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

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, <i>Trypanosoma brucei</i> 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, <b>8</b> (NPD-008) and <b>9</b> (NPD-039), were potent (<i>K</i>_i = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC₅₀ = 5.5 and 6.7 μM, respectively). Treatment of parasites with <b>8</b> caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of <i>T. brucei</i> cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis