Benjamin G. Tehan

Discovery of 1,2,4-Triazine Derivatives as Adenosine A(2A) Antagonists using Structure Based Drug Design

Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A2A receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson’s disease.

Biophysical Fragment Screening of the β1-Adrenergic Receptor: Identification of High Affinity Arylpiperazine Leads Using Structure-Based Drug Design

Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein–ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.

Unifying Family A GPCR Theories of Activation

Several new pairs of active and inactive GPCR structures have recently been solved enabling detailed structural insight into the activation process, not only of rhodopsin but now also of the β2 adrenergic, M2 muscarinic and adenosine A2A receptors. Combined with structural analyses they have enabled us to examine the different recent theories proposed for GPCR activation and show that they are all indeed parts of the same process, and are intrinsically related through their effect on the central hydrophobic core of GPCRs. This new unifying general process of activation is consistent with the identification of known constitutively active mutants and an in-depth conservational analysis of significant residues implicated in the process.

Biophysical Mapping of the Adenosine A2A Receptor

A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (KD, kon, and koff) of receptor–ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein–ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A2A receptor are presented.

Water Network Perturbation in Ligand Binding: Adenosine A2A Antagonists as a Case Study

Recent efforts in the computational evaluation of the thermodynamic properties of water molecules have resulted in the development of promising new in silico methods to evaluate the role of water in ligand binding. These methods include WaterMap, SZMAP, GRID/CRY probe, and Grand Canonical Monte Carlo simulations. They allow the prediction of the position and relative free energy of the water molecule in the protein active site and the analysis of the perturbation of an explicit water network (WNP) as a consequence of ligand binding. We have for the first time extended these approaches toward the prediction of kinetics for small molecules and of relative free energy of binding with a focus on the perturbation of the water network and application to large diverse data sets. Our results support a qualitative correlation between the residence time of 12 related triazine adenosine A(2A) receptor antagonists and the number and position of high energy trapped solvent molecules. From a quantitative viewpoint, we successfully applied these computational techniques as an implicit solvent alternative, in linear combination with a molecular mechanics force field, to predict the relative ligand free energy of binding (WNP-MMSA). The applicability of this linear method, based on the thermodynamics additivity principle, did not extend to 375 diverse A(2A) receptor antagonists. However, a fast but effective method could be enabled by replacing the linear approach with a machine learning technique using probabilistic classification trees, which classified the binding affinity correctly for 90% of the ligands in the training set and 67% in the test set.

Fragment and Structure-Based Drug Discovery for a Class C GPCR: Discovery of the mGlu5 Negative Allosteric Modulator HTL14242 (3-Chloro-5-[6-(5-fluoropyridin-2-yl)pyrimidin-4-yl]benzonitrile).

Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.

Mutagenic Mapping Suggests a Novel Binding Mode for Selective Agonists of M1 Muscarinic Acetylcholine Receptors

Point mutations and molecular modeling have been used to study the activation of the M1 muscarinic acetylcholine receptor (mAChR) by the functionally selective agonists 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl]-piperidine (AC-42), and 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1), comparing them with N-desmethylclozapine (NDMC) and acetylcholine (ACh). Unlike NDMC and ACh, the activities of AC-42 and 77-LH-28-1 were undiminished by mutations of Tyr404 and Cys407 (transmembrane helix 7), although they were reduced by mutations of Tyr408. Signaling by AC-42, 77-LH-28-1, and NDMC was reduced by L102A and abolished by D105E, suggesting that all three may interact with transmembrane helix 3 at or near the binding site Asp105 to activate the M1 mAChR. In striking contrast to NDMC and ACh, the affinities of AC-42 and 77-LH-28-1 were increased 100-fold by W101A, and their signaling activities were abolished by Y82A. Tyr82 and Leu102 contact the indole ring of Trp101 in a structural model of the M1 mAChR. We suggest the hypothesis that the side chain of Trp101 undergoes conformational isomerization, opening a novel binding site for the aromatic side chain of the AC-42 analogs. This may allow the positively charged piperidine nitrogen of the ligands to access the neighboring Asp105 carboxylate to activate signaling following a vector within the binding site that is distinct from that of acetylcholine. NDMC does not seem to use this mechanism. Subtype-specific differences in the free energy of rotation of the side chain and indole ring of Trp101 might underlie the M1 selectivity of the AC-42 analogs. Tryptophan conformational isomerization may open up new avenues in selective muscarinic receptor drug design.

