Small Molecule Antagonists of RAGE‐DIAPH1: Novel Therapeutic Opportunities in Metabolic and Chronic Disease (2018)

Our previous work has shown that the cytoplasmic tail (ct) of RAGE is essential for RAGE ligand‐mediated signal transduction and consequent modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1, or DIAPH1. Given the complex, multi‐ligand nature of ligands binding to the extracellular domains of RAGE, we sought to discover small molecule inhibitors of the interaction of ctRAGE with DIAPH1. Prompted by our identification of 13 small molecules that block the interaction of ctRAGE‐DIAPH1, we pursued structure‐activity relationship experiments to identify analogues potentially able to delay or prevent the progression of RAGE‐related chronic diseases, such as diabetes. In vitro binding studies Native tryptophan fluorescence experiments were conducted using 1mM of compound dissolved in phosphate buffer and DMSO. 10 nM ctRAGE solution was titrated from 0.1 nM–100 μM and the dissociation constants, K d , were estimated from the changes in peak fluorescence intensities as a function of the free compound concentration. NMR experiments were performed operating at 1 H frequencies of 500 MHz and 700 MHz. NMR samples contained 50 μM of [ U‐ 15 N] ctRAGE and 10 μM of the compound. All spectra were collected at 298K, which yielded high quality NMR spectra of [ U‐ 15 N] RAGE tail. Compound binding to ctRAGE was identified as at least a 0.01 ppm change in the ctRAGE chemical shift. Small molecule leads were identified and referred to as NYU 1–5 (Table 1). Ex vivo biological activity assays Inhibition of smooth muscle cell (SMC) migration induced by RAGE ligands after incubation with indicated NYU compounds. SMCs were grown to confluence and starved overnight. The next morning, the medium was removed, compounds were added and the monolayer was wounded using a p200 pipette tip and allowed to incubate for 1.5h. Following incubation, compounds were removed and fresh medium containing the RAGE ligand, CML‐AGE (10 μg/ml) was added for 4h. Images were taken, measured and an area ingrowth of effective migrating cells was calculated (Table 1). In vivo studies (A) Delayed type hypersensitivity. Female CF‐1 mice were sensitized over the left inguinal lymph node with a methylated bovine serum albumin emulsion. On day 19 and 20 after sensitization, mice received, by oral gavage, the test compounds (5 mg/kg/bw) or vehicle twice daily. Post‐final compound injection, mBSA was injected into the left hind paw. Inflammation was assessed using a semiquantitative scoring system (Figure 1). (B) Myocardial infarction in diabetic mice. Male C57BL/6 mice were rendered diabetic with STZ and were diabetic for 2 months. Mice were subjected to left anterior descending coronary artery occlusion followed by reperfusion (LAD/reperfusion). After 48h, the hearts were excised. TTC and Evan's blue staining was used to measure infarct area. Mice received a total of 7 doses of either VEHICLE or an NYU compound (Figure 2). In summary, refined compounds that inhibit the interaction of ctRAGE with DIAPH1, which exhibit in vitro and in vivo inhibition of RAGE‐dependent molecular processes, present attractive scaffolds for the development of therapeutics against RAGE‐mediated diseases, such as those linked to diabetic complications and chronic inflammation. We conclude that these identified compounds hold significant potential as druggable scaffolds for further development and testing for the treatment of RAGE‐related disorders. Support or Funding Information R24 ‐1R24DK103032 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .