Inhibition from the inside
Inhibiting the receptor for advanced glycation end products (RAGE) has been shown to reduce complications of diabetes. Instead of targeting the extracellular domain of RAGE, here, Manigrasso et al. developed a small molecule, called RAGE229, able to inhibit the interaction between the cytoplasmic tail of RAGE and Diaphanous-1, thus completely inhibiting the intracellular RAGE signaling. The small molecule reduced complications of diabetes in mouse models, including impaired wound healing, ischemia, and kidney disease by reducing diabetes-associated inflammation. The results suggest that targeting the intracellular RAGE domain could be an effective strategy to block pathologic RAGE signaling.
Abstract
The macro- and microvascular complications of type 1 and 2 diabetes lead to increased disease severity and mortality. The receptor for advanced glycation end products (RAGE) can bind AGEs and multiple proinflammatory ligands that accumulate in diabetic tissues. Preclinical studies indicate that RAGE antagonists have beneficial effects on numerous complications of diabetes. However, these antagonists target the extracellular domains of RAGE, which bind distinct RAGE ligands at diverse sites in the immunoglobulin-like variable domain and two constant domains. The cytoplasmic tail of RAGE (ctRAGE) binds to the formin, Diaphanous-1 (DIAPH1), and this interaction is important for RAGE signaling. To comprehensively capture the breadth of RAGE signaling, we developed small-molecule antagonists of ctRAGE-DIAPH1 interaction, termed RAGE229. We demonstrated that RAGE229 is effective in suppressing RAGE-DIAPH1 binding, Förster resonance energy transfer, and biological activities in cellular assays. Using solution nuclear magnetic resonance spectroscopy, we defined the molecular underpinnings of the interaction of RAGE229 with RAGE. Through in vivo experimentation, we showed that RAGE229 assuaged short- and long-term complications of diabetes in both male and female mice, without lowering blood glucose concentrations. Last, the treatment with RAGE229 reduced plasma concentrations of TNF-α, IL-6, and CCL2/JE-MCP1 in diabetic mice, in parallel with reduced pathological and functional indices of diabetes-like kidney disease. Targeting ctRAGE-DIAPH1 interaction with RAGE229 mitigated diabetic complications in rodents by attenuating inflammatory signaling.
INTRODUCTION
The receptor for advanced glycation end products (RAGE), a member of the immunoglobulin (Ig) superfamily, binds to and transduces the signals triggered by a diverse group of ligands. The protein encoded by AGER, RAGE, is composed of three extracellular domains: one Ig-like variable (V)–type domain followed by two Ig-like constant (C)–type domains, named C1 and C2; a single transmembrane–spanning domain; and a short, highly charged cytoplasmic tail (ct) domain or ctRAGE. The receptor is named for its ligand families, the advanced glycation end products (AGEs), which accumulate in obesity, diabetes, aging, and oxidative and inflammatory stresses (1–3). RAGE is also a receptor for the S100/calgranulins (such as S100A12 and S100B); amphoterin, also known as high mobility group box 1 (HMGB1); amyloid-beta peptide; phosphatidylserine (PS); and lysophosphatidic acid (LPA), as examples (4–9). Although it was earlier shown that RAGE ligands bound at the V-type domain (and/or to the V-C1 domain), numerous studies have demonstrated that certain RAGE ligands may bind on the C1- and C2-type extracellular domains that do not involve ligation of the Ig-like V-type domain (10, 11).Given the evidence that the ctRAGE is required for RAGE ligand–stimulated signaling (12, 13), it was logical to probe the mechanisms by which this domain recruited signal transduction cascades to modulate gene expression and cellular properties. Toward this end, a yeast two-hybrid assay, used to discover ctRAGE binding molecules, identified Diaphanous-1 (DIAPH1) as a putative interacting partner (14). Immunoprecipitation experiments confirmed this interaction and identified that ctRAGE bound to the formin homology 1 (FH1) domain