Allosteric Site Inhibitor Disrupting Auto-Processing of Malarial Cysteine Proteases

Falcipains are major haemoglobinases of Plasmodium falciparum required for parasite growth and development. They consist of pro- and mature domains that interact via ‘hot-spot’ interactions and maintain the structural integrity of enzyme in zymogen state. Upon sensing the acidic environment, these interactions dissociate and active enzyme is released. For inhibiting falcipains, several active site inhibitors exist, however, compounds that target via allosteric mechanism remains uncharacterized. Therefore, we designed and synthesized six azapeptide compounds, among which, NA-01 & NA-03 arrested parasite growth by specifically blocking the auto-processing of falcipains. Inhibitors showed high affinity for enzymes in presence of the prodomain without affecting the secondary structure. Binding of NA-03 at the interface induced rigidity in the prodomain preventing structural reorganization. We further reported a histidine-dependent activation of falcipain. Collectively, for the first time we provide a framework for blocking the allosteric site of crucial haemoglobinases of the human malaria parasite. Targeting the allosteric site could provide high selectivity and less vulnerable to drug resistance.


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The pyrazine carboxylic acid (0.124 g, 1.0 mmol) was dissolved in dry DCM and added to 200µl of NMM, EDCI.HCl (0.191g,1.0 mmol) followed by the addition of Pyr-xxx-NHNH2 (0.277 g, 1.0 mmol) and reaction was stirred for 24 h. The progress of the 23 reaction was monitored by using TLC. The reaction was worked up as described in (1) to give Pyr-xxx-NH-NH-Pyr, which were 24 purified by column chromatography over silica gel (60-120 mesh) using 5% methanol in chloroform to yield the white powder of     The synthesis of Pyr-Gpn-OH was synthesized according to the procedure reported in the literature 2 . Briefly pyrazine-2-carboxylic 27 acid (3.0 mmol, 0.372 g) was dissolved in dry DCM (3.0 ml) and NMM (0.2 ml) was added, followed by Gpn-OMe. HCl (3.0 28 mmol, 0.666 g) and EDC. HCl; 3.0 mmol, 0.576 g) under ice-cold conditions. The reaction mixture was stirred at room temperature 29 for 12 hr. After completion of the reaction, water (5.0 ml) was added and the reaction mixture was extracted with DCM (3×5 ml).

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To generate the structural model of residues 165-222 by homology modeling method, we searched the template crystal structure in Structure of remaining mature FP2 (PDB: 1YVB) and FP3 (PDB: 3BWK) domains were taken as the seed structure, respectively.
using Z-DOPE value, and further, top scorers were evaluated for stereochemical refinement by PROCHECK 9 , and finally, best 1 model was selected for subsequent studies.

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To monitor the equilibration, we measured the structural deviations of mature domain during simulations by calculating root mean 28 square deviation (RMSD) with respect to starting crystal structure (Fig. S4c). The obtained RMSD fluctuated at average value of 29 1.5 °A during these five simulations (Fig. S4c). This observed deviation is considered as quite small in MD simulations because 30 molecules relaxes and fluctuates due to the applied 300K and 1 atm temperature, and pressure, respectively. On considering this 31 quite small structural deviation, the modelled segment has negligible impact on the active domain and, therefore, the obtained MD 32 trajectories were considered for further binding interaction studies. During the simulations, a cavity was appeared in proximity of an essential hydrophobic cluster (Fig. 1) and at the pro/mature 35 domain interface (Fig. S4b). Additionally, a small cavity was also observed in the prodomain near an essential R185-E221 salt-36 bridge interaction. Therefore, one of the cavities could be a potential binding site for the designed compounds. We designed a 37 protocol by combining structural and energetic approach to determine the ensemble of structures for docking calculations (Fig. S5).

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At first, initial 25 ns of the above obtained five trajectories was discarded by considering as an equilibration phase and further these 39 trajectories were concatenated. The obtained single combined trajectory was used as the input structures for the protocol.

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and an energetic approach was used to select a representative structure from each cluster.

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In the second part of protocol, folding free energy of structures were calculated from concatenated MD trajectory using FoldX 46 program 20 as follows. Structures from trajectory were extracted at 250 ps time-step and their potential energy was minimized using steepest descent method by GROMACS with a force tolerance of 100 kJ mol -1 nm -1 . These structures were further energy minimized and folding free energy were calculated using FoldX programme 21 .
of docked complexes. These force-field parameters were also used for all MD simulations described below.

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After the energy calculation by MM-PBSA method, all poses were sorted by the calculated binding energy, and top 19 complexes 24 including two additional complexes with alternate binding sites are listed in Table S2. To select the most likely native binding pose 25 from these 21 poses, we further performed high temperature ligand MD simulations as described below.  Table S3. Moreover, average binding energy of top six complexes 38 were calculated using MM-PBSA (Table S3).

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Impact of ligand sampling on FP2 conformation: To determine the impact of ligand sampling on FP2 structure in top six 40 complexes, root mean square deviation (RMSD) with respect to its starting structure as a function of time was calculated (Fig. S7).        Structure of S228-L229-R230 residues and E64 are extremely similar and these three residues blocks the active site cavity in whole enzyme. It is highly unlikely that any ligand will bind to active site in presence of prodomain because it has to displace S228-L229-R230 residues from the active site cavity. Therefore, NA-03 most likely acts as allosteric inhibitor as it is binding away from active site. Figure S11. Multiple Sequence alignment of prodomain from cysteine proteases. (a) The sequences of cysteine protease from plasmodium species were aligned by the Clustal Omega server 32,33 . The prodomain region was selected from the alignment for mapping pH sensing histidine residue across the prodomains and coloured according to conservation of residues at each position in the alignment by CLC sequence viewer (https://www.qiagenbioinformatics.com/products/clc-sequence-viewer). Residue numbers are labelled according to the FP2 (Q9N6S8_PLAFA) sequence. The highly conserved residues are in blue letters. The alignment shows that there are four histidine residues positions in FP2 prodomain (H161, H194, H199, H220) and among them H199 is highly conserved across the cysteine protease prodomain of other plasmodium species also. The conserved and pH sensing H199 position is marked by blue arrow and other Histidine position by black arrow. (b) The pairwise alignment of FP2 (Q9N6S8_PLAFA) and FP3 (Q8IIL0_PLAF7) shows the relative residues in FP3 with respect to FP2 histidine residue positions. FP3 have T167, R200, H205 and E226 residues with respect to FP2 histidine residues (H161, H194, H199, and H220). 1   Table S1: List of frame number selected for ensemble docking. Representative frame from each cluster was selected for the 2 ensemble docking. The representative frame has lowest folding free energy in the respective cluster.