Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease.

The COVID-19 pandemic has highlighted the urgent need for the identification of new antiviral drug therapies for a variety of diseases. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2, while other related human coronaviruses cause diseases ranging from severe respiratory infections to the common cold. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of a range of human coronavirus diseases. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cell-based experimental assessment reveals several clinically-relevant drug repurposing candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

Differential ability of proinflammatory and anti-inflammatory macrophages to perform macropinocytosis.

Macropinocytosis mediates the uptake of antigens and of nutrients that dictate the regulation of cell growth by mechanistic target of rapamycin complex 1 (mTORC1). Because these functions differ in proinflammatory and anti-inflammatory macrophages, we compared the macropinocytic ability of two extreme polarization states. We found that anti-inflammatory macrophages perform vigorous macropinocytosis constitutively, while proinflammatory cells are virtually inactive. The total cellular content of Rho-family GTPases was higher in anti-inflammatory cells, but this disparity failed to account for the differential macropinocytic activity. Instead, reduced activity of Rac/RhoG was responsible for the deficient macropinocytosis of proinflammatory macrophages, as suggested by the stimulatory effects of heterologously expressed guanine nucleotide-exchange factors or of constitutively active (but not wild-type) forms of these GTPases. Similarly, differences in the activation state of phosphatidylinositol 3-kinase (PtdIns3K) correlated with the macropinocytic activity of pro- and anti-inflammatory macrophages. Differences in PtdIns3K and Rho-GTPase activity were attributable to the activity of calcium-sensing receptors (CaSRs), which appear to be functional only in anti-inflammatory cells. However, agonists of PtdIns3K, including cytokines, chemokines, and LPS, induced macropinocytosis in proinflammatory cells. Our findings revealed a striking difference in the macropinocytic ability of pro- and anti-inflammatory macrophages that correlates with their antigen-presenting and metabolic activity.

Allosteric modulation in monomers and oligomers of a G protein-coupled receptor.

The M2 muscarinic receptor is the prototypic model of allostery in GPCRs, yet the molecular and the supramolecular determinants of such effects are unknown. Monomers and oligomers of the M2 muscarinic receptor therefore have been compared to identify those allosteric properties that are gained in oligomers. Allosteric interactions were monitored by means of a FRET-based sensor of conformation at the allosteric site and in pharmacological assays involving mutants engineered to preclude intramolecular effects. Electrostatic, steric, and conformational determinants of allostery at the atomic level were examined in molecular dynamics simulations. Allosteric effects in monomers were exclusively negative and derived primarily from intramolecular electrostatic repulsion between the allosteric and orthosteric ligands. Allosteric effects in oligomers could be positive or negative, depending upon the allosteric-orthosteric pair, and they arose from interactions within and between the constituent protomers. The complex behavior of oligomers is characteristic of muscarinic receptors in myocardial preparations.

Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[ß,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins.

Efficacy as an intrinsic property of the M(2) muscarinic receptor in its tetrameric state.

Muscarinic and other G protein-coupled receptors exhibit an agonist-specific heterogeneity that tracks efficacy and commonly is attributed to an effect of the G protein on an otherwise homogeneous population of sites. To examine this notion, M2 muscarinic receptors were purified from Sf9 cells as monomers devoid of G protein and reconstituted as tetramers in phospholipid vesicles. In assays with N-[(3)H]methylscopolamine, seven agonists revealed a dispersion of affinities indicative of two or more classes of sites. Unlabeled N-methylscopolamine and the antagonist quinuclidinylbenzilate recognized one class of sites; atropine recognized two classes with a preference that was the opposite of that of agonists, as indicated by the effects of N-ethylmaleimide. The data were inconsistent with an explicit model of constitutive asymmetry within a tetramer, and the fit improved markedly upon the introduction of cooperative interactions (P < 0.00001). Purified monomers appeared to be homogeneous or nearly so to all ligands except the partial agonists pilocarpine and McN-A-343, where heterogeneity emerged from intramolecular cooperativity between the orthosteric site and an allosteric site. The breadth of each dispersion was quantified empirically as the area between the fitted curve for two classes of sites and the theoretical curve for a single class of lower affinity, which approximates the expected effect of GTP if a G protein were present. The areas measured for 10 ligands at reconstituted tetramers correlated with similar measures of heterogeneity and with intrinsic activities reported previously for binding and response in natural membranes (P ≤ 0.00002). The data suggest that the GTP-sensitive heterogeneity typically revealed by agonists at M2 receptors is intrinsic to the receptor in its tetrameric state. It exists independently of the G protein, and it appears to arise at least in part from cooperativity between linked orthosteric sites.

Binding of orthosteric ligands to the allosteric site of the M(2) muscarinic cholinergic receptor.

The M(2) muscarinic receptor has two topographically distinct sites: the orthosteric site and an allosteric site recognized by compounds such as gallamine. It also can exhibit cooperative effects in the binding of orthosteric ligands, presumably to the orthosteric sites within an oligomer. Such effects would be difficult to interpret, however, if those ligands also bound to the allosteric site. Monomers of the hemagglutinin (HA)- and FLAG-tagged human M(2) receptor therefore have been purified from coinfected Sf9 cells and examined for any effect of the antagonist N-methyl scopolamine or the agonist oxotremorine-M on the rate at which N-[(3)H]methyl scopolamine dissociates from the orthosteric site (k(obsd)). The predominantly monomeric status was confirmed by coimmunoprecipitation and by cross-linking with bis(sulfosuccinimidyl)suberate. Both N-methyl scopolamine and oxotremorine-M acted in a cooperative manner to decrease k(obsd) by 4.5- and 9.1-fold, respectively; the corresponding estimates of affinity (log K(L)) are -2.55 +/- 0.13 and -2.29 +/- 0.14. Gallamine and the allosteric ligand obidoxime decreased k(obsd) by more than 100-fold (log K(L) = -4.12 +/- 0.04) and by only 1.1-fold (log K(L) = -1.73 +/- 0.91), respectively. Obidoxime reversed the effect of N-methyl scopolamine, oxotremorine-M, and gallamine in a manner that could be described by a model in which all four ligands compete for a common allosteric site. Ligands generally assumed to be exclusively orthosteric therefore can act at the allosteric site of the M(2) receptor, albeit at comparatively high concentrations.

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles.

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.