Malaria is one of the most deadly diseases infecting humans. Advances in elimination and vector control have reduced the global malaria burden in the past decade; however, the emerging threat of drug resistance and suboptimal vaccine efficacies threaten global eradication efforts. Unlocking novel drug and vaccine targets while simultaneously mitigating spread of resistant strains seems to be the need of the hour. Protein-protein interactions (PPIs), an integral part of host-pathogen cross-talk and parasite survival, have only recently emerged as promising drug targets. Large PPI networks (interactome) are being developed to better our understanding of various parasite biochemical pathways. In this chapter, we throw light on several newly characterized protein-protein interactions between the host (humans) and parasite (plasmodium) in key processes such as hemoglobin degradation, enzyme regulation, protein export, egress, invasion, and drug resistance and further discuss their viability for development as novel chemotherapeutic targets.
- drug resistance
- protein-protein interactions
- host-parasite interactions
Malaria is one of the deadliest diseases to affect humans, with the latest WHO reports indicating ~445,000 deaths in 2017 alone . More alarmingly, despite decades of advances in controlling the malaria epidemic, death rates caused by malaria seem to have plateaued in the past 3 years, indicating drug resistance and re-emergence. Drug resistance to the current frontline antimalarials have been confirmed by many recent studies and steadily observed over increasing geographic coverage . Thus, it is of utmost importance to develop novel antimalarials, with different modes of action and distinct targets, if possible in conjunction, to check the onslaught of malaria. This chapter looks at such potential scope for antimalarial drug development: disruption of protein-protein interactions in the malaria parasite
2. PPIs: the basics
Protein-protein interactions (PPIs) constitute the fundamental backbone required for occurrence of any biological event. They are defined as the residue level interactions between either the same protein (dimers, trimers, or other multimers) or diverse proteins (protein complexes). These basic interactions are necessary for a myriad of functions such as kinase signaling, receptor binding, proteolytic digestion, apoptosis regulation, and antigen-antibody interactions [3, 4]. Disruptions in the protein interaction networks (PINs) as a result of PPI inhibition have been shown to cause several diseases where either single or multiple biochemical pathways are affected . Owing to their fundamental roles in almost every process imaginable, PPIs have emerged as attractive therapeutic targets in several diseases. Several forms of cancer were also shown to have dysregulated protein interaction networks (PINs) . Similarly, PPI disruptions have been observed in several autoimmune as well as parasitic diseases. Small peptides that infiltrate cellular defenses and specifically bind to target structures are already in development. Taken together, targeting PPIs though challenging can provide a novel understanding of biochemical processes as well as uncover new ways to combat diseases like malaria.
PPIs can be generally categorized into several groups depending on their function or the type of interactions. They include internal (hot-spots) or external (surface), obligate (permanent) or non-obligate (transient), stabilizing or destabilizing, ability to induce conformational changes in either of the partner molecules, peptide-protein or peptide-peptide interactions, and contiguous or discontiguous epitope binding  (Figure 1).
Some types of PPIs such as membrane PPIs can be difficult to characterize. While dedicated techniques like the split-ubiquitin membrane yeast two-hybrid (MYTH) system were developed to specifically detect membrane protein interactions , these techniques are still considered time-consuming and labor-intensive. Such bottlenecks make it hard to generate a complete picture of the membrane interactome. Even for reliable bioinformatic models for detection of membrane PPIs to be developed, there need to be large sets of positive, false-positive, as well as negative data to accurately train such models, which are currently unavailable for membrane PPIs . Thus, decoding membrane PPIs even through bioinformatic approaches remains challenging.
3. PPIs in malaria
Malaria traditionally has been treated using inhibitors which target the broad spectrum proteasome offering several advantages as compared to specific protein inhibitors. Specific inhibitors had comparatively low efficacy in vivo. Also, inhibitors targeting a specific protein/ligand could potentially inhibit parasite growth only in stages when the target proteins are expressed. Broad spectrum antimalarials, such as the current frontline drugs artemisinins (ARTs) and their combination therapies (ACTs), target and break down various cellular pathways including but not limited to hemozoin formation, DNA repair, and mitochondria machinery, which make them highly potent within short exposure times [10, 11]. However, exposure to various cellular targets leads to the rapid development of drug resistance. While resistance to chloroquine worldwide was observed after ~40 years of continued use, resistance to ARTs was achieved in a relatively short span of a decade, from its inception in late 1990s to the first reported resistance in 2008 . While this rapid emergence resistance was partially attributed to suboptimal drug regimens and poor administrative practices, the same could be attributed to earlier drugs as well. Thus, compounds that are specific/flexible to the target protein are the need of the hour. This section deals with and summarizes current knowledge about crucial PPIs in various biochemical pathways of the malaria parasite
3.1 Hemoglobin hydrolysis
Hemoglobin hydrolysis is one of the most targeted pathways for treatment as it is fundamental for parasite survival and involves numerous proteins . Majority of earlier and currently used drugs disrupt multiple protein interactions. Several studies have been conducted recently that target individual PPIs and design inhibitors based on those interactions. Our lab has previously identified a “hot-spot” region in falcipains, the principal hemoglobinases of
Falcipains, owing to their crucial role in Hb degradation, are considered as attractive chemotherapeutic targets. Several inhibitors were designed based on the interactions of the FP2 and the active site inhibitor E64. Molecular dynamics (MD) simulations indicated that two sets of residues, namely, recruiter groups A (rgA) and B (rgB) (rgA (D170, Q171, C168, G169, A151, and G230); rgB (K76, N77, and N81)) of FP2 are primarily involved in the initial binding with E64 about 80% and 14% of the time, respectively, before finally proceeding to bind with the active site residues . Efforts elsewhere have focused on selective inhibition of falcipains rather than indiscriminate inhibition including its human host cathepsin isoforms. While the S1 and S3 subsite residues have been conserved across
Falcipains also contain a domain at their C-terminal called the hemoglobin binding domain (Hb domain), a β-hairpin loop which protrudes away from the active site. Deletion of this 14 amino acid domain ablated the ability of falcipains to degrade Hb, thus indicating a necessary role of this domain in Hb capture prior to degradation at the active site . Our lab recently published another study that identified crucial protease-substrate PPIs within this domain. A functionally conserved single amino acid position in both falcipains (Glu185 in FP2 and Asp194 in FP3) was found to be essential for Hb interactions, with activated falcipain mutants unable to degrade Hb even with accessibility to the active site. Molecular docking results indicated both the residues interacted with Hb-α as well as Hb-β subunits with interactions mediated primarily through this position (Figure 2). A specific inhibitor which could target this position could have potential applications in arresting the parasite hemoglobin degradome .
Hemoglobin degradation as a source for parasite growth was also shown to be dependent on the hemoglobin tetramer composition. Children (<5 years) have different Hb subunits (HbF, α2γ2) as compared to adults (HbA, α2β2), and malaria mortality rates have consistently indicated child mortality to be higher (61% of deaths in 2017) . The essential amino acid isoleucine (I) was found to be a main differentiating factor as it is absent in both α and β chains but present in γ subunit and makes up to 99% of encoded proteins in
Both invasion and egress are important events in the erythrocytic stage and are responsible for the malaria symptoms including chills and fever. The process of invasion requires a host of proteins to be secreted from its apical organelles including rhoptry bodies and micronemes, among others, and is precisely coordinated. The parasite initially aligns the merozoite apical region toward the host erythrocyte and forms a tight junction at the apex, progressing as the moving junction (MJ) pushes the parasite into the erythrocyte, with the erythrocyte surface forming a ring around the engorged parasite, which would later become the parasitophorous vacuolar membrane (PVM) .
While the process of moving junction (MJ) formation and important players involved in the process were well elucidated in
3.3 Protein export
The protein export element (PEXEL) comprising of the conserved sequence RxLxE/Q/D is found in the N-terminus of ~300 proteins bound for export in the
PfEMP1 is one the most studied exported protein as it is one of the exported in abundant quantities to the outer surface and thus is an attractive target along with others such as circumsporozoite protein (CSP). Immunoprecipitation (IP) and mass spectrometry studies of a GFP-tagged minimal section of PfEMP1 (PfEMP1B) identified novel targets in different cellular components including parasitophorous vacuole (PV), Maurer’s clefts, the plasmodium translocon of exported proteins (PTEX) translocon, and a novel exported protein-interacting complex (EPIC). Several new interacting partners including parasitophorous vacuole protein-1 (PV1), PV2, and exported protein-3 (EXP3) have been identified, all of whom localized to the newly described EPIC . Finally, a comprehensive pathway of PfEMP1 export has been suggested, where PfEMP1 is initially translocated to the ER and then trafficked to PTEX machinery and out of PVM with the aid of EPIC, where finally it is received by host erythrocytic chaperonin complex, the TCP-1 ring complex (TRiC), and transported to erythrocyte surface .
IP assays coupled with truncated-construct interaction assays have helped identify few prominent PMV-partner PPIs. The
3.4 PPIs mediating drug resistance
The emerging threat of resistance to the current frontline drug artemisinin (ART), and its combination therapies (ACTs), is a cause of great concern. The mechanism of ART action, though generally agreed to generate free radical species which disrupt several essential pathways in the parasite, is highly debated. Immunoprecipitation studies with chemically tagged ART analogue (AP1) revealed that ART interacts with over a dozen proteins and is predominantly activated by free heme rather than free ferrous ions.
Structural analysis and MD simulations of PfK13 indicated the presence of two evolutionarily highly conserved domains in the broad-complex, tramtrack, and bric à brac (BTB) domain and in the shallow binding pocket formed by the six propeller domain repeats. These domains displayed a different electrostatic surface potential unlike the rest of the bottom PfK13 face and are rich in highly conserved arginine and serine residues. While the BTB domain was shown to be involved in the recruitment of a scaffold protein Cullin, the propeller domain pocket binds to the substrate molecules for further ubiquitination. MD simulations showed that the validated PfK13 markers such as C580Y and R539T mutants induced a significant structural destabilization in the shallow pocket region as compared to wild type while maintaining the overall structural integrity. Specifically, the C580Y mutant disrupted a disulfide bridge (C532-C580) and additional H-bonds created with neighboring residues not present in wild type strains. In the case of R539T mutant, it was shown to have substantially less H-bond interactions along with a complete loss of a salt bridge interaction with E606 while also losing another inter-blade H-bond interaction . Overall, these mutants lead to significantly diminished levels of functional protein which cause diminished substrate interaction, ultimately promoting the PfPI3P-mediated unfolded protein response pathway. Our own group, through Co-IP and mass spectrometric studies, has identified several novel proteins including Trx-like mero protein (PF3D7_1104400), pyridoxal kinase (PF3D7_0616000), trafficking protein particle complex subunit 3 (TRAPP), and putative (PF3D7_0418500) that interact with PfK13 and could potentially have a role in stress-mediated response (Atul et al., 2019, unpublished).
4. PPI inhibition: peptidomimetics and design
While several strategies have been employed for PPI inhibition, bioinformatics-based drug design has been at the forefront to design specific PPI inhibitors. Structure-based drug design, where the solved structure of an enzyme in complex either with an inhibitor or a natural substrate was used to design inhibitors, was popularized in the 1990s. However, rational design for PPI inhibitors needs to overcome some common hurdles such as low proteolytic stability, analyzing extensive libraries of candidate molecules, and in some cases low ligand efficiency when compared to standard active site inhibitors. Thus, several strategies are employed in modern drug synthesis to overcome these problems. Short linear peptides tend to have lower conformational stability; thus cyclization of the peptide is preferred which rigidifies the structure in an active configuration. Certain modifications in the backbone of the peptide such as backbone extension, side-chain shifting to nitrogen atoms (peptoids), and altering the stereochemistry can also be applied. Peptoids can easily fold into helices or other structures as they consist of repeated nitrogen-substituted glycine units that give an added advantage of mimicking the peptide structure and function. Stereochemistry of a compound can be changed by using D-peptides instead of L-peptides as they are more susceptible to proteolytic degradation and are one of the most common strategies to develop potent bioactive compounds. Another modification involves using β-peptides; peptides with amino group bound at β-carbon instead of α-carbon for each amino acid, often called as foldamers, could confer additional proteolytic stability both in vitro and in vivo. A class of oligopeptides (<80 residues in length) called as miniproteins can also be utilized as they have a rigid, well-defined three-dimensional structure. The 19 kDa fragment of merozoite surface protein 1 (MSP1), the fragment that is finally displayed on the merozoite surface after several processing steps, was fused along with a glycosylphosphatidylinositol (GPI) tag used to create a miniprotein, which was successfully targeted by antibodies specific to the miniprotein and inhibited erythrocyte invasion . Substantial effort has been made to develop rational strategies in designing PPI inhibitors for target proteins that have no well-defined binding site (so-called “hot-spot”) and, thus, have previously been considered undruggable (Figure 4).
As described in the earlier section, Villa et al. designed and synthesized peptidomimetics belonging to 1,2,3-triazoles, specifically 1,4-disubstituted 1,2,3-triazoles. These compounds mimicked the contacts made by PfAtg3 template structure containing the residues W-L-L-P, as this template was shown to have the majority of interactions with PfAtg8. Of the four compounds synthesized, compound 2 (C2) exhibited prominent inhibition (IC50–3.8 μM) in vitro, while C1 had better inhibitory effects in vivo .
Natural inhibitors of proteases are one of the best studies substrate groups in malaria as they are highly specific, stable, and reversible.
Protein-protein interactions play roles of utmost importance in the growth and survival of any organism. Thus, focused targeting of such interactions specific to parasite can help produce robust and effective drugs. Recent research has indicated a renewed interest in targeting PPIs in the field of malaria. Various PPIs in pathways essential for parasite survival, erythrocyte invasion/egress, drug resistance, and others have been elucidated. These new classes of peptidomimetic compounds would form the future defense against an ever-increasing resistant parasite threat. Targeting PPIs offers several advantages over active site inhibition as ‘hot-spots’ are more flexible as compared to the active site and thus can be more selective in terms of drug interactions. In contrary to active site, the interactions at allosteric sites and exosites in an enzyme occur away from the active site; thus they tend to fall under less drug pressure and are less likely to develop resistance.
We thank NIMR, New Delhi, for providing basic infrastructure facilities. We also thank Indian Council of Medical Research for providing fellowship assistance to Mr. Rahul (45/16/2019-Bio/BMS).
Conflict of interest
The authors declare no conflict of interest.