Design, synthesis and biological characterization of PfGAPDH inhibitors

Malaria has a tremendous health, social and economic impact on people living in tropical and subtropical regions of the world. WHO estimated 214 million cases and 438 000 deaths in 2015 and 3.2 billion people are globally at risk of infection1. Available treatments are becoming progressively less effective, mainly because parasites are constantly acquiring resistance toward the drug in use. Therefore, there is an urgent need of developing therapeutic agents acting on new targets. Plasmodium parasites (P. falciparum is the most lethal), in their amastigote phase, produce energy only through glycolysis. In this pathway, GAPDH catalyses the first energy productive step and thus represent a valuable target for drug discovery. Known GAPDH inhibitors that bind to the active site are small nonselective molecules (iodoacetamide, 3-bromopyruvate), which irreversibly react with cysteine residues of the enzyme, including the catalytic one. We designed and synthesized the first series of inhibitors characterized by a 3-bromoisoxazoline warhead2, inspired by 3-bromoacivicin, a known inhibitor of reactive cysteine containing enzymes3. The compounds were assayed for the inhibitory activity on the P. falciparum isolated enzyme showing a biphasic irreversible inactivation. MS/MS studies of the digested protein demonstrated the mild reactivity of the exploited warhead, which only reacts with catalytic Cys (activated by a His residues). The human ortholog of GAPDH is only partially inhibited (up to 25%). We explained this through a negative cooperativity that prevents the alkylation of all the four monomers of hGAPDH, resulting in selectivity among the two isoforms of GAPDH4. Compounds were also assayed on cultures of the bloodstream form of P. falciparum and human cell lines in order to determine their in vitro antiparasitic activity and toxicity. The goal of this project is to obtain compounds with improved potency and pharmacokinetic properties, suitable for in vivo studies on animal model of P. falciparum infections. 1. http://www.who.int/mediacentre/factsheets/fs094/en/ 2. Bruno, S.; Pinto, A.; Paredi, G.; Tamborini, L.; De Micheli, C.; La Petra, V.; Marinelli, L.; Novellino, E.; Conti, P.; Mozzarelli, A. J. Med. Chem. 2014, 57(17), 7465-7471. 3. a) Conti, P.; Pinto, A.; Wong, P.E.; Major, L.L.; Tamborini, L.; Iannuzzi M.C.; De Micheli, C.; Barrett, M.P.; Smith, T.K. ChemMedChem 2011, 6(2), 329-333. b) Tamborini, L.; Pinto, A.; Smith, T.K.; Major, L.L.; Iannuzzi, M.C.; Cosconati, S.; Marinelli, L.; Novellino, E.; Lo Presti, L.; Wong, P.E.; Barrett, M.P.; De Micheli, C.; Conti, P. ChemMedChem 2012, 7(9), 1623-1634. 4. Bruno, S.; Margiotta, M.; Pinto, A.; Cullia, G.; Conti, P.; De Micheli, C.; Mozzarelli, A. Bioorg. Med. Chem. 2016, 24(12), 2654-2659.