**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 [5]. 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) [6]. 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 [7] (**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 [8], 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

#### **Figure 1.**

*Categorization of PPIs based on types of interactions. (A) Internal, where the site of interaction lies buried inside such as "hot-spots", (B) external, where proteins interact at the surface, (C) where a peptide could induce conformational changes upon binding, (D) interactions can be stabilized or dissociated based on the type of cofactor/compound binding, (E) whether the epitope binding is contiguous or not, and (F) if both interacting partners are peptides/small molecules.*

**91**

*Protein-Protein Interactions in Malaria: Emerging Arena for Future Chemotherapeutics*

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 [9]. Thus, decoding membrane PPIs even through bioinformatic

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 [12]. 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 *Plasmodium falciparum*.

Hemoglobin hydrolysis is one of the most targeted pathways for treatment as it is fundamental for parasite survival and involves numerous proteins [13]. 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 *P. falciparum* [14]. Falcipains contain a pro and a mature domain, with the pro domain bound to and blocking access to the active site in the mature domain. The interactions between these two domains, termed "hot-spot" interactions, dissociate under acidic conditions and are essential for hemoglobinase activity. This specific hot-spot was identified at the interface of pro and mature domains in falcipain-2 (FP2) and falcipain-3 (FP3). The study further demonstrated that synthetic compounds, NA01 and NA03, specifically bound to this hot-spot region and stabilized these interactions. Thus, even in the presence of an acidic environment (pH - 5.5), pro-mature domains remained intact,

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 [16]. Efforts elsewhere have focused on selective inhibition of falcipains rather than indiscriminate inhibition including its human host cathepsin isoforms.

*DOI: http://dx.doi.org/10.5772/intechopen.89217*

approaches remains challenging.

**3. PPIs in malaria**

**3.1 Hemoglobin hydrolysis**

rendering them inactive [15].

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 [9]. Thus, decoding membrane PPIs even through bioinformatic approaches remains challenging.
