Design and synthesis of novel enzyme inhibitors as potential antiparasitic agents

Background Protozoan parasitic diseases, such as Malaria and Human African Trypanosomiasis (HAT), have a tremendous health, social and economic impact on people living in tropical and subtropical regions of the world. While around 3.8 billion people are at risk of infection, available treatments are unsatisfactory, mainly due to constantly increasing drug resistance. Hence, there is an urgent need of developing new chemotherapeutic agents acting on new molecular targets, such as essential parasitic enzymes. Aims The aims of the present project are: 1) Synthesis of competitive inhibitors of Trypanosoma brucei isoform of N5,N10-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (TbFolD), a crucial enzyme involved in the folate pathway. 2-3) Synthesis of covalent inhibitors of reactive cysteine-containing enzymes, namely Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) and rhodesain (TbCatL), which are central in energy metabolism and virulence, respectively. The covalent inhibitors designed in the present project are characterized by the 3-bromo isoxazoline nucleus as an innovative electrophilic warhead. Methods 1) Synthesis: a) 1,3-dipolar cycloaddition reactions of bromo-nitrile oxide with suitable alkenes; b) condensation/cyclization reactions; c) coupling reactions; d) amino acid protecting groups; 2) Purification, structure assignment and purity assessment: a) flash chromatography; b) crystallization; c) 1H and 13C-NMR; d) HPLC and chiral HPLC; e) determination of chiroptical properties; f) MS analysis; 3) Biochemical and pharmacological assays: a) enzyme inhibition assays; b) identification of the binding site for covalent inhibitors through MALDI/TOF analysis; c) in vitro activity against parasites and human cell lines. Results 1) TbFolD inhibitors(1) We started synthesizing LY374571 (1), a known human FolD inhibitor, with the aim of using it as a lead compound: interestingly, following the published synthesis protocol we unambiguously obtained compound 2, with a structure different from the one reported in the literature. In order to understand the structural requirements for an improved activity, we performed a structure-activity relationship study replacing the glutamate tail with other α-, β- and γ-amino acids (compounds 4a-g). Compound 2 showed a Ki of 1.1 µM toward TbFolD, in the same range of 3 (Ki=8.5 µM), another human known FolD inhibitor; the affinity increased two times in the case of 4b and 4d (Ki=0.48 and 0.54 µM) while the others showed similar or higher Ki values. When assayed in vitro on the bloodstream form of T. brucei, unfortunately, only 2 showed a modest antiparasitic activity (IC50=49 µM), comparable to the one of 3 (IC50=57 µM), but with a higher selectivity index (4 versus 1.8) when assayed on THP1 monocytes. In collaboration with Prof. W. Hunter, we were able to get the first X-ray structure of TbFolD in presence of 2 and NADP+, which allowed us to rationalize the different range of affinity of the synthesized compounds and will guide the design of new inhibitors in the future. 2) PfGAPDH inhibitors(2) The catalytic cysteine residue of GAPDH is inhibited by a number of compounds that alkylate thiols. We previously demonstrated that the 3-Br-isoxazoline nucleus is able to react with the catalytic Cys of cytidine triphosphate synthetase (CTPS), a glutamine amidotransferase. I herein evaluated the strength of this warhead for the irreversible inactivation of PfGAPDH by testing 3-Br-acivicin and a series of potential inhibitors of general structure 5, with distinct substitution patterns at the 5 position to modulate the reactivity (Figure 2). The compounds are able to inhibit PfGAPDH with a biphasic fashion through selective covalent binding to the catalytic Cys residue. The differences in activity of the tested inhibitors were rationalized on the basis of the electron density maps and covalent docking studies. Interestingly, under the same experimental conditions that led to a fast and complete inhibition of the protozoan enzyme, the human orthologue was only 25% inhibited, with the alkylation of a single catalytic cysteine within the tetramer, thus evidencing an interesting selectivity profile(3). The new inhibitors were also tested for their antiparasitic activity against chloroquine-sensitive and chloroquine resistant P. falciparum strains and some of them showed a promising activity. 3) TbCatL inhibitors(4) A further application of the 3-bromoisoxazoline warhead in the design of covalent enzymatic inhibitors is represented by the development of new TbCatL inhibitors obtained by coupling this warhead to a proper peptide-like recognition moiety.(4) Some low micromolar inhibitors were identified, among which 6a,b and 7 showed also an interesting antiparasitic activity. Conclusions We described new inhibitors of parasitic enzymes using both a non-covalent (for TbFolD) and covalent (for PfGAPDH and TbCatL) approach. Covalent inhibitors were designed exploiting the reactivity of the 3-Br-isoxazoline nucleus and properly tuning the recognition moiety to confer affinity and selectivity for the selected target enzymes. The biological results were rationalized through a careful analysis of enzyme-cofactor-inhibitor co-crystal structure or covalent docking studies. These data represent a strong basis for further developments of new, more effective, antiparasitic agents. References (1)Eadsforth, T. C.; Pinto, A.; Luciani, R.; Tamborini, L.; Cullia, G.; De Micheli, C.; Marinelli, L.; Cosconati, S.; Novellino, E.; Lo Presti, L.; Cordeiro da Silva, A.; Conti, P.; Hunter, W. N.; Costi, M. P. Characterization of 2,4-diamino-6-oxo-1,6-dihydropyrimidin-5-yl ureido based inhibitors of Trypanosoma brucei FolD and testing for antiparasitic activity. J. Med. Chem. 2015, 58(20), 7938-7948. (2)Bruno, S.; Pinto, A.; Paredi, G.; Tamborini, L.; De Micheli, C.; La Petra, V.; Marinelli, L.; Novellino, E.; Conti, P.; Mozzarelli, A. Discovery of covalent inhibitors of glyceraldehyde 3-phosphate dehydrogenase, a target for the treatment of malaria. J. Med. Chem. 2014, 57(17), 7465–7471. (3)Bruno, S.; Margiotta, M.; Pinto, A.; Cullia, G.; Conti, P.; De Micheli, C.; Mozzarelli, A. Selectivity of 3-bromo-isoxazoline inhibitors between human and Plasmodium falciparum glyceraldehyde 3-phosphate dehydrogenases. Bioorg. Med. Chem. 2016, in press, doi: 10.1016/j.bmc.2016.04.033. (4)Ettari, R.; Pinto, A.; Previti, S.; Tamborini, L.; Angelo, I. C.; La Pietra, V.; Marinelli, L.; Novellino, E.; Schirmeister, T.; Zappalà, M.; Grasso, S.; De Micheli, C.; Conti, P. Development of novel dipeptide-like rhodesain inhibitors containing the 3-bromoisoxazoline warhead in a constrained conformation. Bioorg. Med. Chem. 2015, 23, 7053-7060.