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The immunoproteasome, known to play an important role in MHC class I antigen processing and presentation, have been linked to neurodegenerative diseases and hematological cancers. However, the pathophysiological functions of the immunoproteasome in these diseases are still not very well established. This can be attributed mainly to the lack of appropriate molecular probes that selectively target the immunoproteasome catalytic subunits. Herein, we report the development of a small molecular inhibitor (AM) that selectively targets the major catalytic subunit, LMP2, of the immunoproteasome. We show that the compound covalently modifies the LMP2 subunit with high specificity in human prostate cancer cell. AM was also shown to selectively inhibit the chymotrypsin-like activity of LMP2 subunit. More importantly, the anti-proliferative activity of AM is more pronounced in prostate cancer cells that highly express LMP2 without inducing toxicity in normal cells. These results implicate an important role of LMP2 in regulating cell growth of malignant tumors that highly express LMP2. Subsequently, the modes of action of AM were investigated. Prostate cancer cells that highly express LMP were shown to induce G2/M cell cycle arrest and apoptosis via PARP cleavage when treated with the compound. Similar to epoxomicin, the treatment of AM induced the accumulation of poly-ubiquitination in prostate cancer cells, which indicates the inhibition of proteolysis. However, unlike epoxomicin, the treatment of AM did not appear to inhibit the activation of inflammation. In conclusion, these results suggest that the LMP2 inhibitor, AM, may induce cytotoxicity prostate cancer cells that highly express LMP2 catalytic subunit in similar modes of action as epoxomicin but it does not involve the inflammatory pathway.

Bz-423 is a immunomodulatory 1,4-benzodiazepine that exerts its therapeutic effects in autoimmune mice by selectively inducing apoptosis of pathogenic B and T cells. The overall goal of the studies presented in this dissertation is to understand the cellular and molecular basis for the selectivity observed in vivo. Not only will the findings of these experiments be useful in helping advance Bz-423 to the clinic, but will also provide one of the first comprehensive outlines of the apoptotic signaling pathways induced by a small molecule. Structure-activity studies identified the aromatic side chain at C2 and the C'4 position phenolic hydroxyl group are key elements for target binding and cytotoxic activity. Bz-423 induces an early, rapid production of superoxide (O2-) from mitochondria, and this response is the critical signal initiating apoptosis. The oligomycin sensitivity-conferring protein (OSCP) component of the mitochondrial F1F0-ATPase is the physiologically relevant target for both O2- production and subsequent cell death. JNK is a key molecule involved in Bz-423-induced apoptosis in fibroblasts. JNK activation occurs by a MAPK signaling cascade involving sequential activation of ASK1/MEKK/JNK resulting in activation of the transcription factor, c-Jun. JNK activation causes increased expression of BH3-only proteins triggering the activation of the multidomain pro-apoptotic proteins Bax and Bak. These results provide a detailed description of events during Bz-423-induced apoptosis, from initial O2- production to cytochrome c release and caspase activation. Bz-423 induces apoptosis in a cellular model of pathogenic lupus through a mechanism unique from that observed in fibroblasts. In all cell types examined apoptotic signaling pathways induced by Bz-423 converge on the Bcl-2 family of proteins resulting in activation of Bax and Bak by BH3-only proteins culminating in cytochrome c release from the mitochondria. However, Bz-423-induced apoptosis in pathogenic lupus cells is independent of MAPK signaling and de novo protein synthesis. These differences result in increased kinetics of cell death. The difference in kinetics of Bz-423-induced cell death in different cell lines coupled with its pharmacokinetic profile could help explain why Bz-423 is selective to pathogenic B cells in vivo .

Chagas' disease is an incapacitating illness that afflicts 16--18 million people, killing 21,000 each year in the Americas. Patients infected with Trypanosoma cruzi who develop the chronic phase of the disease become progressively sick and ultimately die, typically from cardiac failure. Currently there is no satisfactory treatment for Chagas' disease. Malaria is also one of the most important infectious diseases worldwide, as over one million people die each year from infections caused by Plasmodium falciparum. Further complicating the treatment of malaria is the increasing resistance to available drugs. There is a great need to develop new chemotherapy for these diseases, and the targeting of essential cysteine proteases of T. cruzi and P. falciparum has shown to be a promising approach. This work describes the design, synthesis, and evaluation of small molecule inhibitors of parasitic cysteine proteases. A focused library of peptidomimetic vinyl sulfonyl inhibitors was synthesized and shown to contain potent irreversible inhibitors of cruzain, the major cysteine protease of T. cruzi. Structure-activity relationships with cruzain were determined. Many compounds displayed increased activity against T. cruzi in whole-cell assays, and several were completely trypanocidal in nature. A general relationship between inhibitor hydrophobicity (as measured by clogP) and trypanocidal activity was also uncovered and shown to be independent of structure. Similar compounds were tested against falcipain-2, the best characterized and most important of the three major cysteine proteases of P. falciparum. Several inhibitors of falcipain-2 and P. falciparum were shown to have IC50 values in the low-nanomolar range. Structure-activity relationships with both the enzyme and the parasite were identified. Cytotoxicity is an important mechanism for the management of autoimmune diseases, such as systemic lupus erythematosus (SLE), and is important in virtually all cancer treatments. The novel benzodiazepine Bz-423 has been identified by Prof. G. D. Glick as a pro-apoptotic agent with great potential in the treatment of such diseases. This work describes the design, synthesis and evaluation of a series of analogs of Bz-423. These benzodiazepines were useful in determining structural correlates of pro-apoptotic activity, and are currently being used to facilitate target identification studies.

