Combined docking and machine learning identify key molecular determinants of ligand pharmacological activity on β2 adrenoceptor

G protein-coupled receptors (GPCRs) are valuable therapeutic targets for many diseases. A central question of GPCR drug discovery is to understand what determines the agonism or antagonism of ligands that bind them. Ligands exert their action via the interactions in the ligand binding pocket. We hypothesized that there is a common set of receptor interactions made by ligands of diverse structures that mediate their action and that among a large dataset of different ligands, the functionally important interactions will be over-represented. We computationally docked ~2700 known β2AR ligands to multiple β2AR structures, generating ca 75 000 docking poses and predicted all atomic interactions between the receptor and the ligand. We used machine learning (ML) techniques to identify specific interactions that correlate with the agonist or antagonist activity of these ligands. We demonstrate with the application of ML methods that it is possible to identify the key interactions associated with agonism or antagonism of ligands. The most representative interactions for agonist ligands involve K972.68×67 , F194ECL2 , S2035.42×43 , S2045.43×44 , S2075.46×641 , H2966.58×58 , and K3057.32×31 . Meanwhile, the antagonist ligands made interactions with W2866.48×48 and Y3167.43×42 , both residues considered to be important in GPCR activation. The interpretation of ML analysis in human understandable form allowed us to construct an exquisitely detailed structure-activity relationship that identifies small changes to the ligands that invert their pharmacological activity and thus helps to guide the drug discovery process. This approach can be readily applied to any drug target

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Kinetic analysis of endogenous β2-adrenoceptor-mediated cAMP GloSensorTM responses in HEK293 cells

BackgroundStandard pharmacological analysis of agonist activity utilises measurements of receptor-mediated responses at a set time-point, or at the peak response level, to characterise ligands by calculation of empirical parameters such as potency (EC50) and efficacy (Emax). However, the occurrence of non-equilibrium conditions and the differential effects of regulatory mechanisms on transient signals may dramatically impact the properties of the response being measured. Here we have analysed the initial kinetic phases of cAMP responses to β2-adrenoceptor (β2AR) agonists in HEK293 cells expressing the endogenous β2AR at extremely low levels.Experimental ApproachThe kinetics of β2AR agonist-stimulated cAMP responses were monitored in real-time, in the presence and absence of antagonists, in HEK293 cells expressing the cAMP GloSensorTM biosensor. EC50 and Emax values were determined at the peak of the agonist GloSensorTM response and compared to kinetic parameters L50 and IRmax values derived from initial response rates.Key ResultsThe partial agonists salbutamol and salmeterol displayed reduced relative IRmax values (with respect to isoprenaline) when compared with their Emax values. Preincubation of β2AR antagonists with distinct receptor dissociation rates had profound effects on the isoprenaline-stimulated Emax parameters. Except for the fast dissociating bisoprolol, application of antagonists produced a large reduction in the isoprenaline peak response due to a state of hemi-equilibrium in this low receptor reserve system. This effect was exacerbated when IRmax parameters were measured. Furthermore, bisoprolol produced a large reduction in isoprenaline IRmax consistent with its short residence time.Conclusions and ImplicationsKinetic analysis of real-time signalling data can improve our understanding of the impact of agonist-antagonist interactions at receptors expressed at low endogenous levels in different tissues. It provides valuable insights into the hemi-equilibria that can occur in low receptor reserve systems with agonist-antagonist interactions, due to incomplete dissociation of antagonist whilst the peak agonist response is developing

Structural basis of the negative allosteric modulation of 5-BDBD at human P2X4 receptors

The P2X4 receptor is a ligand-gated ion channel activated by extracellular ATP. P2X4 activity is associated with neuropathic pain, vasodilation, and pulmonary secretion and is therefore of therapeutic interest. The structure-activity relationship of P2X4 antagonists is poorly understood. Here we elucidate the structure-activity of 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) at human P2X4 by combining pharmacology, electrophysiology, molecular modeling, and medicinal chemistry. 5-BDBD antagonized P2X4 in a noncompetitive manner but lacked effect at human P2X2. Molecular modeling and site-directed mutagenesis suggested an allosteric binding site for 5-BDBD located between two subunits in the body region of P2X4, with M109, F178, Y300, and I312 on one subunit and R301 on the neighboring subunit as key residues involved in antagonist binding. The bromine group of 5-BDBD was redundant for the antagonist activity of 5-BDBD, although an interaction between the carbonyl group of 5-BDBD and R301 in P2X4 was associated with 5-BDBD activity. 5-BDBD could inhibit the closed channel but poorly inhibited the channel in the open/desensitizing state. We hypothesize that this is due to constriction of the allosteric site after transition from closed to open channel state. We propose that M109, F178, Y300, R301, and I312 are key residues for 5-BDBD binding; provide a structural explanation of how they contribute to 5-BDBD antagonism; and highlight that the limited action of 5-BDBD on open versus closed channels is due to a conformational change in the allosteric site.  SIGNIFICANCE STATEMENT: Activity of P2X4 receptor is associated with neuropathic pain, inflammation, and vasodilatation. Molecular information regarding small-molecule interaction with P2X4 is very limited. Here, this study provides a structural explanation for the action of the small-molecule antagonist 5-BDBD at the human P2X4 receptor

ThermoBRET: A Ligand‐Engagement Nanoscale Thermostability Assay Applied to GPCRs

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre‐existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence‐detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable for high‐throughput screening. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2) and the b2‐adrenoceptor (b2AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non‐purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol‐reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm) of Nluc‐fused GPCRs can be determined in non‐purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split‐Nluc (Tm of 87 ⁰C versus 59 ⁰C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and for drug discovery screening