A fragment-based approach to discovery of Receptor for Advanced Glycation End products inhibitors.

The Receptor for Advanced Glycation End products (RAGE) is a pattern recognition receptor that signals for inflammation via the NF-κB pathway. RAGE has been pursued as a potential target to suppress symptoms of diabetes and is of interest in a number of other diseases associated with chronic inflammation, such as inflammatory bowel disease and bronchopulmonary dysplasia. Screening and optimization have previously produced small molecules that inhibit the activity of RAGE in cell-based assays, but efforts to develop a therapeutically viable direct-binding RAGE inhibitor have yet to be successful. Here, we show that a fragment-based approach can be applied to discover fundamentally new types of RAGE inhibitors that specifically target the ligand-binding surface. A series of systematic assays of structural stability, solubility, and crystallization were performed to select constructs of the RAGE ligand-binding domain and optimize conditions for NMR-based screening and co-crystallization of RAGE with hit fragments. An NMR-based screen of a highly curated ~14 000-member fragment library produced 21 fragment leads. Of these, three were selected for elaboration based on structure-activity relationships generated through cycles of structural analysis by X-ray crystallography, structure-guided design principles, and synthetic chemistry. These results, combined with crystal structures of the first linked fragment compounds, demonstrate the applicability of the fragment-based approach to the discovery of RAGE inhibitors.

Divalent Cations and the Divergence of ßγ-Crystallin Function.

The ßγ-crystallin superfamily contains both ß- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate ßγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial ßγ-crystallins bind calcium ions. The ßγ-crystallin from the tunicate Ciona intestinalis (Ci-ßγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-ßγ is valuable for investigating the evolution of the ßγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-ßγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-ßγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-ßγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.