Novel Therapeutic Opportunities for Diabetic Complications using a Small Molecule Antagonist of RAGE/DIAPH1 Interaction

Our previous work has shown that the cytoplasmic tail (ct) of RAGE is essential for RAGE ligand‐mediated signal transduction and modulation of gene expression and cellular properties. RAGE signaling requires interaction of ctRAGE with the intracellular effector, mammalian diaphanous 1 (DIAPH1). Given the complex nature of ligands binding to regions of the extracellular domains of RAGE, we sought to discover small molecule antagonists of the interaction of the ctRAGE with DIAPH1. Prompted by our discovery of 13 small molecules that block this interaction, we synthesized novel compounds and performed S‐A‐R medicinal chemistry studies to identify analogs potentially able to delay or prevent the progression of RAGE‐related diabetic complications. Methods Ex‐Vivo Studies: Ischemia/Reperfusion (I/R) in T1D Mouse Heart: Male Akita mice were tested in the isolated perfused diabetic heart model at age 3M. Hearts were removed and subjected to the perfused heart protocol: 30m normoxia, 30m zero‐flow ischemia followed by 60m reperfusion in the presence of RAGE229 (1 μM) or veh. (DMSO). Following I/R, the % infarct area and lactate dehydrogenase (LDH) in perfusate recovered were measured. In‐Vivo Studies: Wound Healing: At 8W, male (M) and female (F) BTBR. Cg‐ Lep ob /WiscJ ( ob/ob ) mice had 1.5 cm 2 full thickness wounds created. Beginning on D3 and continuing through D10, animals were subjected to either veh. or RAGE229. Samples taken at 14D were used to test expression patterns of DIAPH1. Time to wound closure and % wound closure were measured. In‐Vivo Studies: Diabetic Renal Disease: At 8W, M and F mice ob/ob were placed on RAGE229 diet (30mg/kg/d) or chow (veh) for 16W. Following sacrifice, kidneys were harvested, processed and analyzed. For light microscopy, 10 selected fields were assessed for semi‐quantification of mesangial sclerosis (MS) severity. Glomerular basement membrane (GBM) thickness and % total foot process effacement (FPE) were evaluated by electron microscopy (>8 glom/mouse). Conclusions This improved lead compound, RAGE229, inhibits the interaction of ctRAGE with DIAPH1, resulting in improvements from the diabetic complications. Namely, in the isolated perfused T1D heart, RAGE229 protects from I/R injury, with reduced infarct size and reduced LDH levels. When administered topically, treated wounds had improved wound closure. Furthermore, medicated chow‐treated BTBR ob/ob mice displayed lower MS scores, % FPE and GBM thickness. We conclude that these early proof of concept data show that the inhibition of the interaction of ctRAGE with DIAPH1 prevents the downstream signal transduction mitigiating diabetes‐associated kidney changes, protects from I/R injury in the isolated perfused heart and improves wound healing. The development and optimization of antagonists for RAGE‐DIAPH1 protein‐protein interaction presents an attractive molecular scaffold for the development of therapeutics against RAGE‐mediated diseases. Support or Funding Information 1R24DK103032. RAGE229 in the isolated perfused diabetic heart. Hearts were retrieved from 3 mo old T1D male Akita mice and subjected to 30 mins normoxia, 30 minutes of global no flow ischemia, followed by 60 minutes of reperfusion, RAGE229, 1 μM, or equal volume DMSO vehicle, was perfused 10 minutes prior to global ischemia. A) Infarct size was determined by tissue staining; B) supernatant was collected and subjected to LDH assay analysis. N=3/group; *p<0.05. Figure 1 Expression of DIAPH1 in diabetic wounds and the effects of topical RAGE229. A,D). Expression of DIAPH1 in wound tissue. Male (M) or female (F) ob/ob mice were subjected to wounding; intact skin and wound tissue on D14 was subjected to rt‐qPCR for Diaph1 mRNA transcripts. B–C (M) and E–F (F) ob/ob mice began treatment at D3 with either topical RAGE229 (5 mg/kg/2x day) vs. veh, through D10. % wound closure from and rep. mice shown. M, N=5/6 mice/grp; F, N=3 mice/grp. Mean±SEM; *p<0.05 Figure 2 Diabetes‐associated renal disease is improved following treatment with RAGE229. Male (A–E) and female (F–J) BTBR ob/ob mice were treated with RAGE229 medicated chow (30 mg/kg/d) vs. vehicle chow from 8 weeks until 24 weeks. Mice were sacrificed and kidneys subjected to analyses for mesangial sclerosis, podocyte effacement and thickness of the GBM. Mean ± SEM is shown. N=7–9 mice/group. Figure 3

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