The autoimmune disease systemic lupus erythematosus (SLE) poses formidable therapeutic challenges; neither the exact population of pathogenic lymphocytes nor specific targets for drug development are defined. As such, SLE is treated with immunosuppressive drugs that target all lymphocytes, and treatment with these agents is associated with serious, sometimes fatal complications. Previous studies have identified a pro-apoptotic 1,4-benzodiazepine (Bz-423) that reduces the severity of autoimmune nephritis in (NZB x NZW)F1 mice, a murine model of SLE in which B cells are dominant. To further probe the properties of Bz-423, studies were conducted with MRL/MpJ-Faslpr mice. In this strain, the development of autoimmune disease is dependent on T cells. Administration of Bz-423 to these mice significantly attenuates the progression of autoimmune arthritis and nephritis and concomitantly decreases CD4+ T cells. Parallel experiments demonstrated that Bz-423 neither suppresses normal B and T cell immune responses nor alters normal lymphocyte populations in vivo. Thus, the effects of Bz-423 are specifically concentrated on autoimmune lymphocytes. To define the cellular basis for selectivity, the effect of Bz-423 on receptor-stimulated lymphocytes was examined. In both B and T cells, moderate [Bz-423] that have minimal activity against non-stimulated cells, synergize with antigen receptor stimulation resulting in increased cell death. Moreover, Bz-423 rescues defective activation-induced cell death (AICD) in both autoimmune B and T lymphocytes. Since defective AICD is one mechanism by which autoreactive cells escape peripheral tolerance, the ability of Bz-423 to restore AICD is a plausible mechanism that explains its ability to attenuate autoimmune disease. Studies in Ramos B cells to probe the molecular basis for the synergistic death response revealed that in non-stimulated cells, Bz-423 induces superoxide, which signals cellular changes ultimately leading to cell death. The generation of superoxide is not increased by ligation of the antigen receptor; rather stimulation sensitizes lymphocytes so that Bz-423-generated superoxide signals an apoptotic pathway that is not triggered in non-stimulated cells. Because receptor stimulation is a requirement for the development and maintenance of autoreactive lymphocytes, the pro-apoptotic properties of Bz-423 provide an opportunity to selectively target a central determinant of lupus pathogenesis.

The factors that cause the autoimmune disease systemic lupus erythematosus (SLE) remain poorly understood. As such, treatment of this debilitating and potentially fatal disease is based on global immunosuppression. However, these drugs non-specifically target all lymphocytes, and are accompanied by serious complications that limit treatment. In effort to develop a new therapeutic for SLE without these limitations, a benzodiazepine (Bz-423) possessing novel cytotoxic properties was identified. Administration of Bz-423 to lupus-prone (NZB x NZW)F1 mice reduces the severity of autoimmune kidney disease with similar efficacy to that previously observed for corticosteroids, azathioprine, and cyclophosphamide, the standard treatments for lupus nephritis. However, unlike these therapies, Bz-423 is not associated with systemic toxicities and is not adversely immunosuppressive. In vivo, Bz-423 induces B-cell apoptosis, with some apparent specificity for pathogenic B-lymphocytes. Because of the well-established link between B-cell autoantibody production and kidney disease, the ability of Bz-423 to reduce B-cell numbers is a plausible mechanism explaining its ability to reduce the signs of autoimmune disease. To define its cellular mechanism of action, the effects of Bz-423 were examined using transformed cell lines. Morphological and biochemical evidence demonstrates that Bz-423 triggers apoptosis. Bz-423-induced apoptosis involves collapse of the mitochondrial transmembrane gradient prior the acquisition of other apoptotic features. Mechanistically, these findings suggest that Bz-423 acts through an apoptotic second messenger. Caspase inhibition prevents apoptosis but does not prevent mitochondrial alterations, suggesting that Bz-423 does not act along the signal transduction pathway activated by ligation of apoptotic death receptors. Comparison of Bz-423 to central and peripheral benzodiazepine receptor ligands indicates that only Bz-423 induces apoptosis. By screening a library of structurally related analogs, it is clear that the naphthylalanine side chain of Bz-423 is a critical structural element. These findings demonstrate a unique structure-activity relationship for benzodiazepines. Because of its ability to treat autoimmune kidney disease and to induce apoptosis in a wide spectrum of cancer-cell-derived cell lines, Bz-423 represents a promising new lead compound for the treatment of many human diseases including SLE, rheumatoid arthritis, and cancer.