Biochemical and Structural Characterization of Selective Allosteric Inhibitors of the Plasmodium falciparum Drug Target, Prolyl-tRNA-synthetase

Plasmodium falciparum (Pf) prolyl-tRNA synthetase (ProRS) is one of the few chemical-genetically validated drug targets for malaria, yet highly selective inhibitors have not been described. In this paper, approximately 40,000 compounds were screened to identify compounds that selectively inhibit PfProRS enzyme activity versus Homo sapiens (Hs) ProRS. X-ray crystallography structures were solved for apo, as well as substrate- and inhibitor-bound forms of PfProRS. We identified two new inhibitors of PfProRS that bind outside the active site. These two allosteric inhibitors showed >100 times specificity for PfProRS compared to HsProRS, demonstrating this class of compounds could overcome the toxicity related to HsProRS inhibition by halofuginone and its analogues. Initial medicinal chemistry was performed on one of the two compounds, guided by the cocrystallography of the compound with PfProRS, and the results can instruct future medicinal chemistry work to optimize these promising new leads for drug development against malaria.

: Dose response curve for halofuginone and N-Boc-halofuginone in the [ 3 H]-L-proline aminoacylation incorporation assay (10 nM PfProRS enzyme, 100 nM [ 3 H]-L-proline, 100 M ATP and 400 g/ml yeast tRNA was incubated for 2 h at 25 C; Z' = .67). Lines are fit to a Hill Slope = 1 and IC50 for Halfuginone is calculated to be 0.011 µM and N-Boc-Halofuginone is >3 µM. Error bars indicate the standard deviation of triplicate samples for each experimental condition.  (cc3) Table S2: Percentage inhibition of ProRS enzyme (Pf = Plasmodium, Hs = human) activity by ATP-depletion assay. Full inhibition (100) was equivalent to controls of no enzyme or no substrate and no inhibition (0) was equivalent to all reagents with 2% DMSO (no compound). Percentage inhibition values are the average of measurements in triplicate. The simplified molecular-input line-entry system (SMILES) formula for each compound is indicated in the lower portion of the table.

Figure S3
A. The effects of TCMDC-124506 (tested at 1 M and 5 M), DMSO (0.1% v/v; solvent control) and the spiroindolone KAE609 (50 nM) on the fluorescence of isolated 3D7 trophozoites loaded with the Na +sensitive dye SBFI. An increase in fluorescence ratio corresponds to an increase in the intracellular [Na + ] ([Na + ] i ); the fluorescence ratios corresponding to 130 mM Na + and 0 mM Na + are shown. TCMDC-124506 and KAE609, but not DMSO, gave rise to an increase in [Na + ] i . B. The effects of various compounds on pH i in isolated 3D7 trophozoites. DMSO (0.1% v/v; solvent control) did not increase pH i , and on addition of the Vtype H + pump inhibitor concanamycin A (100 nM; red arrow) the pH i decreased to below the pH of the extracellular solution. TCMDC-124506 (tested at 5 M) and KAE609 (50 nM) caused an increase in pH i and a reduction in the extent of the concanamycin A-induced acidification. In A and B, the traces shown are from a single experiment, and are representative of those obtained in two similar experiments.

Figure S4
Competition of proline and ATP with TCMDC-124506 for PfProRS demonstrates that both compete with TCMDC-124506.

A. IC50 of TCMDC-124506 as a function of proline B. Linear relationship with upward trend indicates competitive behavior of proline with TCMDC-124506
[TCMDC], M  Figure S4 legend: The experiments were run as described for the ATP consumption assay in the Materials and Methods except that the proline or the ATP was varied and the IC50 for TCMDC-124506 was determined for each condition.

Supplemental Experimental Procedures Biology Methodology
Plasmodium falciparum screening Assays against P. falciparum were conducted as previously described (Trager and Jensen 1976;Bennett et al., 2004;Snyder et al., 2007). Cultures of the widely-used malaria reference strain of chloroquine-sensitive Plasmodium falciparum strain 3D7 were maintained in a 5% suspension of human red blood cells (obtained from East of Scotland Blood Transfusion Service, Ninewells Hospital, Dundee) cultured in RPMI 1640 medium (pH 7.3) supplemented with 0.5% Albumax II (Gibco Life Technologies, San Diego, CA), 12 mM sodium bicarbonate, 0.2 mM hypoxanthine and 20 mg/L gentamicin at 37°C, in a humidified atmosphere of 1% O 2 , 3% CO 2 with a balance of nitrogen. Growth inhibition was quantified using a fluorescence assay utilising the binding of SYBR green to double stranded DNA, which emits a fluorescent signal at 528 nm after excitation at 485nm (Plouffe et al., 2008). Mefloquine (potency range 30-60 nM) was used as a drug control to monitor the quality of the assay (Z' = 0.6 to 0.8, Signal to background >3, where Z' is a measure of the discrimination between the positive and negative controls on a screen plate). Compound bioactivity was expressed as EC 50 , the effective concentration of compound causing 50% inhibition of parasite growth.

Thermal Melt Analysis
Color fluorimetry experiments to measure changes in protein thermal stability were conducted as previously described (Vedadi et al., 2006). Serial dilutions of the purified recombinant protein samples were prepared in 96 well PCR plates (Bio-Rad HSP9655) in a buffer containing 100mM HEPES, 150mM NaCl, pH 7.5 and SYPRO Orange Dye (Life Technologies S-6650). Protein stability for protein samples containing the substrates ATP and L-Proline were compared with apo protein samples by measuring an increase in fluorescence as detected on an MJ Research DNA Engine Opticon 2 qPCR thermocycler from 20C to 99C at half degree increments.

In vitro Cell Assay Data Analysis
All data was processed using IDBS ActivityBase ® raw data was converted into per cent inhibition through linear regression by setting the high inhibition control as 100% and the no inhibition control as 0%. Quality control criteria for passing plates were as follows: Z'> 0.5, S:B> 3, %CV (no inhibition control) < 15. The formula used to calculate Z' is .
All EC 50 Curve fitting was undertaken using XLFit version 4.2 using Model 205 with the following 4 parametric ,where A=% inhibition at bottom, B=% inhibition at top, C= EC 50 , D= slope, x= inhibitor concentration and y= % inhibition. If curve did not reach 100% of inhibition, B was fixed to 100 only when at least 50% of inhibition was reached.

Aqueous solubility
The aqueous solubility of the test compounds was measured using laser nephelometry, as described previously (Patterson et al., 2013). Compounds were subject to serial dilution from 10 mg/mL to 0.5 mg/mL in DMSO. An aliquot was then mixed with MilliQ water to obtain an aqueous dilution plate with a final concentration range of 100 -5 μg/mL, with a final DMSO concentration of 1.0%. Triplicate aliquots were transferred to a flat bottomed polystyrene plate which was immediately read on the NEPHELOstar (BMG Lab Technologies). The amount of laser scatter caused by insoluble particulates (relative nephelometry units, RNU) was plotted against compound concentration using a segmental regression fit, with the point of inflection being quoted as the compounds aqueous solubility (μg/mL). Assays were run in triplicate.

Intrinsic Clearance (CLi) experiments
The procedure was carried out as reported previously (Patterson et al., 2013). Test compound (0.5 µM) was incubated with female CD1 mouse liver microsomes (Xenotech LLC TM ; 0.5 mg/mL 50 mM potassium phosphate buffer, pH 7.4) and the reaction started with addition of excess NADPH (8 mg/mL 50 mM potassium phosphate buffer, pH 7.4). Immediately, at time zero, then at 3, 6, 9, 15 and 30 min an aliquot (50 µL) of the incubation mixture was removed and mixed with acetonitrile (100 μL) to stop the reaction. Internal standard was added to all samples, the samples were centrifuged to sediment precipitated protein and the plates then sealed prior to UPLCMSMS analysis using a Quattro Premier XE (Waters Corporation, USA).
XLfit (IDBS, UK) was used to calculate the exponential decay and consequently the rate constant (k) from the ratio of peak area of test compound to internal standard at each timepoint. The rate of intrinsic clearance (CLi) of each test compound was then calculated using the following calculation: CLi (mL/min/g liver) = k x V x Microsomal protein yield Where V (mL/mg protein) is the incubation volume/mg protein added and microsomal protein yield is taken as 52.5mg protein per g liver. Verapamil (0.5 µM) was used as a positive control to confirm acceptable assay performance. Experiments were performed using a single time-course experiment.

Ion homeostasis in the malaria parasite
To measure ion concentrations inside the parasite, P. falciparum trophozoites (3D7 strain) were isolated from their host erythrocytes by brief exposure to saponin (0.05% w/v final concentration), then loaded with either the Na + -sensitive dye SBFI (for measurements of intracellular [Na + ] ([Na + ] i )) (Spillman et al., 2013a) or the pHsensitive dye BCECF (for measurements of intracellular pH (pH i )) (Saliba and Kirk, 1999). Fluorescence measurements and calibrations were performed at 37°C in the same manner described previously (Spillman et Lehane et al., 2014). To test TCMDC-124506, 1 L of a DMSO stock of the compound was added to 1 mL of isolated parasites suspended in a saline solution (125 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 20 mM glucose, 25 mM HEPES; pH 7.10) at a density of ~ 3 × 10 7 parasites mL -1 to give the desired final concentration.
TCMDC-124506 gave rise to the ionic "signature" of PfATP4 inhibition that has been described previously for spiroindolones (Spillman et al., 2013a;Spillman et al., 2013b) and other chemically diverse compounds (Lehane et al., 2014): it caused (i) an increase in the Na + concentration inside the parasite ( Figure S3A), (ii) a cytosolic alkalinisation ( Figure S3B), and (iii) a reduction in the extent of acidification seen following inhibition of the parasite's V-type H + pump with concanamycin A ( Figure S3B).

Chemistry Experimental Section
Chemistry. General. Solvents and reagents were purchased from commercial suppliers and used without further purification. Dry solvents were purchased in sure sealed bottles stored over molecular sieves. Reactions using microwave irradiation were carried out in a Biotage Initiator microwave. Normal phase TLCs were carried out on pre-coated silica plates (Kieselgel 60 F 254 , BDH) with visualisation via UV light (UV254/365 nm) and/or ninhydrin solution. Flash chromatography was performed using Combiflash Companion Rf (Teledyne ISCO) and prepacked RediSep silica gel columns purchased from Teledyne ISCO. Mass-directed preparative HPLC separations were performed using a Waters HPLC (2545 binary gradient pumps, 515 HPLC make up pump, 2767 sample manager) connected to a Waters 2998 photodiode array and a Waters 3100 mass detector. Preparative HPLC separations were performed with a Gilson HPLC (321 pumps, 819 injection module, 215 liquid handler/injector) connected to a Gilson 155 UV/vis detector. On both instruments, HPLC chromatographic separations were conducted using Waters XBridge C18 columns, 19 x 100 mm, 5 um particle size; using 0.1% ammonia in water or 0.1% formic acid in water (solvent A) and acetonitrile (solvent B) as mobile phase. 1 H NMR spectra were recorded on a Bruker Avance DPX 500 spectrometer ( 1 H at 500.1 MHz), or a Bruker Avance DPX 400 ( 1 H at 400 MHz). Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as the internal reference in all cases. Signal splitting patterns are described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), broadened (br), or a combination thereof. Coupling constants (J) are quoted to the nearest 0.1 Hz. Low resolution electrospray (ES) mass spectra were recorded on a Bruker Daltonics MicroTof mass spectrometer, run in positive mode. LC-MS analysis, HRMS analysis and chromatographic separation were conducted with a Brucker Daltonics MicrOTOf mass spectrometer or an Agilent Technologies 1200 series HPLC connected to an Agilent Technologies 6130 quadrupole LC/MS, where both instruments were connected to an Agilent diode array detector. The column used was a Waters XBridge column (50 mm × 2.1 mm, 3.5 μm particle size,) and the compounds were eluted with a gradient of 5 to 95% acetonitrile/water +0.1% Ammonia. All final compounds showed chemical purity ≥ 95% as determined by the UV chromatogram (190-450nm) obtained by LC-MS analysis and NMR. Unless otherwise stated herein reactions have not been optimized. Compounds 3 and 17 were purchased from Maybridge and tested without further purification after purity was determined to be ≥ 95% by the UV chromatogram (190-450nm) obtained by LC-MS analysis.
General procedure A. A mixture of ethyl 2,2,2-trifluoroacetate (1 g, 7.0 mmol) and the corresponding acetonitrile (1 eq) in dry THF (5 mL) was added to a stirred suspension of NaH (1.5 eq) in dry THF (5 mL) under nitrogen at 40 o C over the course of 30 min. After stirring at 40 o C for 2 hours, the reaction mixture was poured into water and acidified with HCl 3N and extracted with diethyl ether. The combined organic layers were dried over MgSO 4 and concentrated to give a crude material that was triturated using a mixture of CH 2 Cl 2 /petroleum ether and the solid was filtrated to obtain a first fraction of the desired compound. The CH 2 Cl 2 / petroleum ethers filtrate was concentrated under reduced pressure to obtain a mixture of product and starting materials. This mixture was triturated again with CH 2 Cl 2 / petroleum ethers to yield a second fraction of the desired product.
General Procedure B. A solution of methylhydrazine (23 mg, 0.5 mmol) and the corresponding 3-oxobutanenitrile (1 eq) in methanol (1 mL) was heated at 100 o C for 1h under microwave irradiation. The solution was evaporated under reduced pressure and the resulting crude material purified by flash column chromatography using a silica cartridge eluting with 40% of ethyl acetate in heptane. The desired fractions were concentrated under reduced pressured to give the desired pyrazole as a solid.
General Procedure C. To a stirred solution of the desired pyrazol-5-amine (0.71 mmol) in THF (1 mL) the corresponding isocyanate (1.1 eq) was added and the reaction mixture was stirred at room temperature overnight. The reaction was concentrate to dryness and the crude material was purified by flash column chromatography using a silica cartridge eluting with 10% of ethyl acetate in heptane to give the desired pyrazol-5-yl-urea.

1-[4-bromo-2-methyl-5-(trifluoromethyl)pyrazol-3-yl]-3-(4-fluorophenyl)urea (11).
To a solution of 10 (1.2 g, 3.9 mmol) in acetonitrile (5 mL), NBS (0.77 g, 4.4 mmol) was added at room temperature. The reaction was stirred at room temperature for 1h. Solvent was evaporated under reduced pressure, the residue was taken up in DCM (10 mL) and washed with water (5 mL). Solvents were removed and product was purified by column chromatography using a 24 g silica cartridge using heptane (A) and ethyl acetate (B) as eluents and the following gradient: 1 min hold 100%A, 15 min ramp to 50% B, 1 min hold 50%B. Fractions containing product were pooled together and solvents were removed under reduced pressure to obtain 11 as a white solid (730 mg

4-bromo-2-methyl-5-(trifluoromethyl)pyrazol-3-amine.
To a solution of commercially available 2-methyl-5-(trifluoromethyl)pyrazol-3-amine (1.6 g, 10 mmol) in acetonitrile (5 mL) in an ice bath, NBS (1.7 g, 10 mmol) was added. The reaction mixture was stirred for 30 min in an ice bath. Solvents were evaporated under reduced pressure, the resulting residue was taken up in DCM (100 mL) and washed with water (20 mL). The organic phase was separated and dried over MgSO 4 . Solvents were removed under reduced pressure. The desired product was purified by column chromatography using a silica cartridge (24 g) and heptane (A) and ethyl acetate (B) as eluents and the following gradient: 1 min hold 100%A, 18 min ramp to 30% B, 1 min hold 30 %B. Fractions containing product were pooled together and solvents were removed under reduced pressure to obtain the title compound as a light pink solid (1.8 g, 73% yield). ¹H NMR ( General Procedure E. To a solution of 4-bromo-2-methyl-5-(trifluoromethyl)pyrazol-3-amine (1 eq), palladium acetate (0.05 eq), 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl (X-Phos) (0.1 eq), and the corresponding boronic acid (1.5 eq) in toluene (3 mL) was added potassium phosphate (1.5 eq) in water (1 mL). The reaction was sealed and the mixture was stirred at 95 o C overnight. Reaction crude was filtered through Celite (5g cartridge) and Celite washed with DCM (10 mL). Reaction was partitioned between DCM (10 mL) and brine (5 mL). The organic layer was dried over MgSO 4 , solvents were evaporated under reduced pressure. The product was purified by column chromatography using silica cartridge (12 g) and heptane (A) and ethyl acetate (B) as eluents.