Modelling studies of bitter taste, glucocorticoid and VEGFR2 receptors and their ligands

Modelling studies of bitter taste receptors and their ligands The human bitter taste receptor gene family (TAS2R) belongs to the Frizzled/Taste2 subfamily of the G-protein coupled receptors (GPCR) superfamily. TAS2R are expressed on the tongue in bitter taste receptor cells co-expressing specific signal transduction components like G??gustducin and are able to detect stimuli of only one taste quality. Recently these receptors have been identified in isolated human airway smooth muscle cells (ASM). Besides, in the literature it has been reported that bitter taste receptor agonists such as saccharin, chloroquine and denatonium can evoke increased intracellular calcium in ASM eliciting relaxation of isolated ASM and dilation of airways greater than that showed by ?-adrenergic receptor agonists. Furthermore, inhaled bitter taste compounds can decrease airway obstruction in a mouse model of asthma. In light of these evidences this novel pathway looks attractive and might be exploited to identify novel compounds to treat asthma and chronic obstructive pulmonary disease (COPD). During the 1st year of my PhD course, chemoinformatics analyses of known bitter tastants aimed at finding common structural features and pharmacophores were performed. Besides, the generation of hTAS2R10, hTAS2R14 and hTAS2R31 receptor models was carried out together with docking studies of a few known bitter agonists in the putative binding sites of these receptor subtypes. Results of these experiments were validated in light of site-directed mutagenesis experiments available in the literature and provided useful suggestion about additional site-directed mutagenesis studies and chimera constructs to further validate these models. Results of these studies were presented as a poster at the Research Gordon Conference on ࢠComputer Aided Drug Designࢠ, held at Mount Snow Resort, West Dover, VT, in 2011. Elucidation of glucocorticosteroids unbinding pathways from the glucocorticoid receptor Glucocorticoids are endogenous steroid hormones that regulate essential biological functions including metabolism, growth, and apoptosis. Glucocorticoids and structurally related drugs represent the most effective anti-inflammatory agents to treat several inflammatory conditions. However, the clinical use of such drugs is hampered by severe side effects. Therefore, the development of novel glucocorticoid receptor (GRs) modulators with increased therapeutic index is impelling. Herein, using steered molecular dynamics (SMD) simulations a detailed picture of the unbinding process of three clinically relevant GR modulators from GR ligand binding domain is provided. The SMD protocol described can be used to prioritize the synthesis of structural analogues on the basis of their potential of mean forces (PMFs) and calculated unbinding energies. Moreover, these results are instrumental to explain at atomic resolution the reduced ability of dexamethasone to activate the naturally occurring mutant I747M-GR, which is implicated in rare familial glucocorticoid resistance, clinically characterized by glucocorticoids insensitivity. Results of these simulations were presented as a poster at the ACS Meeting held in Philadelphia in 2012 and published in the Journal of Medicinal Chemistry in September 2013. Modelling studies of VEGFR2 and its inhibitors Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) is a type III receptor tyrosine kinase (RTK) in the PDGFR family. VEGF preferably binds this receptor and triggers important signaling pathway implicated in the process of vasculogenesis and angiogenesis. VEGFR2 is a clinically validated target to treat Renal Cell Carcinoma (RCC). Recently the crystal structures of sunitinib and sorafenib, two clinically relevant TKIs, bound to the kinase domain of VEGFR2 have been solved together with the enzyme juxtamembrane (JM) domain, highlighting the critical role played by this enzyme portion to form the inactivated state of VEGFR2 kinase which binds these ligands. The construct utilized to generate these VEGFR2 crystal structures was also used to measure the potencies, time dependencies and selectivity of a wide panel of TKI, including sunitinib and sorafenib. Besides, kinetic parameters (koff) were detected for both these inhibitors and the new construct. While sunitinib is a prototypical type IV inhibitor, showing short dissociative half-life from VEGFR2, sorafenib is an exemplar of type II inhibitor, characterized by long dissociative half-life from VEGFR2 receptor. Herein, a preliminary detailed atomistic description of the unbinding process of sorafenib and sunitinib from VEGFR2 was unveiled by using SMD simulations. While sunitinib exits the ATP binding site from the cavity entrance, sorafenib moves towards the JM domain, causing a change in the orientation of the ?C-helix. While no rupture point could be identified in the force profile of sunitinib, analysis of the average force profile of sorafenib reveals that the rupture point involves the water-assisted breakage of sorafenib interaction with the hinge amino acid C919 and subsequently the disruption of the hydrogen bond of the ligand carbonyl urea moiety with D1046 (DFG-out motif). At the same time the hydrogen bond interactions established between the NH ligand urea moiety and E885 in the ?C-helix likely delay ligand exit. Besides, the phenyl ring decorated with chlorine and trifluoromethyl groups makes hydrophobic interactions with some hydrophobic residues in ?C-helix, which would further facilitate the ligand unbinding process. More robust conclusions will be drawn once the simulations of more ligand frames have been performed.