Molecular dynamics simulations to explore the active/inactive conformers of guinea pig β2 adrenoceptor for the selective design of agonists or antagonists

It is well known that guinea pig β₂ adrenoceptors (Gβ₂ARs) and human β₂ adrenoceptors (Hβ₂ARs) have structural similarity. However, only one conformational state of Gβ₂ARs has been studied – the putative inactive state. As adrenoceptors have a repertoire of conformations, and there is evidence that a certain conformation is stabilised as a ligand approaches, the aim of this study was to build four models of Gβ₂ARs by using putative active/inactive Hβ₂AR conformers as a template. We evaluated the accuracy of these models in regard to the binding mode and affinity values of a set of known β₂AR ligands through docking and molecular dynamics simulations. During docking simulations, ligands reached Gβ₂AR sites similar to those reported for Hβ₂ARs. The greatest differences between conformational states were found in the domains (TM5 and TM6) previously suggested as being key to ligand recognition. The coefficients of determination between experimental and calculated affinity values were near to but less than 0.66 in all cases. The highest values were for agonists on the active models and antagonists on the inactive model. The four Gβ₂AR models proved useful for analysing agonist/antagonist activity. The results suggest that the selection of an adequate model is dependent on the intrinsic activity of a given ligand.     

A multivalent approach to the discovery of long-acting β(2)-adrenoceptor agonists for the treatment of asthma and COPD

A multivalent approach was applied to the design of long-acting inhaled β(2)-adrenoceptor agonists. A series of dimeric arylethanolamines based on the short acting β(2)-adrenoceptor agonist albuterol were prepared, varying the nature and length of the linker between the basic nitrogens. None of the C(2)-symmetric dimers demonstrated increased potency, however dimer 5j, derived from 4-phenethylamine, was found to have increased binding potency in vitro relative to the parent monomer. Optimization of this structure led to the identification of 22 (milveterol) which demonstrates high potency in vitro and long duration of action in a guinea pig model of bronchoprotection.     

Design,synthesis and evaluation of dual pharmacology β2-adrenoceptor agonists and PDE4 inhibitors

A novel series of formoterol–phthalazinone hybrids were synthesised and evaluated as dual pharmacology β₂-adrenoceptor agonists and PDE4 inhibitors. Most of the hybrids displayed high β₂-adrenoceptor agonist and moderate PDE4 inhibitory activities. The most potent compound, (R,R)-11c, exhibited agonist (EC₅₀ = 1.05 nM, pEC₅₀ = 9.0) and potent PDE4B2 inhibitory activities (IC₅₀ = 0.092 μM).     

Structure-guided development of dual β2 adrenergic/dopamine D2 receptor agonists

Aiming to discover dual-acting β₂ adrenergic/dopamine D₂ receptor ligands, a structure-guided approach for the evolution of GPCR agonists that address multiple targets was elaborated. Starting from GPCR crystal structures, we describe the design, synthesis and biological investigation of a defined set of compounds leading to the identification of the benzoxazinone (R)-3, which shows agonist properties at the adrenergic β₂ receptor and substantial G protein-promoted activation at the D₂ receptor. This directed approach yielded molecular probes with tuned dual activity. The congener desOH-3 devoid of the benzylic hydroxyl function was shown to be a β₂ adrenergic antagonist/D₂ receptor agonist with K_i values in the low nanomolar range. The compounds may serve as a promising starting point for the investigation and treatment of neurological disorders.     

Investigation of piperazine benzamides as human β3 adrenergic receptor agonists for the treatment of overactive bladder

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human β₃-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established β₃ potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.     

Dual β2-adrenoceptor agonists-PDE4 inhibitors for the treatment of asthma and COPD

We designed and synthesized a novel class of dual pharmacology bronchodilators targeting both β(2)-adrenoceptor and PDE4 by applying a multivalent approach. The most potent dual pharmacology molecule, compound 29, possessed good inhibitory activity on PDE4B2 (IC(50)=0.278 μM, which was more potent than phthalazinone, IC(50)=0.520 μM) and possessed excellent relaxant effects on tracheal rings precontracted by histamine (pEC(50)=9.3).     

Multivalent design of long-acting β2-adrenoceptor agonists incorporating biarylamines

A series of potent β₂-adrenoceptor agonists incorporating a biarylamine secondary binding group was identified. The previously reported milveterol (5), identified by a multivalent approach and containing a typical β₂-agonist primary binding group linked via a phenethylamine linker to a hydrophilic secondary binding group, served as an initiation point. A more hydrophobic set of secondary binding groups was explored, prepared rapidly from a common intermediate by Buchwald–Hartwig amination. TD-5471 (25), a potent and selective full agonist of the human β₂-adrenoceptor, was identified as the most promising agent. It is potent, with slow onset in an in vitro guinea pig trachea model and shows a dose-dependent and long duration of action in an in vivo guinea pig model of bronchoprotection. TD-5471 is structurally differentiated from milveterol and its long duration of action is consistent with a correlation with hydrophobicity observed in other long-acting β₂-agonist discovery programs.     

Development of covalent antagonists for β1- and β2-adrenergic receptors

The selective covalent tethering of ligands to a specific GPCR binding site has attracted considerable interest in structural biology, molecular pharmacology and drug design. We recently reported on a covalently binding noradrenaline analog (FAUC37) facilitating crystallization of the β₂-adrenergic receptor (β₂AR^H2.64C) in an active state. We herein present the stereospecific synthesis of covalently binding disulfide ligands based on the pharmacophores of adrenergic β₁- and β₂ receptor antagonists. Radioligand depletion experiments revealed that the disulfide-functionalized ligands were able to rapidly form a covalent bond with a specific cysteine residue of the receptor mutants β₁AR^I2.64C and β₂AR^H2.64C. The propranolol derivative (S)-1a induced nearly complete irreversible blockage of the β₂AR^H2.64C within 30 min incubation. The CGP20712A-based ligand (S)-4 showed efficient covalent blocking of the β₂AR^H2.64C at very low concentrations. The analog (S)-5a revealed extraordinary covalent cross-linking at the β₁AR^I2.64C and β₂AR^H2.64C mutant while retaining a 41-fold selectivity for the β₁AR wild type over β₂AR. These compounds may serve as valuable molecular tools for studying β₁/β₂ subtype selectivity or investigations on GPCR trafficking and dimerization.     

Discovery of TD-4306, a long-acting β2-agonist for the treatment of asthma and COPD

A multivalent approach focused on amine-based secondary binding groups was applied to the discovery of long-acting inhaled β₂-agonists. Addition of amine moieties to the neutral secondary binding group of an existing β₂-agonist series was found to provide improved in vivo efficacy, but also led to the formation of biologically active aldehyde metabolites which were viewed as a risk for the development of these compounds. Structural simplification of the scaffold and blocking the site of metabolism to prevent aldehyde formation afforded a potent series of dibasic β₂-agonists with improved duration of action relative to their monobasic analogs. Additional optimization led to the discovery of 29 (TD-4306), a potent and selective β₂-agonist with potential for once-daily dosing.     

Relaxation by β3-adrenoceptor agonists of the isolated human internal anal sphincter