Orthosteric and Allosteric Modes of Interaction of Novel Selective Agonists of the M1 Muscarinic Acetylcholine Receptor

Recent years have witnessed the discovery of novel selective agonists of the M1 muscarinic acetylcholine (ACh) receptor (mAChR). One mechanism invoked to account for the selectivity of such agents is that they interact with allosteric sites. We investigated the molecular pharmacology of two such agonists, 1-[3-(4-butyl-1-piperidinyl)propyl]-3,4-dihydro-2(1H)-quinolinone (77-LH-28-1) and 4-n-butyl-1-[4-(2-methylphenyl)-4-oxo-1-butyl] piperidine hydrogen chloride (AC-42), at the wild-type M1 mAChR and three mutant M1 mAChRs. Both agonists inhibited the binding of the orthosteric antagonist [3H]N-methyl scopolamine ([3H]NMS) in a manner consistent with orthosteric competition or high negative cooperativity. Functional interaction studies between 77-LH-28-1 and ACh also indicated a competitive mechanism. Dissociation kinetics assays revealed that the agonists could bind allosterically when the orthosteric site was prelabeled with [3H]NMS and that 77-LH-28-1 competed with the prototypical allosteric modulator heptane-1,7-bis-[dimethyl-3′-phthalimidopropyl]-ammonium bromide under these conditions. Mutation of the key orthosteric site residues Y381A (transmembrane helix 6) and W101A (transmembrane helix 3) reduced the affinity of prototypical orthosteric agonists but increased the affinity of the novel agonists. Divergent effects were also noted on agonist signaling efficacies at these mutants. We identified a novel mutation, F77I (transmembrane helix 2), which selectively reduced the efficacy of the novel agonists in mediating intracellular Ca2+ elevation and phosphorylation of extracellular signal regulated kinase 1/2. Molecular modeling suggested a possible “bitopic” binding mode, whereby the agonists extend down into the orthosteric site as well as up toward extracellular receptor regions associated with an allosteric site. It is possible that this bitopic mode may explain the pharmacology of other selective mAChR agonists.

High end GPCR design: crafted ligand design and druggability analysis using protein structure, lipophilic hotspots and explicit water networks

PurposeG Protein-Coupled Receptors (GPCRs) are a large family of therapeutically important proteins and as diverse X-ray structures become available it is increasingly possible to leverage structural information for rational drug design.We present herein approaches that use explicit water networks combined with energetic surveys of the binding site (GRID), providing an enhanced druggability and ligand design approach, with structural understanding of ligand binding, including a ‘magic’ methyl and binding site mutations, and a fast new approach to generate and score waters.MethodsThe GRID program was used to identify lipophilic and hydrogen bonding hotspots. Explicit full water networks were generated and scored for (pseudo)apo structures and ligand-protein complexes using a new approach, WaterFLAP (Molecular Discovery), together with WaterMap (Schrödinger) for (pseudo)apo structures. A scoring function (MetaScore) was developed using a fast computational protocol based on several short adiabatic biased MD simulations followed by multiple short well-tempered metadynamics runs.ResultsAnalysis of diverse ligands binding to the adenosine A2A receptor together with new structures for the δ/κ/μ opioid and CCR5 receptors confirmed the key role of lipophilic hotspots in driving ligand binding and thus design; the displacement of ‘unhappy’ waters generally found in these regions provides a key binding energy component. Complete explicit water networks could be robustly generated for protein-ligand complexes using a WaterFLAP based approach. They provide a structural understanding of structure-activity relationships such as a ‘magic methyl’ effect and with the metadynamics approach a useful estimation of the binding energy changes resulting from active site mutations.ConclusionsThe promise of full structure-based drug design (SBDD) for GPCRs is now possible using a combination of advanced experimental and computational data. The conformational thermostabilisation of StaR® proteins provide the ability to easily generate biophysical screening data (binding including fragments, kinetics) and to get crystal structures with both potent and weak ligands. Explicit water networks for apo and ligand-complex structures are a critical ‘third dimension’ for SBDD and are key for understanding ligand binding energies and kinetics. GRID lipophilic hotspots are found to be key drivers for binding. In this context ‘high end’ GPCR ligand design is now enabled.

Pharmacology and Structure of Isolated Conformations of the Adenosine A2A Receptor Define Ligand Efficacy

Using isolated receptor conformations crystal structures of the adenosine A2A receptor have been solved in active and inactive states. Studying the change in affinity of ligands at these conformations allowed qualitative prediction of compound efficacy in vitro in a system-independent manner. Agonist 5′-N-ethylcarboxamidoadenosine displayed a clear preference to bind to the active state receptor; inverse agonists (xanthine amine congener, ZM241385, SCH58261, and preladenant) bound preferentially to the inactive state, whereas neutral antagonists (theophylline, caffeine, and istradefylline) demonstrated equal affinity for active and inactive states. Ligand docking into the known crystal structures of the A2A receptor rationalized the pharmacology observed; inverse agonists, unlike neutral antagonists, cannot be accommodated within the agonist-binding site of the receptor. The availability of isolated receptor conformations opens the door to the concept of “reverse pharmacology” whereby the functional pharmacology of ligands can be characterized in a system-independent manner by their affinity for a pair (or set) of G protein–coupled receptor conformations.