Escherichia coli BL21 carrying pDEST17-TcBD2 and pDEST17-TcBD2m (mutant BD2: Y85A and W92A without the ability to bind to acetylated Histone 428) were grown at OD  0,6 and induced with 0.1 mM isopropyl-b-D-thiogalactopyranoside during 4 hours at 37°C as described previously 14. The proteins were purified using Ni-NTA (Thermo FisherTM) following the manufacturer’s instructions. The purified proteins were dialyzed against the previously determined optimal assay buffer: phosphate 0.1 M PH=8, glicerol 1%, DMSO 0.5%, and diluted to a final concentration of 200 ýM. Correct secondary structure of soluble proteins was verified by circular dichroism spectroscopy using a spectropolarimeter (Jasco J-810, Easton, MD, USA).

FP measurement was performed on a BMG PheraStar FS plate reader (BMG Labtech GmbH, Ortenberg, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 675 nm (50 nm bandwidth) and Black 1536-well flat bottom small volume microplates with a non-binding surface from Greiner Bio-One GmbH (Frickenhausen, Germany). Optimal Bromosporine (BSP) probe/TcBD2 interaction conditions were assayed by including in the reaction phosphate medium and seriated dilutions of DMSO (up to 2%), DTT (up to 50 ýg/ml), EDTA (up to 90 ýg/ml), glycerol (up to 10%), BSA (up to 250 ýg/ml), deoxy big-chaps (up to 50 ýg/ml), CHAPS (up to 50 ýg/ml), Zwittergent 3-14 (up to 50 ýg/ml), Triton-X100 (up to 0,5 M), NP40 (up to 0.3 M), Tween-20 (up to 100 ýg/ml) and Pluronic acid (up to 10%). The final conditions of the screening assay were set at a final volume of 8 ýl, 50 nM of the Bromosporine probe (BSP-AF488), and 100 ýM of TcBDF2, phosphate pH=8, glycerol 1 %, and DMSO 0.5 %. Recombinant TcBD2m was used as a negative control of binding. For the screening, 8 microliters of recombinant TcBD2 contained in the assay buffer plus 50 nM of BSP-AF488 were dispensed in 1,536 wells Grenier plates were compounds resuspended in 8 ýL of DMSO, were previously dispensed. The primary screening was performed at a single shot at a final concentration of 100 ýM per well. Further on, to determine the compounds’ potency 11-concentration points in a 1:3 seriated dilutions pattern were stamped in the plate, starting at 100 ýM. Plates were stored frozen at -20°C until used when they were allowed to equilibrate at room temperature before proceeding to the reading. Controls were made where σ+ and σ2 are the standard deviations and μ+ and μ2 are the mean values of the positive and negative controls, respectively. A series of negative and positive controls was measured. For each, positive and negative control, 64 wells were analyzed.

To identify molecules that bind to the TcBDF2 bromodomain (TcBD2), we set up a high-throughput screening (HTS) assay using Fluorescent Polarization (FP). FP is a very sensitive technique that allows quantitative analysis of the interaction between two molecules. Recent developments have allowed this technique to be adapted to be used in HTS format toward finding inhibitors against a myriad of proteins 34,35.

Firstly, we assayed previously used fluorescent probes with an affinity for the mammalian BDs ATAD236, PCAF and GCN537, BRPFs38, Brd9 that is also a promiscuous human Brd binder39, BET BD1 selective40 and BET BD2 selective (Patent WO2014140076). None of these probes showed significant fluorescence polarization when incubated with recombinant TcBD2 under standard conditions, reinforcing the idea that these parasite BDs are very divergent from human ones.

Given that we previously showed that the BD-pan inhibitor Bromosporine (BSP) binds to TcBD2 14, we decided to modify this molecule to use it as a fluorescent probe in the HTS assay. Hence, AlexaFluor-488 i) s t n u n o i t a z il r a o p lii m ( P m 200 150 100 50 200 150 100 50 200 150 100 50 200 150 100 50 200 150 100 50 200 150 100 50 00.01 0.1

1 GLYCEROL (AF488) was coupled to BSP (Fig. S1). BSP was our last choice because we have previously demonstrated that it has no significative trypanocidal activity 13. BSP-AF488 probe was used with the wild-type (TcBD2) and a previously characterized recombinant mutant version of TcBD2 (TcBD2m, unable to bind to its acetylated ligand) to optimize the conditions in a HTS format. We first tested different buffer compositions to obtain a working window of approximately 120 mP, needed to proceed to the screening assay. Optimal conditions for the screening were determined as shown in Figure 1. Screenings were made in a 1,536 plate with a final volume of 8 ýL. The condition selected was: 100 ýM of recombinant TcBD2, 50 mM of BSP-AF488, buffer phosphate 0.1 M, glycerol 1 %, and DMSO 0.5 %. Recombinant TcBD2m was used as a negative control in all plates assayed. As can be observed, there is very low interaction between the probe and BD2m in all conditions tested. The Z’ score, calculated as mentioned in the Methods section, was used as a quality control parameter 41. Once the assay conditions were established, we performed the screening of a small molecule bromodomain-targeted compound set (summarized in Fig. 2A). The set mostly consisted of compounds showing experimental activity against bromodomains or synthesized during various human bromodomain drug discovery programs, and their analogs (total of 28,251 compounds). The library was tested in a single shot format at a final concentration of 100 ýM, in a 1,536-wells plate format. All plates assayed were analyzed using in-house algorithms and the Z’ score (over 0.4) was used as a quality control parameter.

