Fragment-Based Identification of Ligands for Bromodomain-Containing Factor 3 of Trypanosoma Cruzi

Abstract

The <i>Trypanosoma cruzi</i> (<i>T. cruzi</i>) parasite is the cause of Chagas disease, a neglected disease endemic in South America. The life cycle of the <i>T. cruzi</i> parasite is complex and includes transitions between distinct life stages. This change in phenotype (without a change in genotype) could be controlled by epigenetic regulation, and might involve the bromodomain-containing factors 1-5 (<i>Tc</i>BDF1-5). However, little is known about the function of the <i>Tc</i>BDF1-5. Here we describe a fragment-based approach to identify ligands for <i>T. cruzi</i> bromodomain-containing factor 3 (<i>Tc</i>BDF3). We expressed a soluble construct of <i>Tc</i>BDF3 in <i>E. coli</i>, and used this to develop a range of biophysical assays for this protein. Fragment screening identified twelve compounds that bind to the <i>Tc</i>BDF3 bromodomain. Based on this screen, we developed functional ligands containing a fluorescence or <sup>19</sup>F reporter group, and a photo-crosslinking probe for <i>Tc</i>BDF3. These tools compounds will be invaluable in future studies on the function of <i>Tc</i>BDF3 and will provide insight into the biology of <i>T. cruzi</i>.

Small Molecule Ligands of the BET-like Bromodomain, SmBRD3, Affect Schistosoma mansoni Survival, Oviposition, and Development

“…50 89−93 °C, lit. 37 90−92 °C]; 1 H NMR (400 MHz, CDCl 3 ) δ H 4.89 (1H, sp, J 6.3), 4.58 (2H, br s), 1.24 (6H, d, J 6.3); LRMS m/z (ESI + ) 126 ([M + Na] + , 20%). These data are in good agreement with the literature values.…”

“…These data are in good agreement with the literature values. 37,49,50 Isopropyl (E)-But-2-enoylcarbamate (S2). Using a procedure reported by Shadrick et al, 42 crotonoyl chloride (4.8 mL, 50 mmol, 1.1 equiv) was added to a solution of compound S1 (4.6 g, 45 mmol, 1.0 equiv) in anhydrous THF (43 mL) at −78 °C followed by LiHMDS (1 M in THF, 90 mL, 90 mmol, 2.0 equiv).…”

“…These data are in good agreement with the literature values. 37,38,42 Isopropyl ((2S,4R)-6-Bromo-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)carbamate (S4a). Sodium borohydride (229 mg, 6.05 mmol, 0.75 equiv) was added to a solution of compound S3a (2.77 g, 8.07 mmol, 1.0 equiv) in EtOH (143 mL), cooled to a temperature below −10 °C followed by addition of MgCl 2 •6H 2 O (1.81 g, 8.89 mmol, 1.1 equiv) in water (14.3 mL).…”

“…The reaction mixture was stirred at a temperature below 0 °C for 2 h and then warmed to rt and stirred for 1 h. The resulting suspension was poured into a mixture of citric acid (60 mL, 0.5 M in water), HCl (aq) (205 mL, 1 M), and CH 2 Cl 2 (200 mL) and left to stir for 1 h. After this time, the layers were separated, the aqueous layer was extracted with CH 2 Cl 2 (3 × 150 mL), and the combined organic components were dried over MgSO 4 , filtered, and concentrated in vacuo to give compound S4a as a colorless solid (2.60 g, 98%): R f 0.68 (30% EtOAc/toluene); [α] D 25 = +12.9 (c 1.0, MeOH) [lit. 37 − 14.9 (c 1.0, CHCl 3 )], mp 130−136 °C (from toluene) [lit. 42 Chiral AD-H column (90:10 heptane:ethanol, 0.1% DEA, 1.0 mL min −1 ), retention time = 8.1 min (S4a, 94.3% UV), retention time = 11.5 min (opposite enantiomer (S4b), 5.7% UV); 89% e.e.…”

“…These data are in good agreement with the literature values. 37,38,42 Isopropyl ((2S,4R)-1-Acetyl-6-bromo-2-methyl-1,2,3,4-tetrahydroquinolin-4-yl)carbamate (S5a). Compound S4a (2.18 g, 6.36 mmol, 1.0 equiv) was dissolved in CH 2 Cl 2 (60 mL) under N 2 at room temperature.…”