Chemical–biological characterization of a cruzain inhibitor reveals a second target and a mammalian off-target

Summary Inhibition of the Trypanosoma cruzi cysteine protease cruzain has been proposed as a therapeutic approach for the treatment of Chagas’ disease. Among the best-studied cruzain inhibitors to date is the vinylsulfone K777 (1), which has proven effective in animal models of Chagas’ disease. Recent structure–activity studies aimed at addressing potential liabilities of 1 have now produced analogues such as N-[(2S)-1-[[(E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]amino]-3-(4-methylphenyl)-1-oxopropan-2-yl]pyridine-4-carboxamide (4), which is trypanocidal at ten-fold lower concentrations than for 1. We now find that the trypanocidal activity of 4 derives primarily from the inhibition of T. cruzi 14-α-demethylase (TcCYP51), a cytochrome P450 enzyme involved in the biosynthesis of ergosterol in the parasite. Compound 4 also inhibits mammalian CYP isoforms but is trypanocidal at concentrations below those required to significantly inhibit mammalian CYPs in vitro. A chemical-proteomics approach employing an activity-based probe derived from 1 was used to identify mammalian cathepsin B as a potentially important off-target of 1 and 4. Computational docking studies and the evaluation of truncated analogues of 4 reveal structural determinants for TcCYP51 binding, information that will be useful in further optimization of this new class of inhibitors.

S4 other CYP51 inhibitors described recently [1] and, thus, the spectra for the controls shown above are reproduced from the earlier report. Peaks are assigned as a -cholesta-7,24-dien-3-

CYP Inhibition and reversibility studies
Mammalian CYP inhibition values and reversibility studies were conducted by WuXi AppTech (Tianjin, China 300457) according to standard protocols.

Minimum trypanocidal concentration assay
The minimal trypanocidal concentration assay was derived from the established protocol [9] for evaluating "cidal" activity, except that each compound was evaluated at multiple concentrations (typically 7 or 10) ranging from 0 to 50 M. As in the original protocol, T. cruzi infected J744 macrophages were treated with test compound for 27 days (or until T. cruzi parasites appear).

S10
The sterol-containing residue was resuspended in 2 mL of chloroform-methanol (9:1  Reactions were incubated at room temperature for one hour and then resolved by SDS-PAGE. The resulting gel was scanned for fluorescence (Typhoon Imaging System, Molecular Dynamics) and coomassie stained.
Enrichment of 9-labeled proteins and mass spectrometric analysis. C2C12 cells were treated with probe 9, lysed and denatured as described above. The labeled and denatured S11 lysates were sonicated to reduce viscosity and centrifuged to give pellets of the insoluble material. The buffer was then exchanged by using a NAP-5 column (GE Healthcare) equilibrated with 1% SDS in phosphate buffered saline pH 7.4. The samples were then normalized for protein content. The normalized lysates (1 mL, 2 mg/mL) were subjected to a "click" reaction with a cleaveable biotin azide 11 using the general conditions described above.
Samples were enriched using Streptavidin-coupled Dynabeads (Invitrogen). Enriched proteins were eluted with aqueous sodium hydroxide (0.4 N). The solution was neutralized with aqueous hydrochloric acid (0.8 N), and then run on an SDS-PAGE gel. The gel was colloidal coomassie stained.
The competed band at ~35 kDa was cut, and the proteins contained in the bands of interest were digested in gel with trypsin as described previously [13]. Using the protocol we described recently [14], the extracted digests were vacuum-evaporated and resuspended in 10 µL of 0.1% formic acid in water. The digests were separated by nanoflow liquid chromatography using a 75 µm × 150 mm reverse-phase 1.7 µm BEH 130 C18 column (Waters) at a flow rate of 350 nL/min in a NanoAcquity TM Ultra performance UPLC system (Waters). Solvent A was 0.1% formic acid in water and solvent B was 0.1% formic acid in acetonitrile. Following equilibration of the column in 5% solvent B, approximately one-half of each digest (5 µL) was injected, then the organic content of the mobile phase was increased linearly to 40% over 60 min, and then to 50% in 1 min. The liquid chromatography eluate was coupled to a hybrid linear ion trap-Orbitrap mass spectrometer (LTQ-XL, Thermo Scientific, San Jose, CA) equipped with a nanoelectrospray ion source. Spraying was from an uncoated 15 µm inner diameter spraying needle (New Objective, Woburn, MA). Peptides were analyzed in positive ion mode and in information-dependent acquisition mode to automatically switch between MS and MS/MS acquisition. MS spectra were acquired in profile mode using the Orbitrap analyzer in the m/z range between 300 and 1800.
For each MS spectrum, the 6 most intense multiple charged ions over a threshold of 1000 S12 counts were selected to perform CID (collision-induced dissociation) experiments. Product ions were analyzed on the linear ion trap in centroid mode. The CID collision energy was automatically set to 25%. A dynamic exclusion window of 0.5 Da was applied that prevented the same m/z from being selected for 60 seconds after its acquisition.
Mass spectrometry data analysis. Peak lists were generated using PAVA [15]. The peak lists were searched against the murine and human subset of the UniProtKB database as of July 6, 2011 (containing 182779 entries) using ProteinProspector version 5.8.0. Peptide tolerance in searches was 20 ppm for precursor and 0.6 Da for product ions, respectively. Cleavage specificity was selected to Trypsin. Peptides containing two miscleavages were allowed.
Carbamidomethylation of cysteine, acetylation of the N-terminus of the protein, pyroglutamate formation from N-terminal glutamine, and oxidation of methionine were allowed as variable modifications.
The number of modifications was limited to two per peptide. Hits were considered significant when two or more peptide sequences matched a protein entry and the Prospector score was above the significance level. A minimal ProteinProspector protein score of 20, a peptide score of 15, a maximum expectation value of 0.05 and a minimal discriminate score threshold of 0.0 were used for initial identification criteria. For identifications based on one peptide sequence with high scores, the MS/MS spectrum was reinterpreted manually by matching all the observed fragment ions to a theoretical fragmentation obtained by using MS Product (Protein Prospector).  Compounds 1, 2, and 3 were prepared as described previously [16] and TAMRA azide 10 was prepared according to the reported procedure [17]. Compound 9 was prepared as described below and exhibited spectral characteristics consistent with those reported previously [18]. Intermediate 15 was prepared as described previously [19]. All reagents and solvents were purchased from Aldrich Chemical or Acros Organics and used as received. Column chromatography was carried out by using a Biotage SP1 flash-    The requisite pyridine carboxylic acid (1.2 equiv), HATU (1.5 equiv) and diisopropylethylamine (6.0 equiv) are added and the reaction mixture stirred until the coupling is judged complete by LC/MS analysis, typically 12 hours. The reaction mixture is then diluted with ethyl acetate (at least 5x volume) and shaken with an equivalent volume of 50% saturated aqueous NaHCO 3 .

1-oxopropan-2-yl]pyridine-2-carboxamide trifluoroacetate salt (3).
Compound 3 was prepared according to the general procedure except that additional purification by HPLC was required. 10% overall yield from 14 1   The mixture was then cooled in a dry ice-acetone bath, and HATU (92.8 mg, 0.24 mmol, 1.1 equiv) was added in one portion. The reaction was allowed to slowly warm to −40 °C over 3 hours and was then diluted in ethyl acetate (20 mL) and washed with water (5 × 5 mL) and brine (5 mL). The organic layer was dried over sodium sulfate, filtered and concentrated. The crude material was then purified by reverse-phase HPLC, affording 17 mg of compound 2 as a white S19 solid (11% over three steps). 1

S22
General procedure for the synthesis of 12 and 13 from 27.
Intermediate 27 (0.36 g, 1.22 mmol) was dissolved in 5 mL 4:1 THF/water and then lithium hydroxide (0.117 g, 4.86 mmol) was added. The reaction mixture was stirred at room temperature and usually forms a suspension. When the hydrolysis reaction was judged to be complete by LC/MS, the reaction mixturewas adjusted to pH ~4 by slow addition of ~0.8 mL 2N HCl. The solvent was then removed in vacuo, and the crude lithium carboxylate intermediate was used in subsequent coupling reactions without further purification.
The reaction mixture was stirred at room temperature until the reaction was judged complete by LC/MS (typically 2 h). The reaction mixture was then diluted with ethyl acetate (at least 5x volume) and shaken with an equivalent volume of 50% saturated NaHCO 3 . The organic layer was then separated, washed with saturated NaCl, dried (MgSO 4 ), filtered, and concentrated.
The crude product was then purified by automated silica-gel chromatography (ethyl acetate/hexanes) and/or preparative HPLC to afford the final product.

(12).
Prepared according to the general procedure, employing methylamine in the coupling reaction.