In Silico Study Reveals How E64 Approaches, Binds to, and Inhibits Falcipain-2 of Plasmodium falciparum that Causes Malaria in Humans

Even though malaria has been known to man since the year 2700 BC, the malaria problem remains unsolved. And malaria is one of the deadliest diseases (most especially in the Tropics) killing about half a million people annually with the children under the age of 5 years suffering malaria’s worst scourge. These rank malaria as one of the most important public health and clinical health problems till date.

Malaria is caused by P. falciparum (as well as P. ovale, P. vivax, P. malariae, and P. knowlesi to a much smaller extent). For survival in humans, P. falciparum uses some of its proteins called falcipain-1 (FP1), falcipain-2 (FP2), and falcipain-3 (FP3) to break down human hemoglobin. “In other words, the survival of P. falciparum in humans depends on its ability to degrade humans’ hemoglobin.” https://doi.org/10.1038/s41598-018-34622-1 (As a side note, it is the breaking down of the hemoglobin that leads to some of the most important symptoms and complications of malaria in humans.) Therefore, the malaria-causing protozoan, P. falciparum, could be killed by stopping or hindering of its ability to degrade human hemoglobin through its cysteine proteases (mostly FP2). This is why FP2 is an important antimalarial drug target since its inhibition kills P. falciparum (at least in its trophozoite stage). This motivated us to study the atomic details of how E64 (an epoxide that is known to inhibit cysteine proteases) approaches, binds to and inhibits FP2.

Fig. 1. The structure of FP2-E64 complex based on 3BPF in the PDB and the Chemical Structure of E64. The positions of the catalytic residues, Q36, C42, H174, and N204 (Q36 is located behind C42 in the current view), are shown as spheres in (a). The known binding pocket subsites, S1 (green), S2 (grey), S3 (pale blue), and S1’ (orange) are shown as spheres in (b). The position of E64 (the black spheres) relative to the catalytic residues is shown in ©, and relative to the binding pocket subsites is shown in (d). The structure of E64 is shown in (e).

Reproduced from: Figure 1 in In Silico Study Reveals How E64 Approaches, Binds to, and Inhibits Falcipain-2 of Plasmodium falciparum that Causes Malaria in Humans. https://doi.org/10.1038/s41598-018-34622-1.

We used in silico techniques that span the fields of computational chemistry and artificial intelligence (AI). For example, we set up explicitly solvated molecular systems of E64 posed next to FP2 and used unbiased molecular dynamics (MD) simulations to study how E64 approaches FP2 (prior to FP2-E64 binding). We also used unbiased MD Simulations for studying the interactions of E64 with FP2 (following FP2-E64 binding). The binding Gibbs free energy change for the FP2-E64 system was calculated using Adaptively Biased MD (ABMD) Simulations. And the relative contributions of the various components of the complex (mainly the amino acids of FP2) to FP2-E64 binding were studied using a combination of Molecular Mechanics Poisson-Boltzmann Surface Area (MM-PBSA) and AI techniques.

This study establishes how E64 gradually approaches FP2. To approach and bind to FP2, E64 start by interacting with either Aspartic Acid 170 and Glutamine 171, or with Asparagine 81, Asparagine 77, and Lysine 76. The paper published in Scientific Reports https://doi.org/10.1038/s41598-018-34622-1 provides more details of the process and suggests the importance of the charged amino acids in the recruitment of the E64. The paper also shows that E64 tightly binds to FP2 with a binding Gibbs free energy change of −12.2 ± 1.1 kJ/mol. Furthermore, how E64 persistently blocks access to FP2’s catalytic residues as well as the residues of FP2 (such as S41, D234, D170, N38, N173, etc.) that contribute the most towards favorable FP2-E64 binding are also presented in the original paper. The study also shows that in silico mutations of the amino acids we identified as important (such as D234L/A, N173L/A, W43F/A, D234L/A, H174F/A, N38L/A, etc.) cause significant adverse effects on E64-FP2 binding and interactions.

Fig. 2. How E64 approaches and binds to FP2. The results from the 50 independent unbiased explicit-solvent MD simulations (75 ns each, 3,750 ns in all) are summarised here. (inset in a) The orientation of FP2 presented in every other figure and video. The molecular system is rotated by about 90° counter-clockwise to obtain the orientation of the FP2 presented in this Fig. 2. This is done to enhance the visualization of how E64 approaches FP2 presented here in Fig. 2. (a) A typical structure used for each of the explicit-solvent MD simulations wherein E64 is placed 15 Å away from FP2. Some of the residues of FP2 found to be important are shown as colored spheres. A model depicting (approximately) how E64 leaves the source, S, and approaches FP2 via the residues at recruiter region A (RA, then RC) and at RB, and eventually arrives at the target binding site, T is shown in (b).

Reproduced and modified from: Figure 2 in In Silico Study Reveals How E64 Approaches, Binds to, and Inhibits Falcipain-2 of Plasmodium falciparum that Causes Malaria in Humans. https://doi.org/10.1038/s41598-018-34622-1.

Fig. 3. Dynamic interactions between E64 and FP2 prior to E64-FP2 covalent bond formation. The various residues of FP2 that E64 interacts with/contacts the most, and the respective residue-ligand contact profile over the 1500 ns trajectory are shown in (a). Each block is a bin derived from 25.0 ns segment (i.e. 1250 frames) of the trajectory. This allows the contact probability to be calculated for each block. For example, the first block for H174 shows that between 0 ns and 25.0 ns in the trajectory (made up by 1250 frames), E64 contacts H174 about 1100 times (out of the 1250 possible times) giving rise to a probability of ~0.9 which is depicted by the dark blue color. For easy identification, red dots are placed next to the catalytic residues present in panel (a). Examples of poses of E64 relative to FP2 showing E64 persistently blocking the catalytic residues of FP2 (H174, C42, N204, and Q36, which are labeled in panel c) even prior to the formation of a covalent bond between E64 and C42 of FP2 are shown in (b–i). Please, see Supplementary Video S1 for more details.

Reproduced from: Figure 5 in In Silico Study Reveals How E64 Approaches, Binds to, and Inhibits Falcipain-2 of Plasmodium falciparum that Causes Malaria in Humans. https://doi.org/10.1038/s41598-018-34622-1.

More importantly, the findings presented in the study has antimalaria implications. The study’s findings “suggest that hydrogen bonding and electrostatic interactions play important roles in E64-FP2 binding, and that a potential FP2-blocking E64-based/E64-like antimalarial drug should be capable of being both hydrogen-bond donor and acceptor, and/or have the ability to favourably interact with polar amino acids (such as S41, S149, N38, N173, N77, Q171) and with charged amino acids (such as D234, D170, H174) of FP2.” https://doi.org/10.1038/s41598-018-34622-1 It also emerged from the study that “the ability to favourably interact with asparagine (specifically, N173, N77, N38, N81, N86, and N204) and aspartic acid (specifically, D170, and D234), which are examples of amino acids with polar side chains and amino acids with negatively charged side chains respectively, might be extremely important for any potential E64-based antimalarial drug targeted at blocking FP2’s activities.” https://doi.org/10.1038/s41598-018-34622-1

It is worthy of note that in silico techniques make it possible to carry out scientific studies on biophysical mechanisms and biochemical systems at molecular and atomic resolutions in such a great detail that could not be achieved through the conventional (non-in-silico) approaches. Furthermore, in silico techniques speed up the research process and increase the efficiency of drug-discovery pipelines. The understanding of these advantages of in silico techniques encouraged me to join Insilico Medicine where we are working on drug-discovery projects.