Pharmacological Validation of Trypanosoma brucei Phosphodiesterases B1 and B2 as Druggable Targets for African Sleeping Sickness

Abstract

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei, the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors. We disclose that the human PDE4 inhibitor piclamilast, and some of its analogs, show modest inhibition of TbrPDEB1 and B2, and quickly kill the bloodstream form of the subspecies T. brucei brucei. We also report the development of a homology model of TbrPDEB1 that is useful for understanding the compound-enzyme interactions and for comparing the parasitic and human enzymes. Our profiling and early medicinal chemistry results strongly suggest that human PDE4 chemotypes represent a better starting point for optimization of TbrPDEB inhibitors than those that target any other human PDEs.

Repurposing human PDE4 inhibitors for neglected tropical diseases: Design, synthesis and evaluation of cilomilast analogues as Trypanosoma brucei PDEB1 inhibitors

“…Compound 1 showed no effect upon T. brucei growth (EC 50 > 50 µM). This is surprising, given the close concordance we and others observed between TbrPDEB1 enzyme inhibition and cellular growth inhibition with compounds 2 and 3 5, 6 …”

“…We also wished to determine whether a stereochemically simplified headgroup replacement could be achieved. Secondly, a key structural feature of the TbrPDEB1 binding site, predicted by homology modeling and confirmed by crystallography, 5, 15 is a pocket adjacent to the binding site (termed the “parasite-“ or “Ppocket”) that is deeper in comparison to the same region in hPDE4. Thus, guided by the SAR studies of the catechol diethers 2 and 3 reported previously that were intended to explore the “parasite pocket” of the enzyme, we now report exploration of the cyclopentyl ether (Figure 1, red) to longer, chain-extended versions, and we then studied the protein-ligand interactions in silico by carrying out molecular docking using the recently published crystal structure of TbrPDEB1.…”

“…For example, we showed that piclamilast ( 2 , Figure 1), a hPDE4 inhibitor, inhibited TbrPDEB1 and B2 (IC 50 4.7 and 11.4 µM, respectively), which translated to inhibition of T. brucei brucei (Tbb) cell growth (EC 50 = 9.6 µM). 5 At the same time, others identified 3 as a potent TbrPDE inhibitor via a high-throughput screening campaign. 6 This racemic compound remains the most potent TbrPDE inhibitor described to date, despite several further reports.…”

“…T. brucei expresses five cAMP-specific PDEs, two of which (TbrPDEB1 and TbrPDEB2) have been shown to be together essential for parasite proliferation by both RNAi and small molecules. 4, 5 Initial screening and early optimization experiments have uncovered a susceptibility of TbrPDEB1 and B2 to human PDE4 (hPDE4) inhibitor chemotypes, and that the inhibition of these enzymes leads to parasite death in vitro . For example, we showed that piclamilast ( 2 , Figure 1), a hPDE4 inhibitor, inhibited TbrPDEB1 and B2 (IC 50 4.7 and 11.4 µM, respectively), which translated to inhibition of T. brucei brucei (Tbb) cell growth (EC 50 = 9.6 µM).…”

“…5 Thus, for the purpose of studying a wider variety of hPDE4 inhibitors as starting points for TbrPDEB inhibitors, we investigated cilomiliast, 1 , a related hPDE4 inhibitor. Compound 1 (Ariflo, SB-207,499) is an orally active and selective hPDE4 inhibitor developed by GlaxoSmithKline for the treatment of respiratory disorders such as chronic obstructive pulmonary disease (COPD).…”