Conformationally Selective 2-Aminotetralin Ligands Targeting the alpha2A- and alpha2C-Adrenergic Receptors

Abstract

Many important physiological processes are mediated by alpha2A-and alpha2C-adrenergic receptors (α2Rs), a subtype of class A G protein-coupled receptors (GPCRs). However, α2R signaling is poorly understood, and there are few approved medications targeting these receptors. Drug discovery aimed at α2Rs is complicated by the high degree of binding pocket homology between α2AR and α2CR, which confounds ligandmediated selective activation or inactivation of signaling associated with a particular subtype. Meanwhile, α2R signaling is complex and it is reported that activating α2AR is beneficial in many clinical contexts, while activating α2CR signaling may be detrimental to these positive effects. Here, we report on a novel 5-substituted-2aminotetralin (5-SAT) chemotype that, depending on substitution, has diverse pharmacological activities at α2Rs. Certain lead 5-SAT analogues act as partial agonists at α2ARs, while functioning as inverse agonists at α2CRs, a novel pharmacological profile. Leads demonstrate high potency (e.g., EC 50 < 2 nM) at the α2AR and α2CRs regarding Gα i -mediated inhibition of adenylyl cyclase and production of cyclic adenosine monophosphate (cAMP). To help understand the molecular basis of 5-SAT α2R multifaceted functional activity, α2AR and α2CR molecular models were built from the crystal structures and 1 μs molecular dynamics (MD) simulations and molecular docking experiments were performed for a lead 5-SAT with α2AR agonist and α2CR inverse agonist activity, i.e., (2S)-5-(2′-fluorophenyl)-N,N-dimethyl-1,2,3,4-tetrahydronaphthalen-2-amine (FPT), in comparison to the FDA-approved (for opioid withdrawal symptoms) α2AR/α2CR agonist lofexidine. Results reveal several interactions between FPT and α2AR and α2CR amino acids that may impact the functional activity. The computational data in conjunction with experimental in vitro affinity and function results provide information to understand ligand stabilization of functionally distinct GPCR conformations regarding α2AR and α2CRs.

Development of 2-Aminotetralin-Type Serotonin 5-HT1 Agonists: Molecular Determinants for Selective Binding and Signaling at 5-HT1A, 5-HT1B, 5-HT1D, and 5-HT1F Receptors

“…Syntheses of 5-SATs presented in Table have been reported previously. , Affinities of 5-SATs and 5-HT 1 reference compounds were evaluated by radioligand competition binding assays using [ 3 H]­5-CT as the radioligand for 5-HT 1A , 5-HT 1B , and 5-HT 1D receptors and [ 3 H]­5-HT for the 5-HT 1F receptor (Table ). The K D values for [ 3 H]­5-CT at 5-HT 1A , 5-HT 1B , 5-HT 1D were 2.5 ± 0.2, 5.3 ± 0.5, and 0.82 ± 0.1 nM, respectively; the K D for [ 3 H]­5-HT at 5-HT 1F was 7.8 ± 2.3 nM.…”

“…Docks of 5-CT and 5-SAT analogues at 5-HT 1 receptors were developed following a procedure previously used by our lab. , Three-dimensional ligands were constructed in Masetro (Schrodinger, NY) and optimized via an ab initio quantum chemistry method at the HF/6-31G* level, followed by single-point energy calculations of the molecular electrostatic potential for charge fitting with Gaussian 16 . These docks were formulated using the solved cyro-EM structures of 5-HT 1A (PDB code 7E2Y) and 5-HT 1D (PDB code 7E32) and the solved crystal structure of 5-HT 1B (PDB code 4IAR) all in the active state, with omissions of sidechains and loops added and recapitulated using BIOVIA’s Discovery Studio 2017 (Dassault Systems, Waltham, MA).…”

“…Cell Culture techniques followed procedures previously published by our lab. ,,,, Briefly, HEK293T cells were obtained from ATTC (Manassas, VA) and cultured in DMEM supplemented with 10% regular FBS (%v/v) and 1% penicillin (%v/v) at 37 °C in a humidified incubator kept at 5% CO 2 . Cells were passaged at 80% confluency up until passage 20.…”

“…48 hours after transfection with the desired receptor construct, cells were isolated for saturation and competition binding assays as previously described. ,, Saturation and competition radioligand binding assays were conducted in an assay buffer consisting of 50 mM Tris HCl, 10 mM MgCl 2 , and 0.1 mM EDTA in filtered deionized water. Briefly, saturation binding assays were conducted to determine the K D of [ 3 H]­5-CT at the 5-HT 1A , 5-HT 1B , and 5-HT 1D receptor constructs, and [ 3 H]­5-HT was the radioligand for the 5-HT 1F receptor.…”

“…Our lead 5-SAT FPT (Table ) is an agonist at 5-HT 1A , 5-HT 1B , and 5-HT 1D receptors that reduced repetitive behaviors and spontaneous seizures as well as increased prosocial behaviors in a mouse model of fragile X syndrome, the most common monogenetic form of autism spectrum disorder; moreover, FPT was orally active and did not cause sedation . The contribution of each 5-HT 1 receptor subtype regarding FPT preclinical neurotherapeutic activities is unknown, however, and FPT has other receptor activities. − …”