Francesca Deflorian

Examining the Chirality, Conformation and Selective Kinase Inhibition of 3-((3R,4R)-4-methyl-3-(methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropanenitrile (CP-690,550)

Here, we examine the significance that stereochemistry plays within the clinically relevant Janus kinase 3 (Jak3) inhibitor 1 (CP-690,550). A synthesis of all four enantiopure stereoisomers of the drug was carried out and an examination of each compound revealed that only the enantiopure 3R,4R isomer was capable of blocking Stat5 phosphorylation (Jak3 dependent). Each compound was profiled across a panel of over 350 kinases, which revealed a high level of selectivity for the Jak family kinases for these related compounds. Each stereoisomer retained a degree of binding to Jak3 and Jak2 and the 3R,4S and 3S,4R stereoisomers were further revealed to have binding affinity for selected members of the STE7 and STE20 subfamily of kinases. Finally, an appraisal of the minimum energy conformation of each stereoisomer and molecular docking at Jak3 was performed in an effort to better understand each compounds selectivity and potency profiles.

Intracellular allosteric antagonism of the CCR9 receptor

Chemokines and their G-protein-coupled receptors play a diverse role in immune defence by controlling the migration, activation and survival of immune cells. They are also involved in viral entry, tumour growth and metastasis and hence are important drug targets in a wide range of diseases. Despite very significant efforts by the pharmaceutical industry to develop drugs, with over 50 small-molecule drugs directed at the family entering clinical development, only two compounds have reached the market: maraviroc (CCR5) for HIV infection and plerixafor (CXCR4) for stem-cell mobilization. The high failure rate may in part be due to limited understanding of the mechanism of action of chemokine antagonists and an inability to optimize compounds in the absence of structural information. CC chemokine receptor type 9 (CCR9) activation by CCL25 plays a key role in leukocyte recruitment to the gut and represents a therapeutic target in inflammatory bowel disease. The selective CCR9 antagonist vercirnon progressed to phase 3 clinical trials in Crohn’s disease but efficacy was limited, with the need for very high doses to block receptor activation. Here we report the crystal structure of the CCR9 receptor in complex with vercirnon at 2.8 Å resolution. Remarkably, vercirnon binds to the intracellular side of the receptor, exerting allosteric antagonism and preventing G-protein coupling. This binding site explains the need for relatively lipophilic ligands and describes another example of an allosteric site on G-protein-coupled receptors that can be targeted for drug design, not only at CCR9, but potentially extending to other chemokine receptors.

Ligand-Based Homology Modeling as Attractive Tool to Inspect GPCR Structural Plasticity

G protein-coupled receptors (GPCRs) represent the largest family known of signal-transducing molecules. They convey signals for light and many extracellular regulatory molecules. GPCRs have been found to be dysfunctional/dysregulated in a growing number of human diseases and they have been estimated to be the targets of more than 40% of the drugs used in clinical medicine today. The crystal structure of rhodopsin provides the first three-dimensional GPCR information, which now supports homology modeling studies and structure-based drug design approaches. Here, we review our recent work on adenosine receptors, a family of GPCRs and, in particular, on A(3) adenosine receptor subtype antagonists. We will focus on an alternative approach to computationally explore the multi-conformational space of the antagonist-like state of the human A(3) receptor. We define ligand-based homology modeling as new approach to simulate the reorganization of the receptor induced by the ligand binding. The success of this approach is due to the synergic interaction between theory and experiment.

G protein-coupled adenosine (P1) and P2Y receptors: ligand design and receptor interactions

The medicinal chemistry and pharmacology of the four subtypes of adenosine receptors (ARs) and the eight subtypes of P2Y receptors (P2YRs, activated by a range of purine and pyrimidine mono- and dinucleotides) has recently advanced significantly leading to selective ligands. X-ray crystallographic structures of both agonist- and antagonist-bound forms of the A2AAR have provided unprecedented three-dimensional detail concerning molecular recognition in the binding site and the conformational changes in receptor activation. It is apparent that this ubiquitous cell signaling system has implications for understanding and treating many diseases. ATP and other nucleotides are readily released from intracellular sources under conditions of injury and organ stress, such as hypoxia, ischemia, or mechanical stress, and through channels and vesicular release. Adenosine may be generated extracellularly or by cellular release. Therefore, depending on pathophysiological factors, in a given tissue, there is often a tonic activation of one or more of the ARs or P2YRs that can be modulated by exogenous agents for a beneficial effect. Thus, this field has provided fertile ground for pharmaceutical development, leading to clinical trials of selective receptor ligands as imaging agents or for conditions including cardiac arrhythmias, ischemia/reperfusion injury, diabetes, pain, thrombosis, Parkinson’s disease, rheumatoid arthritis, psoriasis, dry eye disease, pulmonary diseases such as cystic fibrosis, glaucoma, cancer, chronic hepatitis C, and other diseases.

Controlling the Dissociation of Ligands from the Adenosine A2A Receptor through Modulation of Salt Bridge Strength.

The association and dissociation kinetics of ligands binding to proteins vary considerably, but the mechanisms behind this variability are poorly understood, limiting their utilization for drug discovery. This is particularly so for G protein-coupled receptors (GPCRs) where high resolution structural information is only beginning to emerge. Engineering the human A2A adenosine receptor has allowed structures to be solved in complex with the reference compound ZM241385 and four related ligands at high resolution. Differences between the structures are limited, with the most pronounced being the interaction of each ligand with a salt bridge on the extracellular side of the receptor. Mutagenesis experiments confirm the role of this salt bridge in controlling the dissociation kinetics of the ligands from the receptor, while molecular dynamics simulations demonstrate the ability of ligands to modulate salt bridge stability. These results shed light on a structural determinant of ligand dissociation kinetics and identify a means by which this property may be optimized.

