling). For GPCRs, bovine rhodopsin has long been the sole experimental structure available. So, a myriad of homology models (HMs) deriving from the breakthrough of Palczewski's work 5 were built and used in virtual screening experiments.

Identification of key residues involved in the activation and signaling properties of dopamine D3 receptor

“…These include residues Asp110 3.32 , Ser192 5.42 , Ser193 5.43 , Trp342 6.48 , Phe346 6.52 , and Thr369 7.39 which had only been previously shown to be important for ligand binding [8][9][10][11][12][14][15][16][17]. A novel finding of this study was the discovery that the non-conserved Ser361 7.31 residue is required for D3 receptor activation by all three agonists.…”

“…3 suggest that the residue Phe345 6.51 distinguishes the PBZI class of compounds from 7-OH-DPAT and is more important for cis-PBZI-induced D3 receptor activation. Modeling studies suggest that while the tetralin ring of 7-OH-DPAT has non-polar interactions with Phe345 6.51 [16], both isomers of PBZI have hydrophobic interactions with Phe345 6.51 (Fig. 4).…”

“…Activation of human D3 receptor signaling by 7-OH-DPAT, cis-and trans-8-OH-PBZI requires interactions with residues in the orthosteric binding site Site-directed mutagenesis and receptor binding studies, as well as the results from D3 receptor crystal structure and homology modeling have identified residues that are part of the orthosteric binding site of D3 receptor [8][9][10][11][12][13][14][15][16][17]. To determine if the residues in the orthosteric binding site are also required for the activation of human D3 receptor signaling by 7-OH-DPAT, cis-and trans-8-OH-PBZI, we individually mutated 10 different residues, including many in the reported orthosteric binding site, such as Asp110 3.32 , Ser192 5.42 , Ser193 5.43 , Trp342 6.48 , Phe345 6.51 , Phe346 6.52 , His349 6 Tolerance property defined as the ratio of 2nd/1st agonist-induced GIRK response and SRT property defined as the response terminate rate following termination of agonist application was determined for cis-PBZI (gray bar) and trans-PBZI (black bar) in AtT-20 cells stably expressing human D3 receptor.…”

“…Different residues in the sixth and seventh transmembrane helices and third extracellular loop of D3 receptor are required for 7-OH-DPAT, cis-and trans-8-OH-PBZI induced activation of signaling Previous mutagenesis and computational modeling studies as well as antagonist-bound D3 receptor crystal structure have identified Phe345 6.51 , His349 6.55 and Thr369 7.39 as residues that are important for ligand binding to D3 receptor [8][9][10][11][12][13][14][15][16][17]; however, it is not known if these residues are important for agonist-induced activation of D3 receptor signaling. In addition, our previous study described a putative ionic lock between Asp187 residue in extracellular loop 2 and His354 residue in extracellular loop 3 in mediating the PD128907-induced tolerance property of human D3 receptors [22].…”

“…The D3 receptor belongs to the D2-like dopamine receptor subfamily and couples via G i/o G-proteins to downstream effectors such as adenylate cyclase V, G-protein coupled inward rectifier potassium (GIRK) channels, mitogenactivated protein kinases (MAPK) and P/Q-type calcium channels [3][4][5][6][7]. Information obtained from the antagonist-bound D3 receptor crystal structure, together with modelling and mutagenesis studies have identified important structural domains and key residues of the D3 receptor involved in ligand binding; however, the residues involved in agonist-induced activation of D3 receptor signaling remains to be determined [8][9][10][11][12][13][14][15][16][17]. In this paper, we used sitedirected mutagenesis to identify residues important for activation of human D3 receptor signaling by three different agonists.…”