Alminoprofen

GPR56 is a widely expressed adhesion GPCR (AGPCR) that has pleotropic roles in brain development, platelet function, cancer, and more. Nearly all AGPCRs possess extracellular regions that bind protein ligands and conceal a cryptic tethered peptide agonist. AGPCR reception of mechanical or shear force is thought to release the tethered agonist permitting its binding to the AGPCR orthosteric site for consequent activation of G protein signaling. This multistep mechanism of AGPCR activation is difficult to target, emphasizing the need for tool compounds and potential therapeutics that modulate AGPCRs directly. We expanded our cell-based pilot screen for GPR56 small molecule activators to screen >200,000 compounds and identified two promising agonists: 2-(furan-2-yl)-1-[(4-phenylphenyl)carbonyl]pyrrolidine, or compound 4, and propan-2-yl-4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, or compound 36. Both compounds activated GPR56 receptors enginered to have impaired tethered agonists and/or be cleavage deficient. Compound 4 activated a subset of group VIII AGPCRs while compound 36 had exclusive specificity for GPR56 among the GPCRs tested. Compound 36 SAR analysis identified an analog with the isopropyl R group replaced with a cyclopentyl ring and the electrophilic bromine replaced with a CF₃ group. Analog 36.40 had 40% increased potency over compound 36 and was 20-fold more potent than synthetic peptidomimetics designed from the GPR56 tethered agonist. The new GPCR56 tool compounds discovered in this screen may be used to further advance understanding of GPR56 function and aid development of AGPCR-targeted therapeutics. SIGNIFICANCE STATEMENT: Adhesion G protein coupled receptors (AGPCRs) are a large, clinically relevant class of GPCRs with no available therapeutics, in part due to their unique mechanism of activation. GPR56 is a widely expressed model AGPCR involved in cancer metastasis, hemostasis, and neuron myelination. In the present study, we identified novel small molecule agonists for GPR56. These molecules are among the most potent identified thus far and may become useful leads in the development of a GPR56-targeted therapeutic.

Brassinosteroids (BRs) are plant steroid hormones that are involved in the regulation of plant growth and development as well as environmental adaptation. The discovery of new BR derivatives is beneficial to their biosynthesis and regulation mechanisms research. However, there are few reports on the methods for exploring new BRs, and the existing methods tend to lack coverage. In this work, we established a comprehensive, highly sensitive and selective structure-oriented method for the screening and structural identification of potential BRs in plants using chemical isotope labeling-assisted liquid chromatography-high resolution mass spectrometry (CL-LC-HRMS). The potential BRs were speculated according to the structural features of the reported BRs, and those speculated BRs containing cis-diol groups were labeled by isotope reagents of 2-methyl-4-phenylaminomethylphenylboronic acid (2-methyl-4-PAMBA) and 2-methyl-4-PAMBA-d₅ to improve the sensitivity and selectivity of MS detection. In addition, the fragmentation of 2-methyl-4-PAMBA-labeled BRs via collision-induced dissociation (CID) led to the generation of reporter ions, which contributed to specific screening of potential BRs. In-depth structure of potential BRs was elucidated by analyzing multistage MS (MSⁿ) fragmentation patterns. Using our proposed method, a total of 16 potential BRs were detected from plant samples including 5 new ones, of which one new BR derivative was identified as 2-deoxy-3-epi-6-deoxy-dolichosterone, and this new BR may be involved in the biosynthesis of BRs as precursor of 6-deoxo-dolichosterone. The method developed in this work is promising for screening and identifying new BR derivatives in plants, thereby supplementing the biosynthesis pathway of BRs.