A Small Molecule Antagonist of RAGE/DIAPH1 Interaction: Novel Therapeutic Opportunities in Metabolic and Chronic Disease
Authors: Manigrasso MIchaele B , Spelios Michael G , Friedman Richard A , DeVita Robert J , Ramasamy Ravichandran , Shekhtman Alexander , Schmidt Ann Marie 
Published: June 2021

Introduction

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, or DIAPH1. Given the complex nature of ligands binding to regions of the extracellular domains of RAGE, we sought to identify small molecule antagonists of the interaction of ctRAGE with DIAPH1. Prompted by our discovery of 13 small molecules that block this interaction, we synthesized novel compounds and performed structure‐activity relationship experiments to help identify refined analogs potentially able to delay or prevent the progression of RAGE‐related chronic diseases.

Methods

Discovery of Candidate Biomarkers

Given the prominent role of RAGE in inflammation, we pursued target biomarkers through the discovery of diabetes‐ and RAGE‐dependent genes in macrophages retrieved from diabetic mouse kidney. Sorted macrophages (CD45+/F4/80+/CD11b+) from kidney cortex were subjected to RNA‐Seq. WT nondiabetic (wtndm), WT diabetic (wtdm), RAGE knockout nondiabetic (rkondm), and RAGE knockout diabetic (rkodm) were studied (n=3). Differential expression was estimated with a significance cutoff of p≤0.001. We identified 17 genes that might contribute to RAGE effects in diabetes‐associated nephropathy and the RNA‐Seq results for 6 genes, which have a role in macrophage inflammation, are shown in Fig 1.

Human THP1 macrophages

Following differentiation, THP1 cells were treated with RAGE ligand CML‐AGE (10 μg/ml) for 6h alone or after 1h pre‐treatment with NYU5 (50 μM). Supernatant was collected and subjected to Luminex assay for detection of the mediators identified in Fig 1. Results were normalized to total DAPI+ cells.

Delayed type hypersensitivity (DTH

Male and female CD1 mice were sensitized over the inguinal lymph node with a methylated bovine serum albumin (mBSA) emulsion. Mice were then fed 1 of 3 concentrations (30mg/kg, 10mg/kg and 3 mg/kg) of NYU5 for 7D. On day 6, mBSA was injected into the left hind paw. The next morning, the paw was scored using a semiquantitative scoring system. Animals were sacrificed and plasma was retrieved.

Diabetes‐induced inflammation

Male and female C57BL/6 mice were rendered diabetic at 6w of age with STZ. Following induction of diabetes, mice were fed 1 of 3 concentrations (30mg/kg, 10mg/kg and 3 mg/kg) of NYU5 for 3M. Animals were sacrificed and plasma was retrieved.

Conclusions

We conclude that these preliminary data suggest that this refined compound inhibits the interaction of ctRAGE with DIAPH1, thus preventing the downstream signal transduction that mediates RAGEs pathological processes. Furthermore, the development of the RAGE‐DIAPH1 antagonist, NYU5, presents an attractive molecular scaffold for the further development of therapeutics against RAGE‐mediated diseases.

Support or Funding Information

NIH 1R24DK103032 and Harrington Discovery Institute

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.