Out of all compounds tested, 471 compounds were considered positive after the first single shot format assay was performed. The cut-off for positive hits was set at (mean + 3SD) or 37% inhibition, having a positive hit rate of 1.7% (Fig. 2B). Next, to determine the compounds’ potency, 11 concentration points in a 1:3 dilution pattern were stamped in a 1,536-plate starting at 100 ýM. Recombinant TcBD2m was used as a negative control. In this assay, 7 compounds showed binding activity of pIC50 ≥ 4,5 against TcBD2 in a dose-response manner (Fig. 2C, Table 1).

The binding of the 7 selected compounds to TcBD2 was verified by differential scanning fluorimetry (DSF) or Thermal shift observing Kds between 1 and 3 µM (Table 1 and Fig. S2). The (most promising) compounds were modeled at the AcK site of TcBDF2 to further validate their potential role as inhibitors (Fig. S46). Importantly, AcK binds in a heavily hydrophobic pocket but contains several well-resolved 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 structural waters in the binding pocket. These water molecules are observed at the base of the pocket in most BRD as well as in the X-ray structure of TcBDF2 (Fig S3) 42,43. AcK recognition is mediated by a direct hydrogen bond to a conserved asparagine (Asn 86 in TcBDF2), which is mimicked by most BRD inhibitors. Moreover, there is also a conserved tyrosine (Tyr43 in TcBDF2) to coordinate the active site bridging water molecule44 (Fig. S3). Both interactions are also conserved in the crystal structure of TcBDF2 with BSP. We evaluated whether the compounds were able to retrieve those interactions at the AcK pocket through molecular modeling (while binding to the AcK pocket). The docking procedure was validated by reproducing the binding mode of bromosporine while maintaining key interactions (Fig. S4). Docking studies revealed that all seven compounds fit into the substrate pocket of TcBDF2. The compounds were able to form a hydrogen bond with Asn86 and/or the water-bridged hydrogen bond with Tyr43 (Fig. S5-S6). It is worth mentioning that compounds 4, 5, 6, and 7 with methylcinnoline as core made pi-stacking interaction with Trp92, and even 4 and 5 can form a hydrogen bond in a similar way to BSP (Fig. S5-6). The docking pose of compound 3 placed the dimethyl-isoxazole-pyridine moiety rotated 180º vertically from the expected position, as is the case with the AcK mimic methylbenzoisoxazole (PDBid 5Y8C)45. The enantiomer compounds 1 and 2 also made a pi-cation interaction with Trp92, where they can also form pi-stacking interactions between the 3-ethyl-5-methyl-triazolopyrimidine core and Trp92 (Fig. S6). Finally, the 7 compounds were tested in an in vitro assay of T. cruzi intracellular amastigote assay using VERO cells as hosts 46 and in an in-house colorimetric assay using epimastigotes of the Dm28c strain that expresses beta-galactosidase from an episomal plasmid (Fig. S7)31. Not surprisingly, none of the compounds showed parasiticidal activity at concentrations below 50 ýM in the two life cycle stages. Additionally, we assessed the toxicity of these compounds in the cell line used for the infection assays using MTT. Our results indicate that none of the compounds exhibited cytotoxicity up to 200 ýM (data not shown).

We have previously assayed the binding of TcBDF2 and TcBDF3 to several BD inhibitors and determined that they have different binding specificities 13,14. Commercial human inhibitors iBET-151 and BSP bind to both bromodomains; but JQ1(+), which binds TcBDF3 with an affinity similar to iBET-151, does not interact with TcBDF2. The compounds from the HTS we tested herein have Kds for TcBD2 between 1 and 3 ýM, however, they have no significative activity against epimastigotes, nor amastigotes in infected cells. This effect could be due, at least in part, to a low potency of the compounds obtained in our screening, a fact that could be associated with the use of BSP as a probe, a compound that, itself, shows low trypanocidal activity. Also, a possible explanation for this lack of activity of TcBDF2 inhibitors could be associated with the multimeric nature of the complexes in which bromodomains are included (see below). 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300