AimsIn this study, responses of β₃-adrenoceptor agonists were examined on human isolated internal anal sphincter (IAS) in order to explore their relaxant effects on hypertonicity of IAS.Main methodsThe relaxant efficacy (E_max) and potency (? logIC₅₀) of BRL37344 and SR58611A, β₃-adrenoceptor agonists, were examined in contracted IAS muscle strips. The presence of β₃-adrenoceptors, and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by Western blotting, epifluorescence microscopy and enzyme immunoassay, respectively.Key findingsBRL37344 and SR58611A relaxed contracted IAS muscle (E_max = 27 ± 3% and 35 ± 3%; -logIC₅₀ = 6.26 ± 0.24 and 4.87 ± 0.13; respectively). These relaxant responses were blocked by SR59230A, a selective β₃-antagonist but not by β₁/β₂-selective antagonists, neuronal inhibitor or inhibition of nitric oxide synthase. The E_max of β₃-agonists was similar to that of β₂-selective agonists but smaller than that of isoprenaline (nonselective agonist) or β₁-selective agonists. BRL37344 (100 μM) increased cAMP (1.5-fold) without cGMP change, and depressed intracellular calcium signal. β₃-Adrenoceptor expression was smaller than that of β₁- and β₂-adrenoceptors.SignificanceThis is the first study demonstrating the presence of β₃-adrenoceptor in human IAS muscle and β₃-mediated relaxation of augmented sphincter tone. However, direct β₃-relaxation appears smaller than that obtained for nonselective agonists which may limit their potential use in the treatment of anorectal hypertonicity disorders.     

Hybrids consisting of the pharmacophores of salmeterol and roflumilast or phthalazinone: Dual β2-adrenoceptor agonists-PDE4 inhibitors for the treatment of COPD

A novel class of dual pharmacology bronchodilators targeting both β₂-adrenoceptor and PDE4 was designed and synthesised by combining the pharmacophores of salmeterol and roflumilast or phthalazinone. All the compounds exhibited better β₂-adrenoceptor agonist activities (pEC₅₀ = 8.47–9.20) than the reference compound salmeterol (pEC₅₀ = 8.3) and good inhibitory activity on PDE4B2 (IC₅₀ = 0.235–1.093 μM).     

Structure based docking and molecular dynamics studies: Peroxisome proliferator-activated receptors –α/γ dual agonists for treatment of metabolic disorders

Abstract

Diabetes is a foremost health problem globally susceptible to increased mortality and morbidity. The present therapies in the antidiabetic class have sound adverse effects and thus, emphasis on the further need to develop effective medication therapy. Peroxisome proliferator-activated receptor alpha-gamma dual approach represents an interesting target for developing novel anti-diabetic drug along with potential anti-hyperlipidimic activity. In the current study, the peroxisome proliferator-activated receptor alpha-gamma agonistic hits were screened by hierarchical virtual screening of drug like compounds followed by molecular dynamics simulation and knowledge-based structure-activity relation analysis. The key amino acid residues of binding pockets of both target proteins were acknowledged as essential and were found to be associated in the key interactions with the most potential dual hit. This dual targeted approach of structure based computational technique was undertaken to identify prevalent promising hits for both targets with binding energy and absorption distribution metabolism excretion prediction supported the analysis of their pharmacokinetic potential. In addition, stability analysis using molecular dynamics simulation of the target protein complexes was performed with the most promising dual targeted hit found in this study. Further, comparative analysis of binding site of both targets was done for the development of knowledge-based structure-activity relationship, which may useful for successful designing of dual agonistic candidates. Abbreviations ADME absorption distribution metabolism excretion

HTVS highthroughput virtual screening

MD molecular dynamics

MMGBSA molecular mechanics generalized bonn solvation accessible

PDB protein data bank

PPAR peroxisome proliferator-activated receptor

RMSD Root mean square deviation

RMSF Root mean square fluctuation

SAR structural activity relationship

SP simple precision

T2DM TypeII diabetes mellitus

XP Extra precision

Communicated by Ramaswamy H. Sarma     

Virtual identification of novel PPARα/γ dual agonists by scaffold hopping of saroglitazar

