Computational investigation of the impact of potential AT2R polymorphism on small molecule binding.

For more than a century, the renin-angiotensin system (RAS) has been acknowledged for playing a crucial part in the physiological control of arterial pressure, as well as sodium and fluid balance. It is now generally acknowledged that one of the receptor of RAS system i.e. angiotensin type 2 receptor (AT2R) functions as a repair system during pathophysiologic circumstances and performs a significant protective role. Efforts have been made previously to design suitable agonist and antagonist molecules to potentially modulate AT2R. One of the agonists and antagonists, named C21 and EMA401, has been studied in a number of pathological conditions. Additionally, a wide panel of single nucleotide polymorphisms (SNPs) has been reported for AT2R, which might potentially affect the efficacy of these molecules. Therefore, computational investigations have been carried out to analyze all the SNPs (1151) reported in NCBI to find potential SNPs affecting the active site of AT2R, as this domain is still unexplored. Structures of these polymorphic forms were modeled, and in silico drug interaction studies with C21 and EMA401 were carried out. The two mutants (rs868939201 and rs1042852794) that significantly affect the binding affinity as that of the wild type were subjected to molecular dynamics simulations. Our analysis of native and mutant AT2R and their complexes with C21 and EMA401 indicated that the occurrence of these mutations affects the conformation of the protein and has affected the binding of these ligand molecules. The study's findings will aid in the development of better, more versatile medications in the near future, and also in vitro and in vivo studies might be planned in accordance with recent findings.Communicated by Ramaswamy H. Sarma.

Functional assay for assessment of agonistic or antagonistic activity of angiotensin AT2 receptor ligands reveals that EMA401 and PD123319 have agonistic properties.

With the discovery of the protective arm of the renin-angiotensin system (RAS), interest has grown in protective RAS-related receptors such as the angiotensin AT2-receptor [AT2R] as potential new drug targets. While it is known that AT2R couple to Gi, it is also apparent that they do not signal via inhibition of adenylyl cyclase/decrease in cAMP, as do many Gi-coupled receptors. Thus, standard commercially-available assays cannot be applied to test for agonistic or antagonistic properties of AT2R ligands. This lack of standard assays has hampered the development of new drugs targeting the AT2R. Therefore, we aimed at developing a reliable, technically easy assay for the determination of intrinsic activity of AT2R ligands, primarily for distinguishing between AT2R agonists and antagonists. We found that measurement of NO release by DAF-FM fluorescence in primary human aortic endothelial cells (HAEC) or in AT2R-transfected CHO cells is a reliable assay for the characterization of AT2R ligands. While testing the assay, we made several novel findings, including: a) C21 is a full agonist at the AT2R (with the same efficacy as angiotensin II); b) C21 has no intrinsic activity at the receptor Mas; c) AT2R-transfected HEK-293 cells are unresponsive to AT2R stimulation; d) EMA401 and PD123319, which are commonly regarded as AT2R antagonists, are partial agonists at the AT2R. Collectively, we have developed and tested an assay based on the measurement and quantification of NO release in HAEC or in AT2R-CHO cells that is suitable for the characterisation of novel and established AT2R ligands.

Emerging therapies in clinical development and new contributions for neuropathic pain. / Terapias emergentes en desarrollo clínico y nuevas aportaciones en dolor neuropático.

Neuropathic pain is very challenging to manage because of the heterogeneity of aetiologies, symptoms, and underlying mechanisms. Conventional oral therapies have been limited by negative factors such as systemic side effects, drug-drug interactions, slow onset of action, the need for titration, multiple daily dosing, as well as the potential risk of addiction, dependence, withdrawal symptoms and abuse. Therefore, new therapeutic perspectives are justified. New drugs that act on different therapeutic targets are currently in preclinical development or in their first phases of clinical development. In this review, focus will be directed specifically on new pharmacological treatments for neuropathic pain for which clinical data are already available, including older and known drugs with new data on their anti-neuropathic activity.

EMA401: an old antagonist of the AT2R for a new indication in neuropathic pain.

EMA401 is an old molecule, synthesized by Parke-Davis in the last century and characterized at that time as an AT2R antagonist. Professor Maree Smith and her group from the University of Queensland (Australia) patented the drug and many related derivatives and other compounds with high affinity for the AT2R for the indication neuropathic pain in 2004, an example of drug repositioning. After some years of university work, the Australian biotech company Spinifex Pharmaceuticals took over further research and development and characterized the S-enantiomer, code name EMA401, and related compounds in a variety of animal models for neuropathic and cancer pain. EMA401 was selected as the lead compound, based on high selectivity for the AT2R and good oral bioavailability (33%). EMA401, however, was only administered once in a chronic neuropathic pain model, and no data have been published in other pain models, or during steady state, although such data were available for the racemate EMA400 and some related compounds (EMA200, EMA400). A pilot phase IIa study demonstrated the efficacy and safety of the drug taken twice daily as two capsules of 50 mg (400 mg/day). In 2015, Novartis took over the clinical development. Two phase IIb studies designed by Spinifex Pharmaceuticals were put on hold, probably because Novartis wanted to improve the clinical design or collect additional preclinical data. Further data are eagerly awaited, especially since EMA401 is first-in-class in neuropathic pain.

Analgesic effect of angiotensin angiotensin Ⅱ type 2 receptor antagonist EMA401 on neuropathic pain in rats and its mechanism / 中国病理生理杂志

AIM:To explore whether angiotensin Ⅱtype 2 receptor antagonist EMA 401 decreases neuropathic pain and the expression of growth-associated protein-43 (GAP-43), protein kinase C (PKC) and calmodulin (CaM) in dorsal root ganglia (DRG) during chronic constriction injury (CCI) in rats.METHODS:SD rats were used to establish CCI model and randomly divided into 4 groups.The rats in model group were given equal volume of normal saline by intra-gastric administration .The rats in low dose ( LD) group were given 5 mg/kg EMA401 by intragastric administration .The rats in middle dose ( MD) group were given 10 mg/kg EMA401 by intragastric administration .The rats high dose ( HD) group were given 20 mg/kg EMA401 by intragastric administration .The rats in sham operation group received equal volume of normal saline by intragastric administration .Thermal withdrawal latency ( TWL ) and mechanical withdrawal threshold (MWT) were measured before operation and 7 d, 14 d and 28 d after CCI.After behavioral test, DRG of lumbar spinal was obtained from each group , and was used to determine Ca 2+concentration by o-cresolphthalein complexone microplating method, and the expression of GAP-43, PKC and CaM at mRNA and protein levels by Western blotting and RT-PCR.RE-SULTS:Compared with model group, EMA401 significantly increased the TWL and MWT (P <0.05).Meanwhile, EMA401 significantly reduced Ca 2+concentration and the expression of GAP-43, PKC and CaM at mRNA and protein levels in the DRG (P<0.05).CONCLUSION:EMA401 may attenuate neuropathic pain of CCI by inhibiting Ca 2+concentra-tion and the expression of GAP-43, PKC and CaM.