Identification of a Novel Subtype-Selective α1B-Adrenoceptor Antagonist.

α1A-, α1B-, and α1D-adrenoceptors (α1-ARs) are members of the adrenoceptor G protein-coupled receptor family that are activated by adrenaline (epinephrine) and noradrenaline. α1-ARs are clinically targeted using antagonists that have minimal subtype selectivity, such as prazosin and tamsulosin, to treat hypertension and benign prostatic hyperplasia, respectively. Abundant expression of α1-ARs in the heart and central nervous system (CNS) makes these receptors potential targets for the treatment of cardiovascular and CNS disorders, such as heart failure, epilepsy, and Alzheimer's disease. Our understanding of the precise physiological roles of α1-ARs, however, and their involvement in disease has been hindered by the lack of sufficiently subtype-selective tool compounds, especially for α1B-AR. Here, we report the discovery of 4-[(2-hydroxyethyl)amino]-6-methyl-2H-chromen-2-one (Cpd1), as an α1B-AR antagonist that has 10-15-fold selectivity over α1A-AR and α1D-AR. Through computational and site-directed mutagenesis studies, we have identified the binding site of Cpd1 in α1B-AR and propose the molecular basis of α1B-AR selectivity, where the nonconserved V19745.52 residue plays a major role, with contributions from L3146.55 within the α1B-AR pocket. By exploring the structure-activity relationships of Cpd1 at α1B-AR, we have also identified 3-[(cyclohexylamino)methyl]-6-methylquinolin-2(1H)-one (Cpd24), which has a stronger binding affinity than Cpd1, albeit with reduced selectivity for α1B-AR. Cpd1 and Cpd24 represent potential leads for α1B-AR-selective drug discovery and novel tool molecules to further study the physiology of α1-ARs.

Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R.

The colony-stimulating factor-1 receptor (CSF1R) is a tyrosine-protein kinase that is a potential target for asthma therapeutics. We have applied a fragment-lead combination approach to identify small fragments that act synergistically with GW2580, a known inhibitor of CSF1R. Two fragment libraries were screened in combination with GW2580 by surface plasmon resonance (SPR). Binding affinity measurements confirmed that thirteen fragments bind specifically to the CSF1R, and a kinase activity assay further validated the inhibitory effect of these fragments. Several fragment compounds enhanced the inhibitory activity of the lead inhibitor. Computational solvent mapping, molecular docking, and modeling studies suggest that some of these fragments bind adjacent to the binding site of the lead inhibitor and further stabilize the inhibitor-bound state. Modeling results guided the computational fragment-linking approach to design potential next-generation compounds. The inhalability of these proposed compounds was predicted using quantitative structure-property relationships (QSPR) modeling based on an analysis of 71 drugs currently on the market. This work provides new insights into the development of inhalable small molecule therapeutics for asthma.

Identification of small molecule inhibitors against MMP-14 via High-Throughput screening.

Matrix metalloproteinases (MMPs) are involved in various cellular events in physiology and pathophysiology through endopeptidases activity. The expression levels and activities of most MMPs remain minimal in the normal conditions, whereas some MMPs are significantly activated in pathological conditions such as cancer and neovascularization. Hence, MMPs are considered as both diagnostic markers and potential targets for therapeutic agents. Twenty-three known human MMPs share a similar active site structure with a zinc-binding motif, resulting in lack of specificity. Therefore, the enhancement of target specificity is a primary goal for the development of specific MMP inhibitors. MMP-14 regulates VEGFA/VEGFR2-system through cleavage of the non-functional VEGFR1 in vascular angiogenesis. In this study, we developed a fluorescence-based enzymatic assay using a specific MMP-14 substrate generated from VEGFR1 cleavage site. This well optimized assay was used as a primary screen method to identify MMP-14 specific inhibitors from 1,200 Prestwick FDA-approved drug library. Of ten initial hits, two compounds showed IC50 values below 30 µM, which were further validated by direct binding analysis using surface plasmon resonance (SPR). Clioquinol and chloroxine, both of which contain a quinoline structure, were identified as MMP-14 inhibitors. Five analogs were tested, four of which were found to be completely devoid of inhibitory activity. Clioquinol exhibited selectivity towards MMP-14, as it showed no inhibitory activity towards four other MMPs.

Mapping the energy landscape of PROTAC-mediated protein-protein interactions.

A principal challenge in computational modeling of macromolecules is the vast conformational space that arises out of large numbers of atomic degrees of freedom. Recently, growing interest in building predictive models of complexes mediated by Proteolysis Targeting Chimeras (PROTACs) has led to the application of state-of-the-art computational techniques to tackle this problem. However, repurposing existing tools to carry out protein-protein docking and linker conformer generation independently results in extensive sampling of structures incompatible with PROTAC-mediated complex formation. Here we show that it is possible to restrict the search to the space of protein-protein conformations that can be bridged by a PROTAC molecule with a given linker composition by using a cyclic coordinate descent algorithm to position PROTACs into complex-bound configurations. We use this methodology to construct potential energy and solvation energy landscapes of PROTAC-mediated interactions. Our results suggest that desolvation of amino acids at interfaces could play a dominant role in PROTAC-mediated complex formation.