The thiazolidinedione class PPARγ agonists as antidiabetic agents are restricted in clinical use because of the side effects such as edema, weight gain, and heart failure. The single and selective agonism of PPARγ is the main cause of side effects. The multi-target cooperative PPARα/γ dual agonist development is a hot topic in the antidiabetic medicinal chemistry field. Saroglitazar is the first approved PPARα/γ dual agonist, available in India for the treatment of diabetic dyslipidemia. It got rid of these side effects. With the aim of finding more protent PPARα/γ dual agonists, the scaffold hopping was used to replace α-o phenylpropionic acid skeleton of saroglitazar with L-tyrosine skeleton. Then, the structural modification was carried out designing 72 compounds. Considering the importance of chirality, opposite configuration of 72 compounds was also studied. 12 compounds with better -cdocker energy were screened by molecular docking. Subsequently, the pharmacokinetic properties and toxicity evaluated by ADMET prediction, 11 of them showed better properties. Comp#L-17-1 and comp#L-3-1 were regarded as representatives to study the binding stability by molecular dynamics (MD) simulations. The MD simulation results of comp#L-17-1-PPARs (α, γ) and comp#L-3-1-PPARs (α, γ) provided structure reference for the research and development of novel PPARα/γ dual agonists.     

Structural basis of C-terminal β-amyloid peptide binding by the antibody ponezumab for the treatment of Alzheimer's disease

Alzheimer's disease, the most common cause of dementia in the elderly and characterized by the deposition and accumulation of plaques, is composed in part of β-amyloid (Aβ) peptides, loss of neurons, and the accumulation of neurofibrillary tangles. Here, we describe ponezumab, a humanized monoclonal antibody, and show how it binds specifically to the carboxyl (C)-terminus of Aβ40. Ponezumab can label Aβ that is deposited in brain parenchyma found in sections from Alzheimer's disease casualties and in transgenic mouse models that overexpress Aβ. Importantly, ponezumab does not label full-length, non-cleaved amyloid precursor protein on the cell surface. The C-terminal epitope of the soluble Aβ present in the circulation appears to be available for ponezumab binding because systemic administration of ponezumab greatly elevates plasma Aβ40 levels in a dose-dependent fashion after administration to a mouse model that overexpress human Aβ. Administration of ponezumab to transgenic mice also led to a dose-dependent reduction in hippocampal amyloid load. To further explore the nature of ponezumab binding to Aβ40, we determined the X-ray crystal structure of ponezumab in complex with Aβ40 and found that the Aβ40 carboxyl moiety makes extensive contacts with ponezumab. Furthermore, the structure-function analysis supported this critical requirement for carboxy group of AβV40 in the Aβ-ponezumab interaction. These findings provide novel structural insights into the in vivo conformation of the C-terminus of Aβ40 and the brain Aβ-lowering efficacy that we observed following administration of ponezumab in transgenic mouse models.     

Binding of agonists and antagonists to the porcine adipose tissue β-adrenergic receptor(s)

1. Affinities of agonists for porcine adipose tissue β-adrenergic receptors, determined by competitive ligand binding with ³H-dihydroalprenolol to crude adipose tissue membranes in vitro, varied from 50 times > to 25 times < than isoproterenol. Affinities for antagonists varied from 8 times > to 1000 times < propranolol.2. Receptor affinity was not related to the ability to stimulate or inhibit lipolysis, or to the agonist or antagonist purported receptor subtype specificity.3. Modeling of ligand-binding data indicated more than one binding site for several ligands. The assignment of β-adrenergic subtypes to the individual binding sites was unclear because this would depend on the individual ligands used to establish binding sites.     

