**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

**97**

**Figure 4.**

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

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 [42]. 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

*Strategies for improving the potency of PPI inhibitors. Schematic showing the various ways peptides can be* 

*modified (highlighted in red) to achieve potency or improve bioavailability.*

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

considered undruggable (**Figure 4**).

#### *Protein-Protein Interactions in Malaria: Emerging Arena for Future Chemotherapeutics DOI: http://dx.doi.org/10.5772/intechopen.89217*

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 [42]. 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**).

#### **Figure 4.**

*Parasitology and Microbiology Research*

putative (PF3D7\_0418500) that interact with PfK13 and could potentially have a

*Role of PfK13 in mediating ART resistance. In the presence of ART, wild PfK13 binds with PfPI3K, which results in PfPI3K ubiquitination and degradation, thus dysregulation of phosphoinositide signaling pathways ultimately leading to parasite death, while in ART-resistant strains, mutated PfK13 does not bind to PfPI3K* 

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

role in stress-mediated response (Atul et al., 2019, unpublished).

*initiating PI3P signaling thus promoting parasite survival. Adapted and modified from [39].*

**4. PPI inhibition: peptidomimetics and design**

**96**

**Figure 3.**

*Strategies for improving the potency of PPI inhibitors. Schematic showing the various ways peptides can be modified (highlighted in red) to achieve potency or improve bioavailability.*

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 [37].

Natural inhibitors of proteases are one of the best studies substrate groups in malaria as they are highly specific, stable, and reversible. *Plasmodium spp.* contain inhibitors of cysteine proteases (ICPs) and endogenous macromolecular inhibitors, which regulate the activity of cysteine proteases. Orthologs of ICPs are also observed in other apicomplexan groups such as *Trypanosoma cruzi*, whose ICP is called as chagasin [43]. Chagasin was shown to potently bind to the active site of both falcipains, FP2 and FP3. More importantly, three loop regions termed BC-, DE-, and FG loops

#### **Figure 5.**

*Crucial interactions between falcipains and ICPs chagasin and falstatin. (A) Solved structure of FP2 and chagasin in that the ICP bound to the active site of falcipain and that (B) three loops BD, DE, and FG are involved in this interactions. (C, D) mutagenesis studies indicated that just the BC loop of falstatin was sufficient for inhibiting both FP2 (C) and FP3 (D), respectively. Adapted and modified from [44, 45].*

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

were shown to be involved in active site inhibition (**Figure 5A,B**) [44]. However, further studies with the *P. falciparum* ICP, falstatin, indicated that unlike other ICPs, just the BC loop was sufficient for falcipain inhibition and that Asn289 of falstatin formed stabilizing hydrogen bonds and hydrophobic interactions (**Figure 5C,D**) [45].

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

We thank NIMR, New Delhi, for providing basic infrastructure facilities. We also thank Indian Council of Medical Research for providing fellowship assistance

, Rajnikant Dixit1

1 Host-Parasite Interaction Biology Group, ICMR-National Institute of Malaria

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Institute of Science, Nirma University, Ahmedabad, Gujarat, India

\*Address all correspondence to: pandey.kailash70@gmail.com;

and Kailash C. Pandey1

\*

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

less drug pressure and are less likely to develop resistance.

**5. Conclusion**

**Acknowledgements**

**Conflict of interest**

**Author details**

to Mr. Rahul (45/16/2019-Bio/BMS).

Rahul Pasupureddy1,2, Sriram Seshadri2

provided the original work is properly cited.

Research, New Delhi, India

kailash.pandey@icmr.gov.in

The authors declare no conflict of interest.

*Protein-Protein Interactions in Malaria: Emerging Arena for Future Chemotherapeutics DOI: http://dx.doi.org/10.5772/intechopen.89217*

were shown to be involved in active site inhibition (**Figure 5A,B**) [44]. However, further studies with the *P. falciparum* ICP, falstatin, indicated that unlike other ICPs, just the BC loop was sufficient for falcipain inhibition and that Asn289 of falstatin formed stabilizing hydrogen bonds and hydrophobic interactions (**Figure 5C,D**) [45].