of DIAPH1; reduction of Diaph1 expression in cultured cells through silencing RNA approaches blocked RAGE ligand–mediated activation of multiple signaling pathways (14). RAGE and DIAPH1 are highly expressed in the human and murine diabetic kidney, and recently, it was shown that deletion of Diaph1 in vivo imparted similar benefits to that of Ager deletion in murine models of vascular injury, cardiac ischemia, and diabetes-associated kidney disease (15–17).Nuclear magnetic resonance (NMR) experiments revealed that the N terminus of ctRAGE domain (residues 2 to 15) is ordered, whereas residues 16 to 43 of the C terminus are disordered and without discrete structure (18). The structure of ctRAGE is dynamic and likely composed of multiple conformers. Accordingly, mutation of amino acid residues Arg5 and Gln6 to alanine (Ala) residues of the N terminus of ctRAGE eliminated critical electrostatic interactions between Arg5 and Glu10 and disrupted the tertiary structure. In distinct work, super-resolution stochastic optical reconstruction microscopy (STORM) and single-particle tracking (SPT) revealed that in the presence of mutation of these same amino acids in ctRAGE or reduction in DIAPH1 expression, cell membrane RAGE clusters and diffusion are modulated, thereby providing additional support for the ctRAGE-DIAPH1 interactions (19).The mutation of Arg5/Gln6 to Ala residues in ctRAGE limited the functional consequences of its interaction with the FH1 domain of DIAPH1 (18). When this mutation was introduced into primary murine aortic smooth muscle cells (SMCs), RAGE ligand S100B-stimulated phosphorylation of AKT (protein kinase B), migration, and proliferation were suppressed compared with cells transfected with control vector. However, these functional responses to non–RAGE ligand platelet-derived growth factor (PDGF) were not affected (18).We previously screened a library of >58,000 known small molecules and identified 13 small-molecule competitive inhibitors of ctRAGE interaction with DIAPH1; these molecules reduced RAGE ligand–mediated signaling and suppressed the acute inflammatory effects of RAGE ligands in vivo (20). In the current study, upon critical review of the properties of these 13 identified antagonists, we aimed to optimize structure-activity relationships (SARs) of one of the classes emerging from that screen, the 2-phenylquinoline scaffold, for testing in murine models of complications of diabetes. We report the discovery of RAGE229, N-(4-(7-cyano-4-(morpholin-4ylmethyl)quinolin-2-yl)phenyl)acetamide, as a ctRAGE-DIAPH1 inhibitor. Compared to vehicle controls, RAGE229 blocked ctRAGE-DIAPH1 interaction and reduced the consequences of inflammation, ischemia, and long-term diabetes in mice with type 1– and/or type 2-like diabetes, as tested in in vivo models of cardiac ischemia, wound healing, and diabetes-associated kidney disease. Collectively, this work identifies a potent antagonist of ctRAGE-DIAPH1 interaction and identifies a potential approach for targeting the consequences of RAGE pathological activation in chronic diseases such as diabetic complications.
RESULTS
Identification of small molecules that bind to ctRAGE derived from the phenylquinoline scaffold: RAGE203, RAGE208, and RAGE229
To identify highly potent antagonists of the ctRAGE-DIAPH1 interaction, the phenylquinoline scaffold (based on compound 11 in the original report) (20) was selected for further development on account of the multiple opportunities for molecule optimization from the 13 small-molecule antagonists of ctRAGE-DIAPH1 interaction. The medicinal chemistry strategy led to the identification of two early-stage molecules, RAGE203 and RAGE208, in which tryptophan fluorescence quenching assays illustrated the following affinity constants of the small molecules to the ctRAGE, KD of 30 ± 10 nM and 24 ± 6 nM, respectively (Fig. 1, A and B). Subsequent SAR studies revealed that improvements were achievable by adding substituents at the quinoline 7 positions, the acetamide and quinoline 4-methylene amino groups, which led to the discovery of RAGE229, KD of 2 ± 1 nM (Fig. 1C)…
