Autoantibodies that bind DNA (anti-DNA) are a hallmark of the autoimmune disorder systemic lupus erythematosus (SLE). A subset of anti-DNA are pathogenic: they mediate an inflammatory response in kidney tissue resulting in renal damage. During the previous project period, we studied the thermodynamic basis of sequence-specific binding by anti-ssDNA mAb 11F8. These studies were predicated on the observations that 11F8 is pathogenic; injecting hybridoma cells that produce 11F8 into normal mice results in nephritis of a nature and severity similar to that seen in human lupus, and the ability of 11F8 to cause disease is linked to its DNA binding properties. In a second research area, we identified a new benzodiazepine (1) that is pro-apoptotic. This compound is remarkably effective in treating the lupus-like disease in the two most clinically relevant polyclonal animal models of SLE. Significantly, treatment is not accompanied by the broad toxicities and side-effects that plague current therapeutic regimes. Based on these findings, the next phase of this grant proposes a series of basic and translation experiments that could directly impact our understanding of the pathology of lupus and the way in which this disorder is diagnosed and treated: (a) Investigate structural aspects of 11F8 recognition through X-ray crystallography, fluorescence resonance energy transfer experiments, and functional group mutagenesis of the 11F8 consensus sequence (Aims 1-2); (b) Use stopped-flow kinetics to investigate the mechanism by which 11F8 discriminates between specific and non-specific ligands (Aim 3); (c) Revert somatic mutations in 11F8 back to germline residues through site-directed mutagenesis to explore the functional significance of affinity maturation in the evolution of the autoimmune response to DNA (Aim 4); (d) Evaluate the utility of the 11F8 consensus sequence (SEL11F8) as a diagnostic probe for lupus (Aim 5); and (e) Use combinatorial chemistry to identify analogs of 1 possessing increased potency in vitro, and test these molecules in vivo (Aim 6).
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