Modeling the interaction of fipronil-related non-competitive antagonists with the GABA β3-receptor

A three-dimensional model of the beta3-homopentamer of the gamma-aminobutyric acid (GABA) receptor/chloride ionophore complex was developed by homology modeling using the cyro-electron microscopy structure of nicotinic acetylcholine as a template. Interactions between the beta3-homopentamer and two classes of fipronil-related non-competitive antagonists were investigated using docking studies. The phenyl groups of these compounds were stabilized by strong hydrophobic and hydrophilic interactions with the rings formed by Thr256 and Ala252. Leu253 and Ile255 were involved mainly in hydrophobic contact with the pyrazole moiety. Different substitution at positions 15, 16 and 17 of the pyrazole ring of fipronil resulted in weakening of the hydrogen bonds and hydrophobic interactions between the beta3-receptor and fipronil-related heterocyclic compounds, which maybe the principal cause of the decreased affinities reported in vitro. Moreover, a good correlation between total binding energies calculated by AutoDock and experimentally determined IC(50) values proved our models to be reasonable in predicting the interaction mode of the antagonist with the GABA beta3-receptor.     

Specific actions of GLP-1 receptor agonists and DPP4 inhibitors for the treatment of pancreatic β-cell impairments in type 2 diabetes

Type 2 diabetes occurs when the β-cells do not secrete enough insulin to counter balance insulin resistance. GLP-1 and GIP are insulinotropic peptides which are thought to benefit to β-cell physiology. On one hand sustained pharmacological levels of GLP-1 are achieved by subcutaneous administration of GLP-1 analogs while transient and lower physiological levels of GLP-1 are attained following DPP4 inhibitor (DPP4i) treatment. On the other hand, DPP4i increase GLP-1 concentration into the portal vein to recruit the gut-to brain-to pancreas axis which is not the case with injected analogs. Hence, these differences between GLP-1 analogs and DPP4i indicate that both strategies could differentially impact β-cell behavior. Here, we summarize the effects of GLP-1 analogs and DPP4i on β-cell physiology. We discuss the possibility that production of signaling molecules, such as cAMP, generated into the β-cells by native GLP-1 or pharmacological GLP-1 analogs may vary and engage different downstream signaling networks. Hence, deciphering which signaling networks are engaged following GLP-1 analogs or DPP4i administration appears to be critical to unveil the contribution of each treatment/strategy to engage β-cell cellular processes.     

Discovery of disubstituted piperidines and homopiperidines as potent dual NK1 receptor antagonists–serotonin reuptake transporter inhibitors for the treatment of depression

This report describes the synthesis, structure–activity relationships and activity of piperidine, homopiperidine, and azocane derivatives combining NK₁ receptor (NK₁R) antagonism and serotonin reuptake transporter (SERT) inhibition. Our studies culminated in the discovery of piperidine 2 and homopiperidine 8 as potent dual NK₁R antagonists-SERT inhibitors. Compound 2 demonstrated significant activity in the gerbil forced swimming test, suggesting that dual NK₁R antagonists-SERT inhibitors may be useful in treating depression disorders.     

Design-driven LO: the discovery of new ultra long acting dibasic β2-adrenoceptor agonists

Starting with the molecular scaffold of the DA₂/β₂ dual agonist sibenadet (Viozan™), a number of molecular changes were incorporated, which were designed to increase the potency and selectivity of the target molecule, and improve its pharmacokinetics. Through this process a novel, high potency, full β₂-agonist with high selectivity and long duration capable of being dosed once daily has been discovered.     

Lipase resolution of new (±)-3-aryloxy-1-halogenopropan-2-ols: Versatile building blocks for β-adrenergic receptor antagonists

Using two commercial immobilized lipases Lipozyme^® TL and Novozym^® 435 effective kinetic resolution of several novel 3-aryloxy-1-halogenopropan-2-ols was achieved by acyl transfer reaction in organic solvents, yielding both enantiomers with 89–99% ee. In preparative resolutions carried out in tert-butyl methyl ether at 25 °C with vinyl acetate as acyl donor enantioselectivity ratio E was from 64 to 99. The resolved enantiomers were successfully used as chiral building blocks in the synthesis of new 1-alkylamino-3-aryloxypropan-2-ols, by nucleophilic halogen substitution with isopropylamine and tert-butylamine. The obtained products will be evaluated in vitro as potential new β-adrenergic receptors antagonists.