Targeting a Subpocket in Trypanosoma brucei Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity

Abstract

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent (Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 μM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.

Imidazole Derivatives as Promising Agents for the Treatment of Chagas Disease

“…The phenomenon of cAMP efflux has been described for mammalian cells, where it is mediated by ABC class transporters (23), but to the best of our knowledge it has not been reported for protozoan cells, although we previously observed it in T. brucei and Leishmania species (J. C. Munday, T. Kalejaiye, and H. de Koning, unpublished results). The increased efflux from the trypomastigotes clearly shows that this mechanism in part compensates for the inhibition of PDE activities in the cells; however, as the cellular levels still increased significantly, we propose that the efflux mechanism was saturated upon treatment with the PDE inhibitors, leading to toxic cAMP levels and cell death, as demonstrated previously for T. brucei (9,10). Our findings demonstrate promising in vitro activity of a series of phenyl-substituted imidazole derivatives, with several being very active against the different parasite forms and strains, especially compound 9, which merits further studies in order to contribute to the identification of novel therapies for CD.…”

“…With this aim, Y strain BTs were incubated with either 2ϫEC 50 or 5ϫEC 50 of this compound for 2.5 h, and both the cellular cAMP content and the released cAMP in the medium were determined. Cultures incubated in parallel without a test compound and with known T. brucei PDE inhibitors (NPD-001 [9] and NPD-008 [10]) served as negative and positive controls, respectively.…”

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…Compound 138 was amongst a cohort of compounds shown to occupy the P‐pocket (PDB ID: https://www.rcsb.org/structure/5G2B) thereby providing selectivity over the human isoform of the enzyme. Exposure of T. brucei to compound 138 resulted in an increase in intracellular cAMP as well as disruption of cellular organisation . From a screen of fragments related to 137 , compound 139 was identified as a hit compound .…”

Hits and Lead Discovery in the Identification of New Drugs against the Trypanosomatidic Infections

“…On the left, the protein is shown as a semi-transparent surface with the ligand in black sticks and the location of the P-pocket marked. On the right side, the protein is shown in the same orientation as cartoon and only the P-pocket residues are shown as a surface with residues labeled according to Blaazer et al (2018). 7.6 μM.…”