Chemical Validation of Methionyl-tRNA Synthetase as a Druggable Target in Leishmania donovani

Methionyl-tRNA synthetase (MetRS) has been chemically validated as a drug target in the kinetoplastid parasite Trypanosoma brucei. In the present study, we investigate the validity of this target in the related trypanosomatid Leishmania donovani. Following development of a robust high-throughput compatible biochemical assay, a compound screen identified DDD806905 as a highly potent inhibitor of LdMetRS (Ki of 18 nM). Crystallography revealed this compound binds to the methionine pocket of MetRS with enzymatic studies confirming DDD806905 displays competitive inhibition with respect to methionine and mixed inhibition with respect to ATP binding. DDD806905 showed activity, albeit with different levels of potency, in various Leishmania cell-based viability assays, with on-target activity observed in both Leishmania promastigote cell assays and a Leishmania tarentolae in vitro translation assay. Unfortunately, this compound failed to show efficacy in an animal model of leishmaniasis. We investigated the potential causes for the discrepancies in activity observed in different Leishmania cell assays and the lack of efficacy in the animal model and found that high protein binding as well as sequestration of this dibasic compound into acidic compartments may play a role. Despite medicinal chemistry efforts to address the dibasic nature of DDD806905 and analogues, no progress could be achieved with the current chemical series. Although DDD806905 is not a developable antileishmanial compound, MetRS remains an attractive antileishmanial drug target.

intersect defining the point where the active enzyme concentration equals the inhibitor concentration. From this analysis, the point of intersection was found to be 780 nM, indicating that 78% of this LdMetRS protein contains active enzyme, therefore in the LdMetRS enzyme reaction containing 50 nM total protein, the active enzyme concentration is 39 nM. (B) Using this defined active enzyme concentration of 39 nM as a constant, DDD806905 is found to return a K i app of 41 nM following fitting to the Morrison equation. All data shown as mean ± SD (n≥3 technical replicates).

SUPPLEMENTARY FIGURE 3: Sequence Alignment of TbMetRS and LdMetRS
Sequence alignment of T.brucei_MetRS (Tb927. 10.1500) and L.donovani_MetRS (LdBPK_210890. 1) showing a high level of sequence conservation. Residues that line the ligand binding site are highlighted with upward arrows. Residues that line the expanded methionine pocket are highlighted with blue arrows and those that line the auxiliary pocket are highlighted with red arrows. The CP domain (TbMetRS residues 354 to 406), which moves significantly to allow the binding of DDD806905, is highlighted in magenta.

SUPPLEMENTARY FIGURE 4: Co-operativity Between ATP and Methionine Binding Pockets
Co-operativity between the LdMetRS methionine and ATP binding sites is highlighted with the methionine K m varying depending on the ATP concentration used in the assay. (A) When ATP is used at a fixed, saturating concentration of 400 µM (10 x K m ), the methionine K m is shown to be 170 µM.
(B) However, if the fixed ATP concentration is reduced to 80 µM (2 x K m ), the methionine K m increases to 1500 µM. All data shown as mean ± SD (n=3 technical replicates).
[Methionine], µM The maximum concentration in blood (C max ) was 2,600 ng/ml and 3,200 ng/ml for day 1 and day 10 respectively, T max was at 2h, AUC (0-8h) was 950,000 ng-min/ml on day 1 and 1,400,000 ng-min/ml on day 10.

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Serum, %  A gradient of 0-50% B (A+500 mM imidazole) was used for the elution. At 5% B a His rich peak was eluted. At 6.8% B the gradient was held to elute the protein.
The protein was dialyzed overnight at 4 °C in Buffer A in the presence of PreScission Protease (PP) (5mg) before the sample was ran over 1 ml of Glutathione Sepharose 4B beads (GE) to remove the protease.
A second 5 ml HiTrap HIS HP (GE) column was performed to remove uncleaved protein before the sample was concentrated for gel filtration. The sample was run on a Superdex 200 26/60 column pre-equilibrated with 25 mM HEPES, 500 mM NaCl, 10% Glycerol, 2 mM DTT, 10 mM L-methionine pH7.0. Purified TbMetRS was concentrated to 10 mg/ml prior to crystallisation.

Size Exclusion Chromatography and Multi Angle Light Scattering (SEC-MALS)
SEC-MALS experiments were performed on a Dionex Ultimate 3000 HPLC system with an inline

LdMetRS Assay Development and Kinetic Parameter Determinations
Activity of the LdMetRS enzyme was determined by monitoring levels of pyrophosphate released during the first step of the enzymatic reaction. The pyrophosphate formed was converted to two inorganic phosphate molecules using a pyrophosphatase enzyme and levels of the resulting phosphate molecules measured using the BIOMOL® Green reagent (Enzo Life Sciences) 2 .
Following optimization of the assay buffer and determination of the enzyme linearity, 384-well plate based assays were carried out at room temperature in a 50 μl reaction volume containing 30 mM

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Assays were performed by adding 25 μl buffer with enzyme to assay plates (with buffer only added to 'no enzyme' control wells) before the reaction was initiated with the addition of a 25 μl mix containing methionine, ATP and pyrophosphatase to all wells. Following a 120 min reaction at room temperature the assay was stopped with the addition of 50 μl BIOMOL® Green. The BIOMOL® Green signal was allowed to develop for 30 min before the absorbance of each well was read at 650 nm using an EnVision multilabel plate reader (PerkinElmer Life Sciences). All liquid dispensing steps were carried out using a Thermo Scientific WellMate dispenser (Matrix).
ActivityBase from IDBS (version 8.0.5.4) was used for data processing and analysis. All IC 50 curve fitting was undertaken using ActivityBase XE (version 7.7.1) from IDBS. A four-parameter logistic dose-response curve was utilized with prefit used for all four parameters.

Mode of Inhibition Studies
To establish the mode of inhibition of DDD806905, data sets (generated using the BIOMOL® Green assay platform previously described) were collected by varying both the inhibitor and substrate concentrations. Using GraFit v6.0 (Erithacus Software) each data set was individually fitted to the Michaelis-Menten equation or the modified high substrate inhibition Michaelis-Menten equation described above (equation 2), and the resulting Lineweaver-Burk plots were examined for diagnostic patterns of competitive, mixed, or uncompetitive inhibition.
Data sets were then globally fitted to the appropriate model (with equations 4 and 5 used for competitive and mixed inhibition respectively). If more than one model appeared possible, then data were fitted to both and examined for significance using the F-test function in GraFit. Diffraction data were measured at Diamond Light Source beamline I02. Data were integrated using XDS 3 and scaled using Aimless 4 . Structure solution was carried out using MOLREP 5 with TbMetRS (4EGA) used as a search model. The structure was refined with REFMAC5 6 from the CCP4 suite 7 , ligand topologies generated using PRODRG 8 and manual model alteration carried out using COOT 9 . Data measurement and model refinement statistics are presented in Supplementary Table 2.

Crystals of
Coordinate files and associated experimental data have been deposited in the Protein Data Bank (PDB) with accession code 5NFH.

Leishmania tarentolae In Vitro Translation
Protein translation in a Leishmania cell extract was studied using a commercially available cell-free protein expression kit (Jena Bioscience). Reactions were carried out in black 384-well, small-volume plates (Greiner) in 10 µl reaction volumes containing 7 µl L. tarentolae cell extract, 1 µl eGFP DNA template and 2 µl nuclease free water ('no DNA' wells were included as negative controls). eGFP expression was monitored over time using a BMG PheraStar plate reader (Excitation 485 nm; Emission 520 nm).

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When test compounds were to be screened, the compound of interest (solubilised in 100% DMSO) was dispensed into assay plates using an Echo® 550 (Labcyte) before L. tarentolae extract and eGFP template DNA were added to assay wells as described above. The reaction was run at room temperature for 120 min before eGFP fluorescence intensity was measured. IC 50 curve fitting was undertaken in GraFit version 6.0 (Erithacus Software) using a four-parameter logistic dose-response curve (equation 6), with s the slope.

HeLa In Vitro Translation
Protein translation in a HeLa cell extract was studied using a commercially available in vitro translation kit (Thermo Scientific). Reactions were carried out in black 384-well, small-volume plates (Greiner) in 10 µl reaction volumes containing 5 µl HeLa cell extract, 1 µl accessory proteins, 2 µl reaction mix, 0.8 µl GFP DNA template and 1.2 µl nuclease free water ('no DNA' wells were included as negative controls). GFP expression was monitored over time using a BMG PheraStar plate reader (Excitation 485 nm; Emission 520 nm).
When testing DDD806905 and the positive control compound cycloheximide, compounds (solubilised in 100% DMSO) were dispensed into assay plates using an Echo® 550 (Labcyte) before HeLa extract, accessory proteins, reaction mix and GFP template DNA were added to assay wells and GFP expression monitored as described above.

Mitochondrial protein synthesis assay
The effect of DDD806905 on mitochondrial protein synthesis was determined by BioDuro using the MitoBiogenesisTM In-Cell ELISA Kit (Abcam). The kit reports the ratio of a nuclear (SDH-A) and a mitochondrially (COX-1) translated protein which is used to determine if there is a mitochondrial specific protein synthesis effect. In the assay the levels of SDH-A and COX-1 are measured in H9C2 cells. These are normalised to DMSO treated cells (vehicle) and then the ratio of normalised SDH-A over COX-1 is calculated. Dose response curves are calculated with this data and an IC 50 is reported.

Knock-out and overexpressor generation
Unsuccessful experiments to generate Leishmania MetRS knock-out or overexpressor cells were carried out following standard methods as described in 14 .

Leishmania Promastigote Assay
Compounds to be tested were pre-dispensed into white, clear bottom 384-well plates (Greiner). For potency determinations, ten-point one in three dilution curves were generated. The top concentration was 50 μM and on each plate a control curve of Amphotericin B was included. LdBOB promastigotes were added to all wells containing compound (5000 cells per well, 50μl) using a Thermo Scientific WellMate dispenser (Matrix). Media only was dispensed into control columns.
After a 68 h incubation at 37°C under 5% CO 2 in a humidified incubator, resazurin was added to each well at a final concentration of 0.5 mM and the plates were incubated for a further 4 h. Plates were then sealed with clear film and resorufin fluorescence was detected using a Victor 3 plate reader (Perkin Elmer) with excitation at 528 nm and emission at 590 nm. Serum shift experiments were S30 carried out as described above using 5%, 10% and 20% FBS in the media. To examine the effect of the methionine concentration on the potency of DDD806905 parasites were grown in the presence of drug and in the presence (or absence) of 20-fold excess methionine (0.3 g/l). IC 50 curve fitting was undertaken in GraFit version 6.0 (Erithacus Software) using a four-parameter logistic dose-response curve (equation 6), with s the slope.

Leishmania Axenic Amastigote Assay
The Leishmania axenic amastigote assay was performed using the method published in Nühs et al 15 .

Leishmania Intracellular Amastigote Assay
The intramacrophage Leishmania assay was performed using a modified version of the method described in De Rycker et al 16

pK a measurements
To measure the pK a of DDD806905, a fast UV-metric method was performed using a Sirius T3 instrument (Sirius Analytical, U.K.). The calculation of the pK a was determined using the Sirius T3 refine software (Sirius Analytical, U.K.). In brief, the UV-metric pK a method is based on measuring pH during an acid-base titration and monitoring UV absorbance shift. The ionisation curve (Bjerrum curve) was then used to determine the pH where exactly 50 % of neutral and 50 % of ionised species was present. DDD806905 must be fully dissolved throughout the titration and so the titration is set to start at the pH where DDD806905 is in its fully ionised state. The appearance of precipitate was monitored throughout the experiment by a turbidity sensing device. DDD806905 was predicted to be poorly soluble (ACD labs) and so the titration was performed in the presence of methanol. The pK a s of DDD806905 was determined at the pH where there was maximal change in UV absorbance with respect to pH. The pH range of the titration was between 2 and 12. The pK a values were then extrapolated to an aqueous pK a using a graphical method based on the work by Yasuda and Shedlovsky 17,18 using the Sirius T3 refine software 19 .

LogD 7.4 measurements
LogD 7.4 values were quantified using a Sirius T3 instrument (Sirius Analytical, U.K.). The lipophilicity assay was a standard potentiometric titration performed in the presence of three varying octanolwater ratios. The instrument software calculated the pH of each data point using mass and charge balance equations and then fitted this theoretical data to the experimental data. The logP value S32 which gave the best fit to the data was then recorded as the logP. Using the logP and an experimentally determined pK a (see above), the software generated a lipophilicity plot showing changes in the logD with increasing pH and from this plot a logD 7.3 and logD 5.6 values were recorded. The unbound fraction (fu) was determined as the ratio of the peak area in buffer to that in plasma.

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Preparation of 2-((3-(((4,6-dichloro-1H-indol-2-yl)methyl)amino)propyl)amino)quinazolin-4(1H)-one dihydrochloride (DDD806905.2HCl): A slurry of 2-(3-aminopropylamino)-3H-quinazolin-4-one (2.39 g, 11.0 mmol) and 4,6-dichloro-1Hindole-2-carbaldehyde (2.39 g, 11.2 mmol) in THF (250 mL) at room temperature needed to be warmed to 55°C in order to obtain a solution. After stirring at 55°C for 60 mins sodium triacetoxyborohydride (4.75 g, 22.4 mmol) was added portionwise and the resulting solution was stirred for 72 h. The reaction was then cooled to room temperature then quenched by careful addition of 25 mL NaOH solution. The reaction was then diluted with brine (100 mL) and diethylether (100 mL) and stirred for 2 h, the organic phase separated, combined with organic extracts of the aqueous layer (3 x 50 mL EtOAc), the combined organics dried (MgSO 4 ) and concentrated in vacuo. The residual gum was subjected to chromatography (SiO 2 , 100% EtOAc moving to 20% 7 N methanolic ammonia in EtOAc) to give 1.85 g of the title compounds as the free base. This solid was then slurried in dry diethylether and treated dropwise with 1 M HCl in diethylether and the resulting precipitate collected by filtration and dried to give the title compound     After stirring at RT for 3 h, complete consumption of starting material was noted and the reaction mixture was partitioned between 2N NaOH (10 mL) and EtOAc (20 mL), a precipitate formed at this point which was filtered off. LC/MS and NMR analysis of the precipitate showed it not to be product.

Preparation of N-(3,5-dichlorobenzyl)-3-((4-oxo-1,4-dihydroquinazolin-2-yl)amino)propanamide
The aqueous layer was further extracted with EtOAc (2 × 20 mL) and the combined organics were washed with brine, dried over MgSO4 and concentrated. 1 H NMR of the crude reaction mixture showed it to be composed mostly of desired product. The residue was dry-loaded onto silica and purified by column chromatography (SiO 2 , 100% EtOAc moving to 10% 7 N methanolic ammonia in EtOAc) to give the title compound as a yellow oil. added and the reaction mixture was allowed to stir at 60 °C for 2 hours. The reaction mixture was allowed to cool to RT and sodium triacetoxyborohydride (231 mg, 1.09 mmol) was added in one portion and then heated again at 60 °C overnight. The main peak in the LC/MS of the crude reaction mixture corresponded to the mass of the desired product. The reaction mixture was allowed to cool to RT and it was partitioned between 2N NaOH (10 mL) and EtOAc (20 mL). The aqueous layer was further extracted with EtOAc (2 × 20 mL) and the combined organics were washed with brine (20 mL) and dried over MgSO 4 and concentrated to give a brown solid. The residue was dry-loaded onto silica and purified by column chromatography (SiO 2 , 100% EtOAc moving 10% MeOH in EtOAc). The fractions containing the desired product were combined, however impurities were still present. The