Elucidation of the target and molecular mechanism of action of the immunomodulatory benzodiazepine, Bz-423.

Abstract

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by non-specific inflammation that can afflict virtually any organ in the body. Patients with severe lupus are more likely to survive now than they were 30 years ago, but the morbidity from disease and treatment is still significant. Consequently, there is significant interest in developing new agents to treat SLE that have greater efficacy and fewer side effects. As part of efforts to identify novel cytotoxic agents, a library of 1,4-benzodiazepines was screened for lymphotoxic members, leading to the identification of the cytotoxic benzodiazepine, Bz-423. Bz-423 ameliorated disease in two lupus models of mice by killing specific subpopulations of disease-causing lymphocytes without adverse toxicities or global immunosuppression. The mechanism of Bz-423-induced cell death was found to be critically dependent on an early superoxide (O₂<super>-</super>) signal. The goal of this work was to identify the molecular target of Bz-423 and further define the molecular mechanism of action of Bz-423, in order to better understand its selectivity and therapeutic efficacy. These experiments revealed that Bz-423 binds to the OSCP subunit of the mitochondrial F₁F₀-ATPase, leading to the generation of mitochondrial O₂<super>-</super> that initiates an apoptotic signaling pathway. Bz-423 inhibits the F₁F₀-ATPase via a novel uncompetitive kinetic mechanism. Many F₁F₀-ATPase inhibitors, like oligomycin, aurovertin, and efrapeptin, are extremely potent and toxic. They have nanomolar binding affinities for the F₁F₀-ATPase, demonstrate mixed kinetic mechanisms of inhibition, and bind to regions in the F₀ or F₁ domains of the F₁F₀-ATPase that share homology with other ATPases. In contrast, Bz-423 has a low micromolar affinity for the F₁F₀-ATPase, demonstrates a novel uncompetitive mechanism of inhibition, and binds the OSCP subunit of the F₁F₀-ATPase, a subunit with no homologue in other ATPases. It is likely that these differences contribute to ability of Bz-423 to target pathogenic lymphocytes without adverse toxicitics. New therapies specifically for the treatment of lupus have not been approved in over thirty years. This work demonstrates a new target and mechanism on which to base the development of new agents and suggests that molecules with properties similar to Bz-423 would be promising leads.