Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness.

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

Mechanistic studies of the immunochemical termination of self-tolerance with unnatural amino acids.

For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.

Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease.

Drak2 is a serine/threonine kinase expressed in T and B cells. The absence of Drak2 renders T cells hypersensitive to suboptimal stimulation, yet Drak2(-/-) mice are enigmatically resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We show in this study that Drak2(-/-) mice were also completely resistant to type 1 diabetes when bred to the NOD strain of mice that spontaneously develop autoimmune diabetes. However, there was not a generalized suppression of the immune system, because Drak2(-/-) mice remained susceptible to other models of autoimmunity. Adoptive transfer experiments revealed that resistance to disease was intrinsic to the T cells and was due to a loss of T cell survival under conditions of chronic autoimmune stimulation. Importantly, the absence of Drak2 did not alter the survival of naive T cells, memory T cells, or T cells responding to an acute viral infection. These experiments reveal a distinction between the immune response to persistent self-encoded molecules and transiently present infectious agents. We present a model whereby T cell survival depends on a balance of TCR and costimulatory signals to explain how the absence of Drak2 affects autoimmune disease without generalized suppression of the immune system.

Immunochemical termination of self-tolerance.

The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.

DRAK2, a lymphoid-enriched DAP kinase, regulates the TCR activation threshold during thymocyte selection.

DAP kinases are a family of serine/threonine kinases known to regulate intrinsic apoptotic processes. DAP-related apoptotic kinase-2 (DRAK2) is highly expressed in lymphoid organs, with differential expression during thymocyte development. Low levels of transcript were observed in CD4/CD8 double-positive (DP) and double-negative populations, whereas single-positive thymocytes possessed elevated levels. Ex vivo stimulation of DP thymocytes with phorbol myristate acetate or antibodies that activate the TCR complex led to the accumulation of DRAK2 in a protein kinase C- and MAP Kinase-dependent fashion. Although DAP kinase family members are thought to potentiate apoptosis, ectopic expression of DRAK2 using retroviral transduction of primary T cells and NIH3T3 fibroblasts failed to decrease rates of survival, suggesting that DRAK2 expression is not sufficient to promote apoptosis. Rather, our results demonstrate that DRAK2 is a primary response gene activated by TCR stimulation in DP thymocytes. Further, we observed that DRAK2 controlled the threshold for calcium signaling in the thymus since positively selected Drak2-deficient thymocytes displayed a reduced requirement for TCR cross-linking. These findings are consistent with a role for DRAK2 in thymocyte selection and lymphoid maturation, and demonstrate that DRAK2 transduces non-apoptotic signals during thymocyte differentiation.

A deficiency in Drak2 results in a T cell hypersensitivity and an unexpected resistance to autoimmunity.

DRAK2 is a member of the death-associated protein (DAP)-like family of serine/threonine kinases. Members of this family induce apoptosis in various cell types. DRAK2, in particular, is specifically expressed in T cells and B cells, and it is differentially regulated during T cell development. To determine whether DRAK2 regulates lymphocyte apoptosis, we produced Drak2(-/-) mice. Contrary to our expectations, Drak2(-/-) T cells did not demonstrate any defects in apoptosis or negative selection; however, T cells from Drak2(-/-) mice exhibited enhanced sensitivity to T cell receptor-mediated stimulation with a reduced requirement for costimulation. These results provide evidence that DRAK2 raises the threshold for T cell activation by negatively regulating signals through the TCR. In contrast to other models of T cell hypersensitivity, Drak2(-/-) mice were remarkably resistant to experimental autoimmune encephalomyelitis (EAE). These results expose a new pathway regulating T cell activation and highlight the intricacies of induced autoimmune disease.

Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity.

The mechanisms governing positive selection of T cells in the thymus are still incompletely understood. Here, we describe a N-ethyl-N-nitrosourea induced recessive mouse mutant, Ms. T-less, which lacks T cells in the peripheral blood because of a complete block of thymocyte development at the CD4(+)CD8(+) stage. Single nucleotide polymorphism mapping and candidate gene sequencing revealed a nonsense mutation in the inositol (1,4,5) trisphosphate 3 kinase B (Itpkb) gene in Ms. T-less mice. Accordingly, Ms. T-less thymocytes do not show detectable expression of Itpkb protein and have drastically reduced basal inositol (1,4,5) trisphosphate kinase activity. Itpkb converts inositol (1,4,5) trisphosphate to inositol (1,3,4,5) tetrakisphosphate, soluble second messengers that have been implicated in Ca(2+) signaling. Surprisingly, Ca(2+) responses show no significant differences between wild type (WT) and mutant thymocytes. However, extracellular signal-regulated kinase (Erk) activation in response to suboptimal antigen receptor stimulation is attenuated in Ms. T-less thymocytes, suggesting a role for Itpkb in linking T cell receptor signaling to efficient and sustained Erk activation.