Evaluation of interactions between the hepatitis C virus NS3/4A and sulfonamidobenzamide based molecules using molecular docking, molecular dynamics simulations and binding free energy calculations.

The Hepatitis C Virus (HCV) NS3/4A is an attractive target for the treatment of Hepatitis C infection. Herein, we present an investigation of HCV NS3/4A inhibitors based on a sulfonamidobenzamide scaffold. Inhibitor interactions with HCV NS3/4A were explored by molecular docking, molecular dynamics simulations, and MM/PBSA binding free energy calculations. All of the inhibitors adopt similar molecular docking poses in the catalytic site of the protease that are stabilized by hydrogen bond interactions with G137 and the catalytic S139, which are known to be important for potency and binding stability. The quantitative assessments of binding free energies from MM/PBSA correlate well with the experimental results, with a high coefficient of determination, R2 of 0.92. Binding free energy decomposition analyses elucidate the different contributions of Q41, F43, H57, R109, K136, G137, S138, S139, A156, M485, and Q526 in binding different inhibitors. The importance of these sidechain contributions was further confirmed by computational alanine scanning mutagenesis. In addition, the sidechains of K136 and S139 show crucial but distinct contributions to inhibitor binding with HCV NS3/4A. The structural basis of the potency has been elucidated, demonstrating the importance of the R155 sidechain conformation. This extensive exploration of binding energies and interactions between these compounds and HCV NS3/4A at the atomic level should benefit future antiviral drug design.

Crystal structure of the α1B-adrenergic receptor reveals molecular determinants of selective ligand recognition.

α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype.

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1.

Cell-cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS1). While several experimental structures show how NT binds NTS1, details about the structural dynamics of NT during and after binding NTS1, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS1 suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS1. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS1, residue Y11 can exist in two χ1 rotameric states, gauche plus (g+) or gauche minus (g-). Since only the g+ Y11 state is observed in all the structures solved to date, we asked if the g- state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS1 with the same affinity as NT10-13 but did not activate NTS1, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS1.

Probing the correlation between ligand efficacy and conformational diversity at the α1A-adrenoreceptor reveals allosteric coupling of its microswitches.

G protein-coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein-binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1A-adrenoreceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of [13CϵH3]methionine-labeled α1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. [13CϵH3]Methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α1A-adrenoreceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

INPHARMA-Based Determination of Ligand Binding Modes at α1 -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity.

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α1A -AR and α1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.

Diazepam is not a direct allosteric modulator of α1-adrenoceptors, but modulates receptor signaling by inhibiting phosphodiesterase-4.

α1A- and α1B-adrenoceptors (ARs) are G protein-coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α1A- and α1B-AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α1-ARs. Here, using thermostabilized, purified, α1A- and α1B-ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α1A- and α1B-ARs. Similarly, in cell-based assays expressing unmodified α1A- and α1B-ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist-stimulated Ca2+ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α1A- and α1B-ARs; however, this was shown to be caused by off-target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

Determinants of Ligand Subtype-Selectivity at α1A-Adrenoceptor Revealed Using Saturation Transfer Difference (STD) NMR.

α1A- and α1B-adrenoceptors (α1A-AR and α1B-AR) are closely related G protein-coupled receptors (GPCRs) that modulate the cardiovascular and nervous systems in response to binding epinephrine and norepinephrine. The GPCR gene superfamily is made up of numerous subfamilies that, like α1A-AR and α1B-AR, are activated by the same endogenous agonists but may modulate different physiological processes. A major challenge in GPCR research and drug discovery is determining how compounds interact with receptors at the molecular level, especially to assist in the optimization of drug leads. Nuclear magnetic resonance spectroscopy (NMR) can provide great insight into ligand-binding epitopes, modes, and kinetics. Ideally, ligand-based NMR methods require purified, well-behaved protein samples. The instability of GPCRs upon purification in detergents, however, makes the application of NMR to study ligand binding challenging. Here, stabilized α1A-AR and α1B-AR variants were engineered using Cellular High-throughput Encapsulation, Solubilization, and Screening (CHESS), allowing the analysis of ligand binding with Saturation Transfer Difference NMR (STD NMR). STD NMR was used to map the binding epitopes of epinephrine and A-61603 to both receptors, revealing the molecular determinants for the selectivity of A-61603 for α1A-AR over α1B-AR. The use of stabilized GPCRs for ligand-observed NMR experiments will lead to a deeper understanding of binding processes and assist structure-based drug design.