Identification of small molecules that bind to SmBRD3 (1,2) and/or SmBRD3( Using the SmBRD3(2) construct detailed above, we employed differential scanning fluorimetry (DSF) to screen 697 compounds, comprising 153 compounds from the PPI-net library, 160 compounds from our in-house library of human and T. cruzi BRD ligands, and 384 compounds from the Maybridge Fragment Library against SmBRD3(2). Analysis of ΔTm values plotted against -log(SD) reveals a number of hits, including the known human BET bromodomain ligand, I-BET726 (Figure ). Selected compounds were then assessed against SmBRD3 (1,2), and interestingly the human BET bromodomain ligands, OXFBD02 (1), and (+)-JQ1 (5), were observed to stabilise ), implying that these compounds bind preferentially to SmBRD3(1) (Figure ). Conversely, BI-2536 ( ) and I-BET726 (8) 38 stabilise both SmBRD3(1,2) and SmBRD3( ), suggesting that these compounds either bind to both SmBRD3 BRDs, or to SmBRD3(2) alone. Biophysical analysis using ITC (Table and Table ) confirmed the DFS results, and revealed that OXFBD02 ( ) and (+)-JQ1 ( ) have Kd values for SmBRD3(1,2) of 683 ± 166 nM and 445 ± 106 nM, respectively, with no binding detected to SmBRD3 (2). The OXFBD02 (1) analogues OXFBD04 (4) and compound 3 showed the same behaviour, but with a higher Kd value of 2950 ± 261 nM and 1750 ± 301 nM, respectively, for SmBRD3 (1,2). I-BET726 (8) has a Kd = 1520 ± 520 nM for SmBRD3 (1,2) and Kd = 1850 ± 361 nM, for SmBRD3(2) consistent with the idea that this compound either binds to both SmBRD3 BRDs or just to SmBRD3(2). I-BET151 (6) and BI-2536 (7) show similar behaviour, but as their SmBRD3(2) affinities are lower, we progressed our studies with I-BET726 (8). Given the similarity of SmBRD3 and the human BET BRDs, we proposed that I-BET726 (8) would occupy the KAc binding pocket of SmBRD3(2) when bound. To probe this assumption, we obtained an X-ray crystal structure of I-BET726 (8) bound to SmBRD3(2) (Figure ). This is the first X-ray crystal structure of a BRD from S. mansoni (PDB code: 7AMC) and it demonstrates that the overall fold of SmBRD3( ) is very similar to that of HsBRD4(1) (Figure ). The Tyr (Y97 and Y139) and Asn (N140) residues that are important for KAc in HsBRD4( ) are conserved in SmBRD3 (2). N339 forms a hydrogen bond with the carbonyl oxygen atom of I-BET726 ( ) and Y296 forms a water-mediated hydrogen bond with the same atom (Figure ). While HsBRD4(1) and HsBRD4(2) possess the WPF shelf, these residues are replaced by H280, F281, and F282 in SmBRD3(2) (Figure ). This change results in the formation of a narrow channel, the HFF cleft, which is defined by F281, E344, V345, and V348, and in which the chlorophenyl group of I-BET726 resides (Figure ). Despite the SmBRD3(2) affinity of I-BET726 (8), the occupancy of the HFF cleft by the chlorophenyl group does not appear to be optimal. As the affinity of many BET bromodomain ligands results from the binding of lipophilic groups to the WPF shelf, we reasoned that developing I-BET726