Peptide Probes for Plasmodium falciparum MyoA Tail Interacting Protein (MTIP): Exploring the Druggability of the Malaria Parasite Motor Complex

Malaria remains an endemic tropical disease, and the emergence of Plasmodium falciparum parasites resistant to current front-line medicines means that new therapeutic targets are required. The Plasmodium glideosome is a multiprotein complex thought to be essential for efficient host red blood cell invasion. At its core is a myosin motor, Myosin A (MyoA), which provides most of the force required for parasite invasion. Here, we report the design and development of improved peptide-based probes for the anchor point of MyoA, the P. falciparum MyoA tail interacting protein (PfMTIP). These probes combine low nanomolar binding affinity with significantly enhanced cell penetration and demonstrable competitive target engagement with native PfMTIP through a combination of Western blot and chemical proteomics. These results provide new insights into the potential druggability of the MTIP/MyoA interaction and a basis for the future design of inhibitors.

Peptide Permeability Experiments: Experiments were conducted, counting 200,000 cells per run. Typically, P. falciparum 3D7 culture was incubated with peptide probes for 3 hours in fresh media, with Hoechst 33342 (3 µg/mL) added for final 30 min. Samples were spun down (800 rpm, 30 s), blood pellet washed in 1XPBS media twice and either run as live samples or fixed with 4% PFA, 0.01% Gluteraldehyde (1 hour) prior to analysis using a BD Fortessa flow cytometer. Analysis was performed using FlowJoV10. Typically the data for the DMSO control was displayed in a dot plot of forward and side scatter areas (FSC-A vs. SSC-A), a population gate was drawn encompassing 25K -150K in FCS-A and 25K -225K in SSC-A. This sub-population was plotted as FSC-A vs. FSC-width, in order to observe single (FSC-W < 100K) and doublet (FSC-W > 100K) cells. A sub-population of single cells was selected and plotted as FAM intensity vs. DAPI intensity. A quadrant was placed on this plot, placing the erythrocytes (DAPI -/FAM -) in Q1 and parasites (DAPI + / FAM -) in Q2, with the vertical line as close to the parasite dots as possible. This population separation was then applied to all wells. As cells took up fluorescein-labelled peptide, they acquired both DAPI and FAM fluorescence intensity and so moved over into the top right quadrant (Q2). A measure of percentage uptake of the peptide probes can then be estimated as a ratio of Q2:(Q1+Q2). For the production of uptake curves, at least 6 experimental replicates were performed and the mean ± SEM was presented. P values compared using unpaired t-test, buffer control vs. peptide.
Recombinant PfMTIPΔ60 labelling: All samples made up to a final volume of 150 µL in FA assay buffer, containing PfMTIPΔ60 (1 µM) and Diazirine containing peptide (5 µM). Protein and peptide was incubated for 30 min, static, on ice. Samples were UV irradiated (365 nm) for various times, experimentally found that 3 min was sufficient. Sample removed and prepared for SDS-PAGE and western blot analysis using standard method above.
Schizont lysate PfMTIP pull down: Typical scale for experimental samples had a parasite lysate Cf = 1 mg/mL. 10X stock of peptides added and made up to Vf = 100 µL with parasite lysis buffer. Probes incubated with lysate solution (on ice, 30 min, static), before UV irradiation (365 nm, 3 min).Protocol then follows typical Tate group chemical proteins procedure for pull down of biotinylated proteins. 30 Samples were precipitated in MeOH:CH2Cl2:H2O (2 vol:0.5 vol:1 vol), precipitate spun down (2 min, 2,000 rpm, RT), liquids removed to leave precipitate disk. Precipitate was resuspended in MeOH (1 mL) and spun down (12,000 rpm, 10 min). Pellet was air-dried and resuspended in 2% SDS/10 mM DTT/1XPBS (10 µL) and diluted 10-fold in 1XPBS. Samples were added to 25 µL of Dynabeads™ MyOne™ Streptavidin C1 beads (Thermo Fisher Scientific) and incubated (RT, 2 hours, mixing). Using magnetic rack -supernatant was removed, kept for SDS-PAGE gel, and beads were washed in 3 x 5 min mixing with 1% SDS in 1XPBS to remove non-specific binders. Proteins were eluted by boiling beads in 15 µL of 1XSDS-PAGE sample loading buffer (95°C/5 min) before analysis by SDS-PAGE and western blot.
Preparation and Submission to QE: When samples were required a solution of TFA (0.5% v/v), MeCN (2% v/v) in H2O (20 µL) was added to the dry supernatant residue and shaken/vortexed for 2 x 10 min each. Samples were centrifuged 15,000 rpm/10 min/4°C and transferred compatible samples vials. LC-MS/MS analysis was performed on an Easy nLC-1000 system coupled to a Q-Exactive mass spectrometer via an easy-spray source (Thermo Fisher Scientific). Tryptic peptide samples were separated with a reverse phase Acclaim PepMap RSLC column 50 cm x 75 µm inner diameter (Thermo Fisher Scientific) using a 2 hour acetonitrile gradient in 0.1% formic acid at a flow rate of 250 nL/min. The Q-Exactive mass spectrometer was operated in data-dependent mode with survey scans acquired at a resolution of 75,000 at m/z 200 (transient time 256 ms). Up to the top 10 most abundant isotope patterns with charge +2 from the survey scan were selected with an isolation window of 3.0 m/z and fragmented by HCD with normalized collision energies of 25 W. The maximum ion injection times for the survey scan and the MS/MS scans (acquired with a resolution of 17,500 at m/z 200) were 250 and 80 ms respectively. The ion target value for MS was set to 106 and for MS/MS to 105.

