During infection in mammals, the protozoan parasite Trypanosoma brucei transforms from a proliferative bloodstream form to a quiescent form that is pre-adapted to host transition. AMP analogs are known to induce quiescence and also inhibit TbTOR4. To examine the role of AMP-activated kinase (AMPK) in regulation of this developmental transition, we characterized trypanosome TbAMPK complexes. Expression of a constitutively active AMPKα1 induces quiescence of the infective form and TbAMPKα1 phosphorylation occurs during differentiation of wild-type pleomorphic trypanosomes to the quiescent stumpy form in vivo. Compound C, a well-known AMPK inhibitor inhibits parasite differentiation in mice. We also provide evidence linking oxidative stress to TbAMPKα1 activation and quiescent differentiation, suggesting that TbAMPKα1 activation balances quiescence, proliferation and differentiation.

Human African Trypanosomiasis is a neglected tropical disease that is lethal if left untreated. Existing therapeutics have limited efficacy and severe associated toxicities. 2-(2-(((3-((1H–Benzo[d]imidazol-2-yl)amino)propyl)amino)methyl)-4,6-dichloro-1H–indol-1-yl)ethan-1-ol (NEU-1053) has recently been identified from a high throughput screen of >42,000 compounds as a highly potent and fast acting trypanocidal agent capable of curing a blood stream infection of T. brucei in mice. We have designed a library of analogs to probe the SAR and improve the predicted CNS exposure of NEU-1053. We report the activity of these inhibitors of Trypanosoma brucei, the efficacy of NEU-1053 in a murine CNS model of infection, and identification of the target of NEU-1053 via X-ray crystallography.

Ataxia-telangiectasia (A-T) is a complex disease arising from mutations in the ATM gene (Ataxia-Telangiectasia Mutated), which plays crucial roles in repairing double-strand DNA breaks (DSBs). Heterogeneous immunodeficiency, extreme radiosensitivity, frequent appearance of tumors and neurological degeneration are hallmarks of the disease, which carries high morbidity and mortality because only palliative treatments are currently available. Gene therapy was effective in animal models of the disease, but the large size of the ATM cDNA required the use of HSV-1 or HSV/AAV hybrid amplicon vectors, whose characteristics make them unlikely tools for treating A-T patients. Due to recent advances in vector packaging, production and biosafety, we developed a lentiviral vector containing the ATM cDNA and tested whether or not it could rescue cellular defects of A-T human mutant fibroblasts. Although the cargo capacity of lentiviral vectors is an inherent limitation in their use, and despite the large size of the transgene, we successfully transduced around 20% of ATM-mutant cells. ATM expression and phosphorylation assays indicated that the neoprotein was functional in transduced cells, further reinforced by their restored capacity to phosphorylate direct ATM substrates such as p53 and their capability to repair radiation-induced DSBs. In addition, transduced cells also restored cellular radiosensitivity and cell cycle abnormalities. Our results demonstrate that lentiviral vectors can be used to rescue the intrinsic cellular defects of ATM-mutant cells, which represent, in spite of their limitations, a proof-of-concept for A-T gene therapy.