The human inhibitors mentioned were also assayed against BDF orthologues from other trypanosomatids with different results. Schulz and coworkers showed that iBET-151 (BET bromodomains inhibitor) induces T. brucei bloodstream form to develop insect-stage features, like the expression of surface procyclin and upregulation of glycolysis enzymes. They found that TbBDF2 and TbBDF3 bind iBET-151 with a low affinity (Kd=225 μM and 175 μM, respectively) and do not bind at all to other BET inhibitors like JQ1(+). iBET-151 was co-crystalized with TbBDF2, and it was found in a completely atypical position, flipped by roughly 180° to the position it binds to human BDs, something that could explain the low affinity of the interaction 21,47. Later, Yang and collaborators assessed 27 compounds obtained by a structure-based virtual screening combined with ITC experiments. They found one compound (GSK2801) that binds with higher affinity to the BD of TbBDF2 (Kd=15 μM) and with lower affinity to the second BD of TbBDF5 (Kd=83 μM). By contrast, GSK2801 does not bind to TbBDF3 or the first BD from TbBDF5 48. BDF5 from Leishmania donovani was also recently assayed against human BD inhibitors 27. LdBDF5 binds to SGC-CBP30, BSP, and I-BRD9, with different affinities for the first or second DB. SGC-CBP30, which showed the higher affinity (Kd=281 nM, for LdBD5.1), also exhibited activity against promastigotes from L. mexicana (IC50=7,16 ýM) and L. donovani (IC50=6,16 ýM).

It is worth mentioning that not always a direct correlation is found between the affinity of the inhibitors and their activity against the parasite in T. cruzi nor T. brucei or Leishmania. For example, iBET-151 and JQ1(+) have IC50 against Dm28c epimastigotes of 6.35 μM and 7.14 μM respectively 13. In contrast, BSP that has a Kd for TcBDF2 similar to iBET-151 was less active against parasites (IC50 > 50 ýM). GSK2801 inhibits the growth of procyclic T. brucei, disrupting the nucleolar localization of TbBDF2, with an IC50 of 1.37 μM, which suggests that GSK2801 has other targets beyond the inhibition of TbBDF2 48. Also, as was mentioned above, the IC50 for SGC-CBP30 against Leishmania promastigotes was significantly lower than the Kd measured for this compound on LdBD5.1.

Staneva and coworkers established that the majority of T. brucei BDFs participate in complexes that include other BDFs 49. A similar situation was reported by Jones and coworkers for LdBDF5 50. In this situation, which seems to be characteristic of trypanosomatids, limited inhibition of only one BD of the complex could be countered by the presence of another one or ones. However, over-expression of dominant negative mutants of one of the BDFs would induce disruption of the whole complex and a more deleterious effect over the parasite, as we have previously reported for TcBDF2 28. This model could also explain the results obtained for other BD inhibitors in other trypanosomatids. Moreover, extrapolating when determining the essentiality of BDFs in these parasites should be made with caution. The activity of bromodomain inhibitors was also determined for other parasites showing intriguing results. Chua and coworkers assayed 42 compounds previously characterized as BD inhibitors for activity against the asexual form of Plasmodium falciparum. All compounds were predicted to be correctly placed into the BD of P. falciparum histone acetyltransferase PfGCN5 and to interact with the conserved asparagine by a hydrogen bond, but these interactions were not experimentally measured. SGC-CBP30, a selective inhibitor of human CREBBP (CBP) and EP300 bromodomains, showed the highest in vitro activity, with an IC50 of 3.2 μM and a selectivity index (calculated using a human HEK 293 cell line) of around 7 24. Finally, Toxoplasma gondii was assayed with L-Moses, a specific inhibitor for the GCN5-family bromodomains 26. L-Moses interferes with the in vitro interaction of GCN5b bromodomain 318 319 with acetylated histone residues and displays potent activity against Toxoplasma tachyzoites infecting HFF cells (IC50 of ~0.6 μM).

All this evidence suggests that maybe targeting only one BD is not the best strategy against trypanosomatids. An alternative could be designing pan-inhibitors against BDs present in nuclear protein complexes taking advantage of the divergence in parasitic BDs vs. human BDs. However, it is hard to conceive that such kind of compound could be designed. Another alternative could be focusing on BDs with novel localizations outside the nucleus that are druggable, at least in T. cruzi, and do not take part in protein complexes with other BDs 29,51.

In summary, we identified 7 hits competitive against TcBDF2 in a fluorescence polarization assay and validated their binding by DSF. The confirmed hits originated from a set of compounds targeting human bromodomains, but despite bearing some features reminiscent of previously published bromodomain inhibitors they are structurally distinct. Except compound 3, all have been tested in various historical hBRD4 assays at GlaxoSmithKline and did not give fitted BRD4 dose-response curves at concentrations up to 50 µM. We are therefore confident that the hits are not generally promiscuous bromodomain inhibitors but may represent specific binders to TcBDF2 with some selectivity over the BET family, the most studied human bromodomain-containing proteins and those whose potential clinical safety risks are best understood. While these hits bind more weakly to TcBDF2 than clinical inhibitors of human BET bind to their targets in vitro (typically at least 100 nM), they represent good starting points for optimization.

This work was supported by Tres Cantos Open Lab Foundation (Tc261) and Agencia Nacional de Ciencia y Tecnologıa from Argentina (PICT-2017-1978, PICT-2020-SERIEA-01704) and Universidad Nacional de Rosario (1BIO490). We would like to thank Dolores Campos for their technical assistance with Vero cell and parasite culture.

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