Data Analysis by MaxQuant:
The .raw data file obtained from each LC-MS/MS acquisition was directly processed with the software MaxQuant version 1.6.0.13 with the peptides being identified from the MS/MS spectra searched against the P. falciparum PlasmoDB database-39 (July 2018) using the Andromeda search engine. Cysteine carbamidomethylation (+57.021 Da) was set as a fixed modification and methionine oxidation (+15.995 Da) and N-terminal acetylation (+42.011 Da) set as variable modifications for the search. The minimum length of a peptide was set to 7 residues and the maximum amount of missed trypsin cleavages was set to 2 and the maximum number of modifications per peptide was set to 5. Peptide and protein FDRs were set to 0.01. Quantification of peptides was allowed from 'razer and unique' peptides carrying no modifications as well as methionine oxidation, N-terminal acetylation or carbamidomethylation. Re-quantification was enabled with a match type of Match from and to. Match between runs was enabled. All other parameters were used as pre-set by the software.
Data Analysis by Perseus: Data outputted from MaxQuant was analysed using Perseus version 1.6.0.7. LFQ Intensity values for the triplicates were loaded into the main columns and number of razor and unique peptide files added to the numerical columns. Protein identifications by MaxQuant based on 'contaminants', 'only identified by site' and 'reverse' were filtered out. LFQ intensity values were then converted to log2 form before further filtering only allowed identification of a protein target if it contained at least two 'valid values' in at least one experimental concentration. Conditions were then categorically annotated into sample type (e.g. Blank, F/D-ArgMy1 or F/D-ArgMy1-Inhib). Missing values were then replaced from normal distribution. Statistical significance was analysed using a Student's t-test (FDR threshold = 0.05, S0 = 1) between "blank" and "probe only" containing samples, e.g. Blank and F/D-ArgMy1-Biotin. This was followed by Significance testing, Student's t-test (FDR threshold = 0.15, S0 = 0.4), between "inhibited sample" and "probe only" e.g. with F-ArgMy1 + F/D-ArgMy1-Biotin and F/D-ArgMy1-Biotin only. Volcano plots were used to display this data Data Analysis by PEAKS: Data outputted from MaxQuant was analysed using PEAKS Studio (ver. 8.5) -Trypsin digest, OrbiOrbi-HCD. Sample files were loaded into PEAKS; De Novo search was performed using pre-set settings with precursor mass = 10 ppm and fragment ion = 0.01 Da. PEAKS search was performed using pre-set settings with precursor mass = 5 ppm and fragment ion = 0.01 Da. With all searches FDR was set to 0.01. P. falciparum IC50 experiments: P. falciparum trophozoite stage culture was separated into triplicates of 90 µL aliquots across the wells of 96 well plate. Dihydroartemisinin (100X stock, 1 µL in 1XPBS, made up to 100 µL with 1XPBS) or peptide probes (10X stock, 10 µL in 1XPBS) were also added to required wells. Parasites were allowed to egress overnight, whereupon media was changed. Surviving ring stage parasites were allowed to mature to trophozoite stage, more easily analysed by flow cytometry. Prior to analysis, cells were incubated with Hoechst 33342 (3 µg/mL, 30 min). Cells were washed twice with 1XPBS, diluted 1 in 10 with 1XPBS into a fresh 96 well plate and analysed using BD Fortessa in 96 well plate mode. Again, analysis performed using FlowJoV10. Two constant values were used in the estimation of in vivo concentration:

Supplementary Figures and Tables
1. The estimated volume of a mature schizont = 80 fL, taken from a literature value derived using confocal microscopy. 31 2. The number of cells per 1.5 mL of 4% haematocrit culture was found to be 3.3 x 10 8 cells, estimated experimentally using a haemocytometer. This was the volume used for each experimental condition, with a 4% parasitemia culture. Therefore, 1.33 x 10 7 parasitised erythrocytes/schizonts were present per 1.5 mL volume.