A suicide inhibitor of nematode trehalose-6-phosphate phosphatases

Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 μM and 0.56 μM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber’s pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 μM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.

• Mobile Phase B: 100% acetonitrile with 0.1% formic acid • Flow Rate: 0.400 ml min -1 • Column temperature: 30 °C • Sample injection volume: 1 µL The elution protocol consisted of a gradient formed from 95% A to 100% B over 4.50 min; then hold at 100% B for 1 min; change to 95% A over 0.5 min; then hold for 1 min. The MS was collecting data for the complete 7 min run. Spectral absorbance data were acquired in the wavelength range from 190 nm to 350 nm with chromatograms constructed using the absorbance at 254 nm.

Screening of compounds by ligand binding assay
The optimal ratio of protein and fluorescence dye for a two-state unfolding curve was optimised by testing a 7×4 matrix of conditions varying the protein concentration from 0.5 to 32 μM, and SYPRO Orange (Invitrogen; Life Technologies, Mulgrave, VIC, Australia) concentration between 5× and 20× in a sample volume of 20 μL with a buffer composed of 100 mM NaCl and 20 mM HEPES (pH 7.5) 1 . For Acey-TPP, the best conditions were determined to contain 0.5 μM protein and 5× SYPRO Orange. DSF experiments were carried out in 96-well plates using a Roche LightCycler 480 (Roche, Basel, Switzerland). Three technical replicates were tested for each ligand, along with three replicates of a protein-buffer and a protein-DMSO mixture per 96-well plate. Each reaction mixture comprised the optimised protein:dye ratio in a total volume of 20 μL. Ligands were added from their stock solutions in DMSO at a final concentration of 2.5-5 μM, with a final DMSO concentration of 5%.

Enzyme end-point assays
Briefly, the phosphatase activity of Acey-TPP and Tcan-TPP was assessed using 500 μM trehalose-6-phosphate and 10 μM enzyme to be tested. Reactions were carried out in a volume of 50 μL in assay buffer (100 mM NaCl, 20 mM TRIS, pH 7.5). Individual compounds were added to the reaction mixtures at a final concentration of 25 μM, followed by incubation for 5 min before reactions were initiated by the addition of substrate (final DMSO concentration of 4%). Reactions were allowed to proceed for 5 min before quenching with 100 μL of BIOMOL ® Green reagent (Enzo Life Sciences, New York, NY, USA). Absorbance at 620 nm was determined using a Biotek ® Synergy 2 plate reader (BioTek, Winooski, VT, USA) after an incubation period of 15 min for colour development. All reactions were set up in triplicate in 96-well plates (Corning, Sigma-Aldrich, NSW, Australia) at 25 °C and control experiments in the absence of enzyme were used to correct for background absorbance. To determine IC 50 values, end-point assays were performed in the presence of increasing concentrations of compound (2.5 nM to 250 μM). Compounds were prepared in stocks of increasing concentration such that only 2 μL was added to reaction wells, thus keeping the DMSO concentration consistent at 4%. Enzyme in the absence of compound (with DMSO only) was run as a control and after correction for background absorbance, all test wells were scaled relative to the enzyme-only control.

Mass spectrometry
To determine possible modification of Tcan-TPP by N-(phenylthio)phthalimide, the protein was diluted to a final concentration of 1 mg mL -1 into a buffer containing 25 μM of 1, 100 mM NaCl, 0.2 mM MgCl 2 and 20 mM TRIS (pH 7.5). Mass spectrometric analyses were conducted at the Australian Proteome Analysis Facility (APAF). For in-solution digestion, 20 μL of the protein solution were mixed with 5 μL of 100 mM triethylammonium bicarbonate (TEAB) and 1 μg trypsin was added. After incubation at 37 C for 5.5 h, 2.5 μL of the digested sample were diluted into 7.5 μL of TEAB buffer. The final sample was subjected to 1D-nano-LC electrospray ionisation (ESI) MS/MS analysis using a model 6600 Sciex mass spectrometer and an Eksigent nanoLC-Ultra HPLC system with HALO C18 analytical (160 Å, 2.7 μm, 200 μm × 20 cm) and trap (160 Å, 2.7 μm, 150 μm × 3.5 cm) columns. The loading buffer contained 2% acetonitrile, 97.9% water and 0.1% formic acid; mobile phases A and B consisted of 99.9% water and 0.1% formic acid, and 99.9% acetonitrile and 0.1% formic acid, respectively. The sample (10 μL) was injected onto a reverse-phase trap for preconcentration and desalted with loading buffer, at 4 μL min -1 for 10 min. The peptide trap was then switched into line with the analytical column. Peptides were eluted from the column using a linear solvent gradient from mobile phase A: mobile phase B (98:2) to mobile phase A: mobile phase B (76:24) over 55 min. The reverse phase nanoLC eluent was subjected to positive ion nanoflow electrospray analysis in an information dependant acquisition mode. A TOF-MS survey scan was acquired (m/z 350-1500, 0.25 second) with the 20 most intense multiply charged ions (2 + -5 + ; exceeding 200 counts per second) in the survey scan sequentially subjected to MS/MS analysis. MS/MS spectra were accumulated for 100 ms in the mass range m/z 100-1800 with rolling collision energy. Dynamic exclusion was set to 30 s. The sequence of Tcan-TPP was added to a database of E. coli proteins (23,043 sequences) and the LC-MS/MS data were searched against this database using ProteinPilot v5.0 (SCIEX) in 'thorough' mode. A custom modification of thiophenyl (C 6 H 5 S, monoisotopic mass: 109.011196 Da) on cysteine was added to the list of modifications.