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.

Evaluation of Molecular Modeling of Agonist Binding in Light of the Crystallographic Structure of an Agonist-Bound A2A Adenosine Receptor

Molecular modeling of agonist binding to the human A(2A) adenosine receptor (AR) was assessed and extended in light of crystallographic structures. Heterocyclic adenine nitrogens of cocrystallized agonist overlaid corresponding positions of the heterocyclic base of a bound triazolotriazine antagonist, and ribose moiety was coordinated in a hydrophilic region, as previously predicted based on modeling using the inactive receptor. Automatic agonist docking of 20 known potent nucleoside agonists to agonist-bound A(2A)AR crystallographic structures predicted new stabilizing protein interactions to provide a structural basis for previous empirical structure activity relationships consistent with previous mutagenesis results. We predicted binding of novel C2 terminal amino acid conjugates of A(2A)AR agonist CGS21680 and used these models to interpret effects on binding affinity of newly synthesized agonists. d-Amino acid conjugates were generally more potent than l-stereoisomers and free terminal carboxylates more potent than corresponding methyl esters. Amino acid moieties were coordinated close to extracellular loops 2 and 3. Thus, molecular modeling is useful in probing ligand recognition and rational design of GPCR-targeting compounds with specific pharmacological profiles.

Pharmacochemistry of the platelet purinergic receptors

Platelets contain at least five purinergic G protein-coupled receptors, e.g., the pro-aggregatory P2Y1 and P2Y12 receptors, a P2Y14 receptor (GPR105) of unknown function, and anti-aggregatory A2A and A2B adenosine receptor (ARs), in addition to the ligand-gated P2X1 ion channel. Probing the structure–activity relationships (SARs) of the P2X and P2Y receptors for extracellular nucleotides has resulted in numerous new agonist and antagonist ligands. Selective agents derived from known ligands and novel chemotypes can be used to help define the subtypes pharmacologically. Some of these agents have entered into clinical trials in spite of the challenges of drug development for these classes of receptors. The functional architecture of P2 receptors was extensively explored using mutagenesis and molecular modeling, which are useful tools in drug discovery. In general, novel drug delivery methods, prodrug approaches, allosteric modulation, and biased agonism would be desirable to overcome side effects that tend to occur even with receptor subtype-selective ligands. Detailed SAR analyses have been constructed for nucleotide and non-nucleotide ligands at the P2Y1, P2Y12, and P2Y14 receptors. The thienopyridine antithrombotic drugs Clopidogrel and Prasugrel require enzymatic pre-activation in vivo and react irreversibly with the P2Y12 receptor. There is much pharmaceutical development activity aimed at identifying reversible P2Y12 receptor antagonists. The screening of chemically diverse compound libraries has identified novel chemotypes that act as competitive, non-nucleotide antagonists of the P2Y1 receptor or the P2Y12 receptor, and antithrombotic properties of the structurally optimized analogues were demonstrated. In silico screening at the A2A AR has identified antagonist molecules having novel chemotypes. Fluorescent and other reporter groups incorporated into ligands can enable new technology for receptor assays and imaging. The A2A agonist CGS21680 and the P2Y1 receptor antagonist MRS2500 were derivatized for covalent attachment to polyamidoamine dendrimeric carriers of MW 20,000, and the resulting multivalent conjugates inhibited ADP-promoted platelet aggregation. In conclusion, a wide range of new pharmacological tools is available to control platelet function by interacting with cell surface purine receptors.

Demystifying the three dimensional structure of G protein-coupled receptors (GPCRs) with the aid of molecular modeling

We review our recent work on adenosine receptors, a family of GPCRs; focusing our attention on A3 adenosine receptor, we have demonstrated that the reciprocal integration of different theoretical and experimental disciplines can be very useful for the successful protein-based design of new, potent and selective receptor ligands.

Molecular Probes for the A2A Adenosine Receptor Based on a Pyrazolo(4,3-e)(1,2,4)triazolo(1,5-c)pyrimidin-5-amine Scaffold

Pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivatives such as SCH 442416 display high affinity and selectivity as antagonists for the human A(2A) adenosine receptor (AR). We extended ether-linked chain substituents at the p-position of the phenyl group using optimized O-alkylation. The conjugates included an ester, carboxylic acid and amines (for amide condensation), an alkyne (for click chemistry), a fluoropropyl group (for (18)F incorporation), and fluorophore reporter groups (e.g., BODIPY conjugate 14, K(i) 15 nM). The potent and A(2A)AR-selective N-aminoethylacetamide 7 and N-[2-(2-aminoethyl)-aminoethyl]acetamide 8 congeners were coupled to polyamidoamine (PAMAM) G3.5 dendrimers, and the multivalent conjugates displayed high A(2A)AR affinity. Theoretical docking of an AlexaFluor conjugate to the receptor X-ray structure highlighted the key interactions between the heterocyclic core and the binding pocket of the A(2A)AR as well as the distal anchoring of the fluorophore. In conclusion, we have synthesized a family of high affinity functionalized congeners as pharmacological probes for studying the A(2A)AR.