Exploring RGD-integrin mimics as a novel therapeutic approach in diabetes

Type 2 Diabetes (T2D) was estimated in 2021 to affect over 480 million people and can lead to the development of cardiovascular disease (CVD), which is the cause of death for 50.3% of people with T2D. Insulin-like growth factor binding protein 1 (IGFBP-1) overexpression has been shown to improve insulin sensitivity, promote nitric oxide production, reduce blood pressure and protect against atherosclerosis. IGFBP-1 contains an Arginine-Glycine-Aspartic acid (RGD) peptide sequence, used in many proteins to bind to integrins, which are transmembrane proteins, primarily involved in the extracellular matrix. IGFBP-1 specifically binds to the α5β1 integrin via a flexible loop which contains the RGD sequence. It has been demonstrated that hexapeptides containing RGD have the same beneficial effects of IGFBP-1. The research presented investigates if small molecules could be used to mimic the effects of IGFBP-1 overexpression. From preliminary ROCS virtual shape screening and subsequent hit validation by a phosphorylation of Akt assay, two series of compounds were designed from the hit matter using docking in Schrodinger Maestro. The first series was zwitterion based, with a carboxylic acid mimicking the aspartate in the endogenous ligand, and a glycine, histidine or arginine at the other terminus. Phenyl, piperidine, pyrazole and alkyl chains were explored as linking groups. High hydrophilicity and poor solubility limited the synthesis; however, seven compounds were submitted for biological analysis. None of the compounds showed increased cell death but none showed an increase from the baseline in a limited phosphorylation of Akt assay. Direct binding to α5β1 was measured with surface plasmon resonance (SPR), with only one compound, 5.6.1, showing a measurable KD of 59 μM ± 0.64-fold. A second series from another hit molecule was designed, replacing the carboxylic acid with a hydantoin group, which is neutral charge at physiological pH but has precedent for metal binding. After the amide linkage of the hydantoin in the hit molecule could not be replicated, an imine linked molecule was produced with a piperazine terminus, compound 6.3.6. No toxicity was detected in a live/dead assay and the molecule and the Boc-protected intermediate had response in the limited phosphorylation of Akt assay. Modifications to the basic terminus and linker were designed and docked, incorporating examples used in literature. Sixteen of these targets were successfully synthesised and analysed using SPR. Five compounds had KD values measured, with the best two compounds 6.5.10 and 6.7.3 showing KD values of 70 μM ± 0.65-fold and 71 μM ± 0.52-fold respectively.