Modelling of substrate-bound B. malayi TPP
As the deposited crystal structures of Bmal-TPP (PDB accession code 4ofz, 5e0o) lacked several residues due to absence of electron density, we generated a model that included residues 63-491 based on the structure deposited as 4ofz. The resultant model was solvated and subjected to a molecular dynamics (MD) simulation of 20 ns to reduce possible bias. Using the completed model of Bmal-TPP, trehalose-6-phosphate was placed in the vicinity of the presumed binding pocket, but without direct interactions of ligand groups with protein amino acid residues. The binding of the substrate was then investigated by an MD simulation of the solvated system for a period of 20 ns. The results showed that the system attained an apparent equilibrium state after ~3 ns (Supplementary Figure S3). After ~7.5 ns, the ligand had manoeuvred itself into a binding pose that remained stable for the remainder of the simulation period. Both MD simulations (Bmal-TPP in water, Bmal-TPP:T6P in water) were carried out with Gromacs 4.6.5 4 using the G43a1 force field and the spc water model; the topology for T6P was calculated using the PRODRG2 server 5 . The ligand was manually placed in the binding site with the phospho group projecting away from the magnesium cofactor. Sodium and chloride ions were added by replacing solvent molecules at sites of high electrostatic potential to ensure a charge-neutral cell and at a concentration of 100 mM. Following an energy minimisation step, a position-restrained dynamics simulation of 20 ps with a time step of 2 fs was performed to equilibrate the solvated protein complex and gradually equilibrate the system at 300 K and 1 bar. Periodic boundary conditions were applied in all three dimensions. Long-range interactions were modelled using the particle mesh Ewald method 6 and a grid spacing of 1.2 Å; the cutoffs for computation of short-range electrostatic and van der Waals interactions were 10 Å and 14 Å, respectively. The temperature was controlled with the V-rescale thermostat 7 and the pressure with the Parrinello-Rahman barostat 8 . All bonds were constrained using the LINCS algorithm 9 . The final MD simulation was performed for 20 ns with a time step of 1 fs. Simulations were performed on a custom-built server with an Intel Xeon E5-1650 Six Core (3.5 GHz) and 32 GB RAM. Analyses were performed with Gromacs tools and automated plots generated with Grace (http://plasmagate.weizmann.ac.il/Grace/).

Animal ethics
For work with T. canis and T. cati (Warsaw University of Life Sciences, Poland), no ethics approval was required as no animals were involved in clinical-diagnostic procedures other than requested for their health and with owner permission. H. contortus (Haecon-5 strain) was maintained in experimental sheep (male; 6-8 weeks of age), maintained helminth-free, and housed at the University of Melbourne, as described previously 10,11 . The use of sheep was approved by the Institutional Animal Care and Use Committee of the University of Melbourne (permit no. 1413429). All animal experiments were performed in accordance with the Australian National Health Medical Research council (Australian code of practice for the care and use of animals for scientific purposes, 7th Edition, 2004, ISBN: 1864962658).

Toxocara larvae assays
For the survival assay, an average number of 150 L3 larvae were incubated in 24-well culture plates with serial dilutions of 1 (100 µM-6.25 µM) in Minimal Essential Medium for 24 h at 37 °C, 5% CO 2 . Control larvae were maintained in 0.4% DMSO in Minimal Essential Medium. The survival of L3s exposed to the compound was assessed at several time points after the start of the incubation using a light microscope (at 40× magnification). Larvae were considered alive if they had a characteristic coiled appearance and were motile; they were considered dead if they appeared straight and immobile even after extended observation 12 .
To assess migration, 150 L3 larvae were incubated in different concentrations of 1 (6.25 µM-100 µM). After 24 h of incubation (37 °C, 5% CO 2 ), an equivalent volume of 1.5% agar was added to each well 13 . The agar was allowed to set prior to the addition of 0.3 ml of phosphate-buffered saline to each well and the plates were again incubated for 24 h at 37 °C, 5% CO 2 . The number of larvae that migrated to the top of the well was counted using a light microscope (at 40× magnification).

SUPPLEMENTARY FIGURE S1 Structure-based amino acid sequence alignment of nematode TPPs
The alignment was generated with SBAL 21 using secondary structure prediction obtained with PSIPRED 22 . Helical structure is indicated in green, β-strands are shown in red and cysteine residues are highlighted yellow. The domain topology is indicated in the second row. Important amino acid residues mentioned in the main text are annotated with residue numbers based on B. malayi TPP.

SUPPLEMENTARY FIGURE S3 Analysis of selected parameters from the molecular dynamics trajectory of Bmal-TPP:T6P
The plots of parameters versus simulation time indicate that the system was stable throughout the entire simulation and protein backbone conformation reached an equilibrium state. Stable binding of the substrate in the binding site is evident from the distance between the active site metal and the phosphorus atom of T6P.

SUPPLEMENTARY FIGURE S4 Mass spectrometric analysis of Tcan-TPP incubated with N-(phenylthio)phthalimide confirms thiophenyl conjugation of the cysteine residues at positions 215 and 415
Peptides detected in the MS experiemnt are coloured in blue, unmatched peptides are shown in grey; cysteine residues are highlighted in yellow. Bold 'C' indicates thiophenyl conjugation and underlined 'M' indicates methionine oxidation.