Identification of Trypanosoma brucei AdoMetDC Inhibitors Using a High-Throughput Mass Spectrometry-Based Assay

Abstract

Human African trypanosomiasis (HAT) is a fatal infectious disease caused by the eukaryotic pathogen Trypanosoma brucei. Available treatments are difficult to administer and have significant safety issues. S-Adenosylmethionine decarboxylase (AdoMetDC) is an essential enzyme in the parasite polyamine biosynthetic pathway. Previous attempts to develop TbAdoMetDC inhibitors into anti-HAT therapies failed due to poor brain exposure. Here, we describe a large screening campaign of two small-molecule libraries (∼400,000 compounds) employing a new high-throughput (∼7 s per sample) mass spectrometry-based assay for AdoMetDC activity. As a result of primary screening, followed by hit confirmation and validation, we identified 13 new classes of reversible TbAdoMetDC inhibitors with low-micromolar potency (IC50) against both TbAdoMetDC and T. brucei parasite cells. The majority of these compounds were >10-fold selective against the human enzyme. Importantly, compounds from four classes demonstrated high propensity to cross the blood-brain barrier in a cell monolayer assay. Biochemical analysis demonstrated that compounds from eight classes inhibited intracellular TbAdoMetDC in the parasite, though evidence for a secondary off-target component was also present. The discovery of several new TbAdoMetDC inhibitor chemotypes provides new hits for lead optimization programs aimed to deliver a novel treatment for HAT.

Synthesis and evaluation of analogs of 5′-(((Z)-4-amino-2-butenyl)methylamino)-5′-deoxyadenosine (MDL 73811, or AbeAdo) – An inhibitor of S-adenosylmethionine decarboxylase with antitrypanosomal activity

“…26 Unfortunately, none of the tested amide or carbamate derivatives retained any meaningful activity in the AdoMetDC enzyme assay, suggesting that the basic amine is essential for activity within this series. This is in agreement with the established mechanism of MDL 73811 ( 1 ) inhibition, which relies on the primary amine for suicide transamination of the catalytic pyruvoyl group.…”

“…26 Briefly, the assay was initiated in a 384-well plate by combining 20 µL of the enzyme solution (50 nM purified T. brucei AdoMetDC/prozyme complex 15 in 100 mM Hepes, pH 7.7, 50 mM NaCl, 5 mM putrescine, 2 mM DTT, 0.1% bovine serum albumin) and 20 µL of the substrate solution (80 µM AdoMet in 100 mM Hepes, pH 7.7, 50 mM NaCl, 5 mM putrescine, 0.02% Nonidet P40 substitute) in the presence of 0.8 µ L of a compound in dimethyl sulfoxide (DMSO). A compound was added to a dry plate using Echo 555 acoustic liquid dispenser (Labcyte, Sunnyvale, CA, USA).…”

“…26 Briefly, the assay was initiated by adding 20 µL of a 100-fold intermediate dilution of a compound in HMI-19 medium to 180 µL of cells at 3000 cells/mL in HMI-19 medium in a 96-well culture plate (CellStar, Greiner Bio-One, Monroe, NC, USA). The final DMSO concentration was 0.1%.…”

“…26 When the two values are within <10 nm/s of each other, only permeability in the presence of the inhibitor is reported. f In vitro metabolic stability presented as a half-life of a compound in human (h) or mouse (m) pooled liver microsomes (S9) measured exactly as previously described. 26 A single value is shown when the half-lives are >90 min or <2.5 min for both species.…”

“…26 When the two values are within <10 nm/s of each other, only permeability in the presence of the inhibitor is reported. …”