**2.3.** *T. brucei* **choline kinase activity assay**

High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 µl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 µl from each well of the working plates was added to 110 µl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2. 3 µg of purified *Tb*CK was added to each well in 30 µl of the same buffer and the plates were mixed and incubated for 5 min at room temperature. A fur‐ ther 30 µl of buffer containing PEP (1 mM final), ATP (0.5 mM final), NADH (0.5 mM final) and pyruvate kinase and lactate dehydrogenase(PK/LDH) (5 units/ml final) was added, and the reaction was started by addition of 30 µl choline (0.5 mM final) to rows 1-11, 30 µl buffer alone was added to row 12 (negative control) and this was used as an intra-plate control (background rate) in conjunction with row 1 (maximal rate). Following mixing the change in absorbance at 341 nm was monitored for 10 min at room temperature. For testing inhibition of the coupling enzymes (PK/LDH), standard buffer conditions were used but the assay con‐ tained 1 mM PEP, 0.1 mM ADP and 0.5 mM NADH. The PK/LDH was titrated to give a change in absorbance of approximately 0.05 absorbance units/min in the absence of inhibi‐ tor.

### **2.4. Differential scanning fluorimetry with** *Tb***CK**

of a 96-well plate, at 10 mM in 5% DMSO, allowing the two outside columns for positive

Large-scale recombinant expression and purification of *Tb*CK was conducted using the con‐ struct pET-15bTEV-*Tb*CK in BL21 Rosetta (DE3) cells as described previously [23], except the cells were grown in tryptone phosphate broth [37], harvested by centrifugation at 3500 g for 20 min at 4°C and affinity purified with either a HisTrap™ FF crude column (enzyme

Briefly, pelleted cells were suspended in buffer A (50 mMTris/HCl, pH 8.0, 300 mMNaCl and 10 mM imidazole) and lysed in the presence of DNase I by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTrap™ FF crude column column (GE Healthcare) pre-loaded with Ni2+. Unbound proteins were re‐ moved by washing the column with 15 column volumes of buffer A containing 32.5 mM imidazole and *Tb*CK was eluted with 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mMTris/HCl, pH 8.0, 300 mMNaCl, glycerol

Alternatively, pelleted cells were suspended in 50 mMTris/HCl, pH 8.0, 300 mMNaCl and 5 mM imidazole and lysed by sonication. The lysate was cleared by centrifugation at 35000 g for 30 min at 4°C and applied to a 1 ml HisTALON Cartridge (Clontech).Unbound proteins were removed by washing the column with 10 column volumes of loading buffer, *Tb*CK was eluted with 15 mM imidazole and a final clearing wash of 250 mM imidazole in the same buffer. Using a PD10 column, *Tb*CK was buffer exchanged into 50 mM HEPES pH 8.0, 300

Typical yields were > 10 mg per litre of bacterial culture,*Tb*CK was stable and freeze thaw‐

High throughput screening of the Maybridge Rule of 3 Fragment Library was carried out at a final test concentration of 0.5 mM in 96-well plates (final assay volume 200 µl) using a spectrophotometric assay that has been described previously [23]. The screened library working plates consisted of compounds arrayed in 96 well plates at 10 mM in 5% DMSO; columns 1 and 12 contained 5% DMSO only. For high throughput screening, 10 µl from each well of the working plates was added to 110 µl of buffer containing 50 mM MOPS (pH 7.8), 150 mMKCl and 6 mM MgCl2. 3 µg of purified *Tb*CK was added to each well in 30 µl of the same buffer and the plates were mixed and incubated for 5 min at room temperature. A fur‐ ther 30 µl of buffer containing PEP (1 mM final), ATP (0.5 mM final), NADH (0.5 mM final) and pyruvate kinase and lactate dehydrogenase(PK/LDH) (5 units/ml final) was added, and the reaction was started by addition of 30 µl choline (0.5 mM final) to rows 1-11, 30 µl buffer alone was added to row 12 (negative control) and this was used as an intra-plate control (background rate) in conjunction with row 1 (maximal rate). Following mixing the change in

and negative controls.

416 Drug Discovery

(15% w/v) and stored at -80°C.

**2.2. Recombinant expression and purification of** *Tb***CK**

mMNaCl and 15% glycerol prior to storage at -80°C.

ing did not lead to any significant loss of activity.

**2.3.** *T. brucei* **choline kinase activity assay**

activity assay) or a HisTALON Cartridge (thermal shift analysis).

Differential scanning fluorimetry was set up in 96 well PCR plates using a reaction volume of 100 µL. Samples contained 2.1 µM *Tb*CK, 6 mM MgCl2, 50 mM HEPES pH 8.0, 80 mMNaCl, 5.25% glycerol (v/v) and 1.4 x Sypro Orange (Invitrogen), Maybridge Ro3 com‐ pounds were screened at 1 mM concentration with a final DMSO concentration of 0.5% (v/ v).Two controls with eight repetitions per plate were used for the thermal shift experiments: 0.5% DMSO; 0.5 mM ATP, 0.5% DMSO.

Differential fluorimetric scans were performed in a realtime PCR machine (Stratagene Mx3005P with software MxPro v 4.01) using a temperature scan from 25°C to 95°C at 0.5°C min-1.Data were then exported to Excel for analysis using "DSF analysis" modified from the template provided by Niesen et al. [38].Tm values were calculated by non-linear regression, fitting the Boltzmann equation to the denaturation curves using GraFit. *Tb*CK Tm, in the presence of 6 mM MgCl2 and 0.5% DMSO, 41.21 ± 0.03°C (n > 60), Tm for *Tb*CK and 0.5 mM ATP = 44.46 ± 0.05°C (n > 60).
