Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy

A molecular model for the P450 enzyme cytochrome P450 C17 (CYP17) is presented based on sequence alignments of multiple template structures and homology modeling. This enzyme plays a central role in the biosynthesis of testosterone and is emerging as a major target in prostate cancer, with the recently developed inhibitor abiraterone currently in advanced clinical trials. The model is described in detail, together with its validation, by providing structural explanations to available site-directed mutagenesis data. The CYP17 molecule in this model is in the form of a triangular prism, with an edge of similar to 55 angstrom and a thickness of similar to 37 angstrom. It is predominantly helical, comprising 13 alpha helices interspersed by six 3(10) helices and 11 beta-sheets. Multinanosecond molecular dynamics simulations in explicit solvent have been carried out, and principal components analysis has been used to reveal the details of dynamics around the active site. Coarse-grained methods have also been used to verify low-frequency motions, which have been correlated with active-site gating. The work also describes the results of docking synthetic inhibitors, including the drug abiraterone and the natural substrate pregnenolone, in the CYP17 active site together with molecular dynamics simulations on the complexes. (C) 2010 Elsevier Ltd. All rights reserved.

CoMFA and homology-based models of the glycine binding site of N-methyl-D-aspartate receptor

Homology modeling was used to build 3D models of the N-methyl-D-aspartate (NMDA) receptor glycine binding site on the basis of an X-ray structure of the water-soluble AMPA-sensitive receptor. The docking of agonists and antagonists to these models was used to reveal binding modes of ligands and to explain known structure-activity relationships. Two types of quantitative models, 3D-QSAR/CoMFA and a regression model based on docking energies, were built for antagonists (derivatives of 4-hydroxy-2-quinolone, quinoxaline-2,3-dione, and related compounds). The CoMFA steric and electrostatic maps were superimposed on the homology-based model, and a close correspondence was marked. The derived computational models have permitted the evaluation of the structural features crucial for high glycine binding site affinity and are important for the design of new ligands.

Status of GPCR modeling and docking as reflected by community-wide GPCR Dock 2010 assessment

The community-wide GPCR Dock assessment is conducted to evaluate the status of molecular modeling and ligand docking for human G protein-coupled receptors. The present round of the assessment was based on the recent structures of dopamine D3 and CXCR4 chemokine receptors bound to small molecule antagonists and CXCR4 with a synthetic cyclopeptide. Thirty-five groups submitted their receptor-ligand complex structure predictions prior to the release of the crystallographic coordinates. With closely related homology modeling templates, as for dopamine D3 receptor, and with incorporation of biochemical and QSAR data, modern computational techniques predicted complex details with accuracy approaching experimental. In contrast, CXCR4 complexes that had less-characterized interactions and only distant homology to the known GPCR structures still remained very challenging. The assessment results provide guidance for modeling and crystallographic communities in method development and target selection for further expansion of the structural coverage of the GPCR universe.

Rhodopsin and the Others: A Historical Perspective on Structural Studies of G Protein-Coupled Receptors

The role of rhodopsin as a structural prototype for the study of the whole superfamily of G protein-coupled receptors (GPCRs) is reviewed in an historical perspective. Discovered at the end of the nineteenth century, fully sequenced since the early 1980s, and with direct three-dimensional information available since the 1990s, rhodopsin has served as a platform to gather indirect information on the structure of the other superfamily members. Recent breakthroughs have elicited the solution of the structures of additional receptors, namely the beta 1- and beta 2-adrenergic receptors and the A(2A) adenosine receptor, now providing an opportunity to gauge the accuracy of homology modeling and molecular docking techniques and to perfect the computational protocol. Notably, in coordination with the solution of the structure of the A(2A) adenosine receptor, the first "critical assessment of GPCR structural modeling and docking" has been organized, the results of which highlighted that the construction of accurate models, although challenging, is certainly achievable. The docking of the ligands and the scoring of the poses clearly emerged as the most difficult components. A further goal in the field is certainly to derive the structure of receptors in their signaling state, possibly in complex with agonists. These advances, coupled with the introduction of more sophisticated modeling algorithms and the increase in computer power, raise the expectation for a substantial boost of the robustness and accuracy of computer-aided drug discovery techniques in the coming years.

Two Arginine-Glutamate Ionic Locks Near the Extracellular Surface of FFAR1 Gate Receptor Activation

Activation of a number of class A G protein-coupled receptors (GPCRs) is thought to involve two molecular switches, a rotamer toggle switch within the transmembrane domain and an ionic lock at the cytoplasmic surface of the receptor; however, the mechanism by which agonist binding changes these molecular interactions is not understood. Importantly, 80% of GPCRs including free fatty acid receptor 1 (FFAR1) lack the complement of amino acid residues implicated in either or both of these two switches; the mechanism of activation of these GPCRs is therefore less clear. By homology modeling, we identified two Glu residues (Glu-145 and Glu-172) in the second extracellular loop of FFAR1 that form putative interactions individually with two transmembrane Arg residues (Arg-183(5.39) and Arg-258(7.35)) to create two ionic locks. Molecular dynamics simulations showed that binding of agonists to FFAR1 leads to breakage of these Glu-Arg interactions. In mutagenesis experiments, breakage of these two putative interactions by substituting Ala for Glu-145 and Glu-172 caused constitutive receptor activation. Our results therefore reveal a molecular switch for receptor activation present on the extracellular surface of FFAR1 that is broken by agonist binding. Similar ionic locks between the transmembrane domains and the extracellular loops may constitute a mechanism common to other class A GPCRs also.

Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC(50) values for inhibition of cAMP, 5.83 ± 0.11; Ca(2+) mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs.

Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor

FFA2 is a G protein-coupled receptor that responds to short chain fatty acids (SCFAs) and has generated interest as a therapeutic target for metabolic and inflammatory conditions. However, definition of its functions has been slowed by a dearth of selective ligands that can distinguish it from the closely related FFA3. At present, the only selective ligands described for FFA2 suffer from either poor potency, altered signaling due to allosteric modes of action, or a lack of function at non-human orthologs of the receptor. To address the need for novel selective ligands, we synthesized two compounds potentially having FFA2 activity and examined the molecular basis of their function. These compounds were confirmed to be potent and selective FFA2 agonists that interact with the orthosteric binding site. A combination of ligand structure-activity relationship, pharmacological analysis, homology modeling, species ortholog comparisons and mutagenesis studies were then employed to define the molecular basis of selectivity and function of these ligands. From this, we identified key residues within both extracellular loop 2 (ECL2) and the transmembrane domain (TM) regions of FFA2 critical for ligand function. One of these ligands was active with reasonable potency at rodent orthologs of FFA2 and demonstrated the role of FFA2 in the regulation of lipolysis in murine 3T3-L1 adipocytes. Together, these findings describe the first potent and selective FFA2 orthosteric agonists and demonstrate key aspects of ligand interaction within the orthosteric binding site of FFA2 that will be invaluable in future ligand development at this receptor.

P2Y(1) antagonists: Combining receptor-based modeling and QSAR for a quantitative prediction of the biological activity based on consensus scoring

P2Y(1) is an ADP-activated G protein-coupled receptor (GPCR). Its antagonists impede platelet aggregation in vivo and are potential antithrombotic agents. Combining ligand and structure-based modeling we generated a consensus model (LIST-CM) correlating antagonist structures with their potencies. We docked 45 antagonists into our rhodopsin-based human P2Y(1) homology model and calculated docking scores and free binding energies with the Linear Interaction Energy (LIE) method in continuum-solvent. The resulting alignment was also used to build QSAR based on CoMFA, CoMSIA, and molecular descriptors. To benefit from the strength of each technique and compensate for their limitations, we generated our LIST-CM with a PLS regression based on the predictions of each methodology. A test set featuring untested substituents was synthesized and assayed in inhibition of 2-MeSADP-stimulated PLC activity and in radioligand binding. LIST-CM outperformed internal and external predictivity of any individual model to predict accurately the potency of 75% of the test set.

Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment:Meeting new challenges

Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems.