Identification of selective inhibitors of phosphofructokinase as lead compounds against trypanosomiasis

(eng) The protist Trypansoma brucei is the causative agent of Human African Trypanosomiasis (HAT), a disease that is endemic to sub-Saharan Africa and is responsible for approximately 50,000 deaths each year worldwide. Current therapies employed to treat HAT generally suffer from poor selectivity profiles, and consequently, lead to high rates of deleterious side effects. Furthermore, growing resistance to these drugs has highlighted a need for the identification of novel therapies. The glycolytic enzyme phosphofructokinase (PFK) has been recognized as a potential therapeutic target in the fight against the bloodstream form of T. brucei due to the reliance of this parasite on the metabolism of glucose as its sole mechanism for the generation of adenosine triphosphates (ATP). The importance of this enzyme has been confirmed by genetic validation, and thus, its inhibition may represent a novel strategy for the treatment of HAT. Currently, no inhibitors of Tb PFK have been described that possess ideal drug-like qualities or submicromolar potency in either enzymatic or live parasite culture assays. As a result, in collaboration with Professor Malcolm Walkinshaw of the University of Edinburgh, a quantitative High Throughput Screening (qHTS) campaign was initiated at the NIH Chemical Genomics Center to identify novel small molecule inhibitors of Tb PFK. The primary screen utilized a firefly luciferase-coupled assay specifically designed to monitor the production of ADP in the kinase reaction. This work, together with subsequent medicinal chemistry efforts, has identified a novel small molecule inhibitor (ML251) of Tb PFK, which is described herein to possess potent activity in enzymatic assays (IC50 = 370 nM) and toxicity against cultured T. brucei (ED50 = 16.3 M). This compound has been subjected to a panel of in vitro absorption, distribution, metabolism, and excretion (ADME) assays and delivers a promising profile in this respect. Finally, cytotoxicity evaluations against MRC-5 human lung fibroblast cells and KB-3-1 HeLa cells, in addition to assessment against purified Bacillus stearothermophilus PFK, indicate that ML251 displays a high degree of selectivity for the targeted enzyme