Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated Multidrug-Resistance Phenotypes in Agents of Neglected Tropical Diseases

*Nivedita Jaishankar, Sangeetha Muthamilselvan and Ashok Palaniappan*

#### **Abstract**

Mammalian ABCB1 P-glycoprotein is an ATP- dependent efflux pump with broad substrate specificity associated with cellular drug resistance. Homologous to this role in mammalian biology, the P-glycoprotein of agents of neglected tropical diseases (NTDs) mediates the emergence of multidrug-resistance phenotypes. The clinical and socioeconomic implications of NTDs are exacerbated by the lack of research interest among Big Pharma for treating such conditions. This work aims to characterise P-gp homologues in certain agents of key NTDs, namely (1) Protozoa: *Leishmania major,Trypanosoma cruzi*; (2) Helminths: *Onchocerca volvulus, Schistosoma mansoni*. Based on structural modelling of the organismal P-gp homologues, potential antibiotics targeting these structures were identified based on similarity and repurposing of existing drugs. Docking studies of the Pgp receptor—antibiotic ligand complexes were carried out and the most tenable target-ligand conformations assessed. The interacting residues were identified, and binding pockets studied. The in silico studies yielded measurements of the relative efficacy of the new drugs, which need experimental validation. Our studies could lay the foundation for the development of effective synergistic or new therapies against key neglected tropical diseases. The potential mechanisms of multidrug resistance emergence in *E. coli* were examined.

**Keywords:** P-glycoprotein, neglected tropical diseases, multidrug resistance, homology modeling, receptor-ligand docking, differential ligand affinity, synergistic effects, leishmaniasis, trypanosomiasis, onchocerciasis, schistosomiasis

#### **1. Introduction**

#### **1.1 Multidrug resistance (MDR)**

Bacterial evolution has been constrained to respond to the selection pressure of antibiotics and combined with their reckless use and has led to the emergence of varied defenses against antimicrobial agents. The main mechanisms whereby the bacteria develop resistance to antimicrobial agents include enzymatic inactivation, modification of the drug target(s), and reduction of intracellular drug concentration by changes in membrane permeability or by the over expression of efflux pumps [1]. Multidrug resistance efflux pumps are recognized as an important component of resistance in both Gram-positive and Gram-negative bacteria [2]. Some bacterial efflux pumps may be selective for one substrate or transport antibiotics of different classes, conferring a multiple drug resistance (MDR) phenotype. With respect to efflux pumps, they provide a self-defense mechanism whereby antibiotics are extruded from the cell interior to the external environment. This results in sublethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level target-based resistance [3]. Therefore, efflux pumps are viable antibacterial targets, and identification and development of potent efflux pump inhibitors is a promising and valid strategy potential therapeutic agents that can rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. The world is searching for new tools to combat multidrug resistance.

*1.3.1 Leishmaniasis*

*Leishmania*.

*1.3.2 Onchocerciasis*

*1.3.3 Schistosomiasis*

resistance-associated proteins.

*1.3.4 Trypanosomiasis*

**129**

ulating intracellular drug concentrations.

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

by avoiding being bitten by flies.

contained IVM-resistant *O. volvulus* worms.

Leishmaniasis is a disease caused by parasites of the Leishmania type. It is spread by the bite of certain types of sandflies [12]. The disease can present in three main ways: cutaneous, mucocutaneous, or visceral leishmaniasis [13]. The cutaneous form presents with skin ulcers, whereas the mucocutaneous form presents with ulcers of the skin, mouth, and nose [12]. Leishmaniasis is transmitted by the bite of infected female phlebotomine sand flies [14] which can transmit the protozoa

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

Gammaro et al. [12] first reported that the overexpression of P-glycoprotein in *Leishmania* species was responsible for the drug resistance of the organisms against drugs such as methotrexate. The multidrug resistance has been associated with several ATP-binding cassette transporters including MRP1 (ABCC1) and P-

glycoprotein (ABCB1). Wyllie et al. [15] demonstrated the presence of metal efflux pumps in the cell membrane of all *Leishmania* species. Soares et al. [16] reported that natural or synthetic modulators of human P-glycoprotein such as flavonoids restore sensitivity to pentamidine, sodium stibogluconate, and miltefosine by mod-

Onchocerciasis, also known as river blindness, is a disease caused by infection with the parasitic worm *Onchocerca volvulus* and is transmitted by the bite of an infected black fly of the *Simulium* type. Symptoms include severe itching, bumps under the skin, and blindness. It is the second most common cause of blindness due to infection, after trachoma, according to WHO. Usually, many bites are required before infection occurs. A vaccine against the disease does not exist. Prevention is

Ivermectin (IVM) is a semisynthesized macrocyclic lactone that belongs to the avermectin class of compounds. It is administered en masse and but is effective only against microfilariae [17]. Bourguinat et al. [11] have found evidence of IVM resistance in *Onchocerca volvulus*. The clinical trial sampled patients before and after IVM treatment over a period of 3 years. The nodules collected from the patients

Schistosomiasis is a disease caused by infection with one of the species of *Schistosoma* helminthic flatworms known as flukes belonging to the class Trematoda of the phylum Platyhelminthes. There are three main species of *Schistosoma* associated with human disease: *Schistosoma mansoni* and *Schistosoma japonicum* cause intestinal schistosomiasis, and *Schistosoma haematobium* causes genitourinary schistosomiasis. Other *Schistosoma* species have been recognized less commonly as agents of intestinal schistosomiasis in humans [18]. Pinto-Almeida et al. [19] demonstrated that drug resistance by *Schistosoma mansoni* to praziquantel (commonly employed drug) is mediated by efflux pump proteins, including P-glycoprotein and multidrug

The trypanosomiasis consists of a group of diseases caused by parasitic protozoa

of the genus *Trypanosoma*. There are two main parasites such as *Trypanosoma brucei*, which causes the sleeping sickness or human African trypanosomiasis and *Trypanosoma cruzi*, which causes the Chagas' disease or American trypanosomiasis

#### **1.2 P-glycoprotein**

P-glycoprotein is a mammalian multidrug-resistance protein belonging to the ATPbinding cassette (ABC) superfamily [4]. It is an ATP-dependent efflux pump encoded by the MDR1 gene and is primarily found in epithelial cells lining the colon, small intestine, pancreatic ductules, bile ductules, kidney proximal tubes, the adrenal gland, and the blood-testis and the blood-brain barrier [5]. This efflux activity of P-glycoprotein, coupled with its wide substrate specificity, is responsible for the reduction in bioavailability of drugs as it extrudes all foreign substances such as drugs and xenobiotics out of the cells. ATP hydrolysis provides energy for the efflux of drugs from the inner leaflet of the cell membrane [6, 7]. This protein is believed to have evolved as a defense mechanism against toxic compounds and prevent their entry into the cytosol [8].

P-glycoprotein confers resistance to a wide range of structurally and functionally diverse compounds, which has resulted in the emergence of multidrug resistance in medically relevant microorganisms. The pharmacodynamic role of P-glycoprotein in parasitic helminths has widespread clinical and socioeconomic implications, exacerbating the problem of neglected tropical diseases (NTDs) whose causative agents are helminths and protozoa.

Sheps et al. [9] reported that 15 P-glycoproteins are present in *Caenorhabditis elegans*, and Laing et al. [10] reported that 10 homologous P-glycoproteins were present in *Haemonchus contortus*. A bioinformatic and phylogenetic study conducted by Bourguinat et al. [11] on the *Dirofilaria immitis* genome identified three orthologous ABC-B transporter genes. These genes are suspected to be responsible for the P-glycoprotein-mediated drug extrusion of melarsomine in *D. immitus* and other parasites.

#### **1.3 Neglected tropical diseases**

Neglected tropical diseases (NTDs) encompass 17 bacterial, parasitic, and viral diseases that prevail in tropical and subtropical conditions in 149 countries and affect more than 1 billion people worldwide, according to WHO.

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

#### *1.3.1 Leishmaniasis*

**1. Introduction**

**1.2 P-glycoprotein**

other parasites.

**128**

**1.3 Neglected tropical diseases**

**1.1 Multidrug resistance (MDR)**

Bacterial evolution has been constrained to respond to the selection pressure of antibiotics and combined with their reckless use and has led to the emergence of varied defenses against antimicrobial agents. The main mechanisms whereby the bacteria develop resistance to antimicrobial agents include enzymatic inactivation, modification of the drug target(s), and reduction of intracellular drug concentration by changes in membrane permeability or by the over expression of efflux pumps [1]. Multidrug resistance efflux pumps are recognized as an important component of resistance in both Gram-positive and Gram-negative bacteria [2]. Some bacterial efflux pumps may be selective for one substrate or transport antibiotics of different classes, conferring a multiple drug resistance (MDR) phenotype. With respect to efflux pumps, they provide a self-defense mechanism whereby antibiotics are extruded from the cell interior to the external environment. This results in sublethal drug concentrations at the active site that in turn may predispose the organism to the development of high-level target-based resistance [3]. Therefore, efflux pumps are viable antibacterial targets, and identification and development of potent efflux pump inhibitors is a promising and valid strategy potential therapeutic agents that can rejuvenate the activity of antibiotics that are no longer effective against bacterial pathogens. The world is searching for new tools to combat multidrug resistance.

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

P-glycoprotein is a mammalian multidrug-resistance protein belonging to the ATPbinding cassette (ABC) superfamily [4]. It is an ATP-dependent efflux pump encoded by the MDR1 gene and is primarily found in epithelial cells lining the colon, small intestine, pancreatic ductules, bile ductules, kidney proximal tubes, the adrenal gland, and the blood-testis and the blood-brain barrier [5]. This efflux activity of P-glycoprotein, coupled with its wide substrate specificity, is responsible for the reduction in bioavailability of drugs as it extrudes all foreign substances such as drugs and xenobiotics out of the cells. ATP hydrolysis provides energy for the efflux of drugs from the inner leaflet of the cell membrane [6, 7]. This protein is believed to have evolved as a defense mechanism against toxic compounds and prevent their entry into the cytosol [8].

P-glycoprotein confers resistance to a wide range of structurally and

by Bourguinat et al. [11] on the *Dirofilaria immitis* genome identified three

affect more than 1 billion people worldwide, according to WHO.

whose causative agents are helminths and protozoa.

functionally diverse compounds, which has resulted in the emergence of multidrug resistance in medically relevant microorganisms. The pharmacodynamic role of P-glycoprotein in parasitic helminths has widespread clinical and socioeconomic implications, exacerbating the problem of neglected tropical diseases (NTDs)

Sheps et al. [9] reported that 15 P-glycoproteins are present in *Caenorhabditis elegans*, and Laing et al. [10] reported that 10 homologous P-glycoproteins were present in *Haemonchus contortus*. A bioinformatic and phylogenetic study conducted

orthologous ABC-B transporter genes. These genes are suspected to be responsible for the P-glycoprotein-mediated drug extrusion of melarsomine in *D. immitus* and

Neglected tropical diseases (NTDs) encompass 17 bacterial, parasitic, and viral diseases that prevail in tropical and subtropical conditions in 149 countries and

Leishmaniasis is a disease caused by parasites of the Leishmania type. It is spread by the bite of certain types of sandflies [12]. The disease can present in three main ways: cutaneous, mucocutaneous, or visceral leishmaniasis [13]. The cutaneous form presents with skin ulcers, whereas the mucocutaneous form presents with ulcers of the skin, mouth, and nose [12]. Leishmaniasis is transmitted by the bite of infected female phlebotomine sand flies [14] which can transmit the protozoa *Leishmania*.

Gammaro et al. [12] first reported that the overexpression of P-glycoprotein in *Leishmania* species was responsible for the drug resistance of the organisms against drugs such as methotrexate. The multidrug resistance has been associated with several ATP-binding cassette transporters including MRP1 (ABCC1) and Pglycoprotein (ABCB1). Wyllie et al. [15] demonstrated the presence of metal efflux pumps in the cell membrane of all *Leishmania* species. Soares et al. [16] reported that natural or synthetic modulators of human P-glycoprotein such as flavonoids restore sensitivity to pentamidine, sodium stibogluconate, and miltefosine by modulating intracellular drug concentrations.

#### *1.3.2 Onchocerciasis*

Onchocerciasis, also known as river blindness, is a disease caused by infection with the parasitic worm *Onchocerca volvulus* and is transmitted by the bite of an infected black fly of the *Simulium* type. Symptoms include severe itching, bumps under the skin, and blindness. It is the second most common cause of blindness due to infection, after trachoma, according to WHO. Usually, many bites are required before infection occurs. A vaccine against the disease does not exist. Prevention is by avoiding being bitten by flies.

Ivermectin (IVM) is a semisynthesized macrocyclic lactone that belongs to the avermectin class of compounds. It is administered en masse and but is effective only against microfilariae [17]. Bourguinat et al. [11] have found evidence of IVM resistance in *Onchocerca volvulus*. The clinical trial sampled patients before and after IVM treatment over a period of 3 years. The nodules collected from the patients contained IVM-resistant *O. volvulus* worms.

#### *1.3.3 Schistosomiasis*

Schistosomiasis is a disease caused by infection with one of the species of *Schistosoma* helminthic flatworms known as flukes belonging to the class Trematoda of the phylum Platyhelminthes. There are three main species of *Schistosoma* associated with human disease: *Schistosoma mansoni* and *Schistosoma japonicum* cause intestinal schistosomiasis, and *Schistosoma haematobium* causes genitourinary schistosomiasis. Other *Schistosoma* species have been recognized less commonly as agents of intestinal schistosomiasis in humans [18]. Pinto-Almeida et al. [19] demonstrated that drug resistance by *Schistosoma mansoni* to praziquantel (commonly employed drug) is mediated by efflux pump proteins, including P-glycoprotein and multidrug resistance-associated proteins.

#### *1.3.4 Trypanosomiasis*

The trypanosomiasis consists of a group of diseases caused by parasitic protozoa of the genus *Trypanosoma*. There are two main parasites such as *Trypanosoma brucei*, which causes the sleeping sickness or human African trypanosomiasis and *Trypanosoma cruzi*, which causes the Chagas' disease or American trypanosomiasis

[20]. These diseases are transmitted by several arthropod vectors such as *Glossina* and *Triatominae*. Chaga's disease causes 21,000 deaths per year mainly in Latin America [21]. Benznidazole and Nifurtimox, only available drugs, however, have limited efficacy in the advanced stages of the disease [22]. Liu et al. [23] and Rappa et al. [24] concluded that *Trypanosoma cruzi* develops resistance to the drugs after prolonged treatment. It was shown that this happens due to the overexpression of the MDR1 gene, at high levels of the drug, which accumulates in the cells over time. Campos et al. [25] demonstrated that the drug resistance is continued throughout the life cycle of the worm.

were modeled for each organism, using multiple templates. The templates having high sequence similarity with the target sequences were given preference. The

The validity of the structures was checked using Procheck, an open source tool

The ligand data set was created by surveying the literature to determine the drugs which the pathogenic helminths are both sensitive and resistant to. Drug resistance which was conferred via efflux pump activity was given importance. This set of ligands was created for each efflux pump, comprising known and potential antibiotics. The canonical *simplified molecular-input line-entry system* (SMILES) of each drug was retrieved from the PubChem database. The PDB model of each antibiotic was then generated using MarvinView by converting the canonical

The efflux pump proteins and ligands were individually docked using the AutoDock Version 4.2.6 suite of programs [32]. The software consists of two main programs: AutoGrid, which precalculates a set of grid points on the receptor, and AutoDock, which docks the ligand to the receptor through the grids. The PDB files of the P-glycoprotein structures and the ligands were modified through the addition of Gasteiger charges, followed by the addition and merging of hydrogen atoms to each structure. These modified structures were then saved as PDBQT files using the AutoDock tools. A uniform grid box was then defined and centered in the internal binding cavity of each P-glycoprotein structure, and the affinity maps were generated using AutoGrid. This procedure was repeated for each protein-drug complex.

**2.7 Molecular docking of the helminthic efflux pumps with known and**

Each drug was individually docked with each target protein using AutoDock 4.2.6. The local search algorithm used was the Lamarckian genetic algorithm, set to its default parameters. The docking parameters were set to 250,000 cycles per run

used to assess the reliability of the protein structure. It is a part of the SWISS-MODEL server. The structures were refined using energy-minimization protocols, and the least energetic structure corresponding to each protein was chosen for docking studies. The criteria used to assess the quality of the structure include model geometry and the Ramachandran plot. The Ramachandran plot describes the rotation of the polypeptide backbone around the N-C<sup>α</sup> (ϕ) and C-C<sup>α</sup> (ψ) bonds. It provides an overview of the distribution of the torsion angles over the core, allowed, generous, and disallowed regions. The three main parameters used to

models were built, and the PDB files of the structures were obtained.

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

**2.4 Structure validation**

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

select the structures were:

3.Taxonomy

SMILES [31].

1.Overall Ramachandran value

2.Phylogenetic tree distance

**2.5 Creation of the ligand dataset**

**2.6 Protein and ligand preparation**

**potential antibiotics**

**131**

#### **2. Methods**

The methodology is essentially similar to that in our earlier study on Pglycoproteins in priority infectious agents [26].

#### **2.1 Determining the full helminthic complement of efflux pump proteins homologous to mammalian P-glycoprotein**

The protein sequence of the human P-glycoprotein (P08183) was obtained from the SWISS-PROT database. The position-specific iterated BLAST (PSI-BLAST) was performed against a search set of nonredundant protein sequences in the organism of interest, using hPGP as the query. Through a PSI-BLAST search, a large set of related proteins are compiled. It is used to identify distant evolutionary relationships between protein sequences [27]. In the algorithm, parameters were set with an E-value of 0.001, and the scoring matrix BLOSUM62 was used. This step was performed on all four organisms of interest (*Leishmania major*, *Onchocerca volvulus*, *Schistosoma mansoni*, and *Trypanosoma cruzi*). Hundreds of hits were obtained for Pglycoprotein, and these results were prioritized according to predetermined parameters such as medical relevance, annotation status, and the presence of conserved regions. Sequences having a high percentage of sequence identity and query coverage were prioritized. Specific UniProt searches of these protein sequences were performed using the accession number. The results were analyzed, and the Pglycoprotein sequence of each organism was finalized.

#### **2.2 Multiple sequence alignment**

The templates chosen for multiple sequence alignment (MSA) were 4M1M (*Mus musculus*), 4F4C (*Caenorhabditis elegans*), 3WME (*Cyanidioschyzon merolae*), 2HYD (*Staphylococcus aureus*), 3B5Z (*Salmonella enterica*). These five metazoan, algal, and bacterial templates were used due to their high sequence identity with the hPGP sequence. The target sequences and the five templates were aligned using ClustalX 2.1 [28]. MSA was performed in order to infer the homology and evolutionary relationship between the sequences of the biological data set. The clustering algorithm used was Neighbor Joining (NJ). The phylogenetic distance between the target sequence and the templates was calculated.

#### **2.3 Homology modeling**

The chosen P-glycoprotein sequences were used as target sequences for modeling using software such as SWISS-MODEL. SWISS-MODEL is an open-source, structural bioinformatics tool used for the automated comparative modeling of three-dimensional protein structures [29, 30]. Several P-glycoprotein structures

were modeled for each organism, using multiple templates. The templates having high sequence similarity with the target sequences were given preference. The models were built, and the PDB files of the structures were obtained.

#### **2.4 Structure validation**

[20]. These diseases are transmitted by several arthropod vectors such as *Glossina* and *Triatominae*. Chaga's disease causes 21,000 deaths per year mainly in Latin America [21]. Benznidazole and Nifurtimox, only available drugs, however, have limited efficacy in the advanced stages of the disease [22]. Liu et al. [23] and Rappa et al. [24] concluded that *Trypanosoma cruzi* develops resistance to the drugs after prolonged treatment. It was shown that this happens due to the overexpression of the MDR1 gene, at high levels of the drug, which accumulates in the cells over time. Campos et al. [25] demonstrated that the drug resistance is continued throughout

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

The methodology is essentially similar to that in our earlier study on P-

**2.1 Determining the full helminthic complement of efflux pump proteins**

The protein sequence of the human P-glycoprotein (P08183) was obtained from the SWISS-PROT database. The position-specific iterated BLAST (PSI-BLAST) was performed against a search set of nonredundant protein sequences in the organism of interest, using hPGP as the query. Through a PSI-BLAST search, a large set of related proteins are compiled. It is used to identify distant evolutionary relationships between protein sequences [27]. In the algorithm, parameters were set with an E-value of 0.001, and the scoring matrix BLOSUM62 was used. This step was performed on all four organisms of interest (*Leishmania major*, *Onchocerca volvulus*, *Schistosoma mansoni*, and *Trypanosoma cruzi*). Hundreds of hits were obtained for Pglycoprotein, and these results were prioritized according to predetermined parameters such as medical relevance, annotation status, and the presence of conserved regions. Sequences having a high percentage of sequence identity and query coverage were prioritized. Specific UniProt searches of these protein sequences were performed using the accession number. The results were analyzed, and the P-

The templates chosen for multiple sequence alignment (MSA) were 4M1M (*Mus musculus*), 4F4C (*Caenorhabditis elegans*), 3WME (*Cyanidioschyzon merolae*), 2HYD (*Staphylococcus aureus*), 3B5Z (*Salmonella enterica*). These five metazoan, algal, and bacterial templates were used due to their high sequence identity with the hPGP sequence. The target sequences and the five templates were aligned using ClustalX 2.1 [28]. MSA was performed in order to infer the homology and evolutionary relationship between the sequences of the biological data set. The clustering algorithm used was Neighbor Joining (NJ). The phylogenetic distance between the

The chosen P-glycoprotein sequences were used as target sequences for modeling using software such as SWISS-MODEL. SWISS-MODEL is an open-source, structural bioinformatics tool used for the automated comparative modeling of three-dimensional protein structures [29, 30]. Several P-glycoprotein structures

the life cycle of the worm.

glycoproteins in priority infectious agents [26].

**homologous to mammalian P-glycoprotein**

glycoprotein sequence of each organism was finalized.

target sequence and the templates was calculated.

**2.2 Multiple sequence alignment**

**2.3 Homology modeling**

**130**

**2. Methods**

The validity of the structures was checked using Procheck, an open source tool used to assess the reliability of the protein structure. It is a part of the SWISS-MODEL server. The structures were refined using energy-minimization protocols, and the least energetic structure corresponding to each protein was chosen for docking studies. The criteria used to assess the quality of the structure include model geometry and the Ramachandran plot. The Ramachandran plot describes the rotation of the polypeptide backbone around the N-C<sup>α</sup> (ϕ) and C-C<sup>α</sup> (ψ) bonds. It provides an overview of the distribution of the torsion angles over the core, allowed, generous, and disallowed regions. The three main parameters used to select the structures were:


#### **2.5 Creation of the ligand dataset**

The ligand data set was created by surveying the literature to determine the drugs which the pathogenic helminths are both sensitive and resistant to. Drug resistance which was conferred via efflux pump activity was given importance. This set of ligands was created for each efflux pump, comprising known and potential antibiotics. The canonical *simplified molecular-input line-entry system* (SMILES) of each drug was retrieved from the PubChem database. The PDB model of each antibiotic was then generated using MarvinView by converting the canonical SMILES [31].

#### **2.6 Protein and ligand preparation**

The efflux pump proteins and ligands were individually docked using the AutoDock Version 4.2.6 suite of programs [32]. The software consists of two main programs: AutoGrid, which precalculates a set of grid points on the receptor, and AutoDock, which docks the ligand to the receptor through the grids. The PDB files of the P-glycoprotein structures and the ligands were modified through the addition of Gasteiger charges, followed by the addition and merging of hydrogen atoms to each structure. These modified structures were then saved as PDBQT files using the AutoDock tools. A uniform grid box was then defined and centered in the internal binding cavity of each P-glycoprotein structure, and the affinity maps were generated using AutoGrid. This procedure was repeated for each protein-drug complex.

#### **2.7 Molecular docking of the helminthic efflux pumps with known and potential antibiotics**

Each drug was individually docked with each target protein using AutoDock 4.2.6. The local search algorithm used was the Lamarckian genetic algorithm, set to its default parameters. The docking parameters were set to 250,000 cycles per run

and 10 runs per protein-drug complex, to obtain the 10 best poses for each complex. The best pose was defined as the conformation having the least binding energy. The 10 poses obtained for each receptor-ligand pair were clustered at 2.0 *Å* r.m.s. to validate the convergence to the best pose. The AutoDock was run, and the PDBQT file of the best pose of each docked complex was generated.

The results were analyzed to verify whether the pathogenic strain could develop resistance to known antibiotics using efflux pump activity and if the novel antibiotics could be effective against the development of such resistance.

#### **2.8 Calculation of differential ligand binding affinity**

The differential binding affinities of the repurposed ligands were determined using the conventionally used drugs as a baseline. A lower value is indicative of a more stable complex. The differential affinity of the potential drug for a given efflux pump protein relative to the known drug is estimated as the difference in the binding energies of the known and potential drugs, as given by Eq. (1):

$$
\Delta\Delta\mathbf{G}\_{\text{invest.known}} = \Delta\mathbf{G}\_{\text{bind,potential}} - \Delta\mathbf{G}\_{\text{bind,known}} \tag{1}
$$

Max Scores, but low query coverage. These protein sequences were also found to be unannotated. For these reasons, the proteins which had a lower Max Score in comparison to other results, but satisfied other parameters, were chosen.

**Sequence length**

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

*Leishmania major* P-glycoprotein 1341 36% 98% 767 *Onchocerca volvulus* P-glycoprotein 1278 37% 97% 776

*Trypanosoma cruzi* P-glycoprotein 1034 29% 30% 79.7

**% of identity**

SMDR2 1254 40% 98% 889

**Query coverage**

**Max score**

Certain metazoan, algal and bacterial crystal structures shown in **Table 2** were

Each target protein sequence was aligned with the set of chosen templates using ClustalX 2.1. The MSA between Leishmania major and the 4M1M and 4F4C templates showed the highest sequence identity, as shown in **Figure 1**. Additionally, the

phylogenetic distances between the sequences were calculated using the NJ

The chosen P-glycoprotein sequences of the organisms were used as target sequences for homology modeling using the SWISS-MODELER. Each protein was modeled using several templates, and the predetermined templates were used if they were found to have a fairly high GMQE score. Each modeled structure was

Global Model Quality Estimation (GMQE) is a score that provides an estimation of the quality of the alignment. It is expressed as a value between 0 and 1, where the reliability of the model is directly proportional to the score. The GMQE of the homology models are found to be (mostly) between 0.60 and 0.70 for all organisms, with the exception of *Trypanosoma cruzi*, which gave scores in the range 0.29–0.52. The templates 4M1M, 4F4C, and 3WME were found to be comparatively more reliable. Hence, only the protein structures modeled using these templates were

**3.2 Template selection and multiple sequence alignment**

selected as potential templates for homology modeling [38].

saved as a PDB file. The results are summarized in **Table 4**.

**Template Organism** 4M1M *Mus musculus* 4F4C *Canorhabditis elegans* 3WME *Cyanidioschyzon merolae* 2HYD *Staphylococcus aureus* 3B5Z *Salmonella enteric*

algorithm (**Table 3**).

**3.3 Homology modeling**

**Organism Name of**

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

*Schistosoma mansoni*

**Table 1.**

**protein**

*PSI-BLAST results of the target organisms using hPGP as the query.*

used for further validation studies.

*Templates chosen for multiple sequence alignment.*

**Table 2.**

**133**

where ΔΔGinvest.known = differential ligand affinity, kcal/mol; ΔGbind = free energy of binding, kcal/mol.

#### **2.9 Identification of interacting residues in each docked complex**

The best pose of each docked complex was viewed using RasMol [33], and all interacting residues within a radius of 4.5 *Å* of the ligand were selected. The PDBQT file of each restricted complex was saved as a PDB file. The interacting residues of each docked complex were then analyzed.

#### **3. Results and discussion**

Extensive literature searches on Neglected Tropical Diseases (NTDs) showed that leishmaniasis, onchocerciasis, schistosomiasis, and trypanosomiasis have started exhibiting multidrug resistance, mediated by P-glycoprotein efflux pumps [11, 12, 25, 34]. New drugs targeting NTD's are undergoing clinical trials [35–37], and efforts are being taken to uncover the mechanisms of drug resistance employed by the causative helminths.

The sequence identity of each helminthic P-glycoprotein with the human Pglycoprotein (hPGP) which was retrieved from the UniProt database (UniProt ID: P08183) was determined by running a PSI-BLAST.

#### **3.1 Psi-blast analysis**

The PSI-BLAST was performed on each target organism using hPGP as the query. The results were refined according to predetermined parameters such as medical relevance, annotation status, and the presence of conserved regions. The chosen efflux pump protein sequences were shown in **Table 1**.

The top hits of each PSI-BLAST were analyzed, and the hit having the highest Max Score was chosen only in the case of *Leishmania major* and *Onchocerca volvulus.* These protein sequences were fully annotated and had high sequence identities over a large portion of the protein sequence. The top hits of the PSI-BLAST of *Schistosoma mansoni* and *Trypanosoma cruzi* with hPGP yielded results having high


*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

**Table 1.**

and 10 runs per protein-drug complex, to obtain the 10 best poses for each complex. The best pose was defined as the conformation having the least binding energy. The 10 poses obtained for each receptor-ligand pair were clustered at 2.0 *Å* r.m.s. to validate the convergence to the best pose. The AutoDock was run, and the PDBQT

The results were analyzed to verify whether the pathogenic strain could develop resistance to known antibiotics using efflux pump activity and if the novel antibi-

The differential binding affinities of the repurposed ligands were determined using the conventionally used drugs as a baseline. A lower value is indicative of a more stable complex. The differential affinity of the potential drug for a given efflux pump protein relative to the known drug is estimated as the difference in the

where ΔΔGinvest.known = differential ligand affinity, kcal/mol; ΔGbind = free

The best pose of each docked complex was viewed using RasMol [33], and all interacting residues within a radius of 4.5 *Å* of the ligand were selected. The PDBQT file of each restricted complex was saved as a PDB file. The interacting residues of

Extensive literature searches on Neglected Tropical Diseases (NTDs) showed that leishmaniasis, onchocerciasis, schistosomiasis, and trypanosomiasis have started exhibiting multidrug resistance, mediated by P-glycoprotein efflux pumps [11, 12, 25, 34]. New drugs targeting NTD's are undergoing clinical trials [35–37], and efforts are being taken to uncover the mechanisms of drug resistance employed

The sequence identity of each helminthic P-glycoprotein with the human Pglycoprotein (hPGP) which was retrieved from the UniProt database (UniProt ID:

The PSI-BLAST was performed on each target organism using hPGP as the query. The results were refined according to predetermined parameters such as medical relevance, annotation status, and the presence of conserved regions. The

The top hits of each PSI-BLAST were analyzed, and the hit having the highest Max Score was chosen only in the case of *Leishmania major* and *Onchocerca volvulus.* These protein sequences were fully annotated and had high sequence identities over

*Schistosoma mansoni* and *Trypanosoma cruzi* with hPGP yielded results having high

ΔΔGinvest*:*known ¼ ΔGbind,potential � ΔGbind,known (1)

file of the best pose of each docked complex was generated.

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

**2.8 Calculation of differential ligand binding affinity**

energy of binding, kcal/mol.

**3. Results and discussion**

by the causative helminths.

**3.1 Psi-blast analysis**

**132**

each docked complex were then analyzed.

P08183) was determined by running a PSI-BLAST.

chosen efflux pump protein sequences were shown in **Table 1**.

a large portion of the protein sequence. The top hits of the PSI-BLAST of

otics could be effective against the development of such resistance.

binding energies of the known and potential drugs, as given by Eq. (1):

**2.9 Identification of interacting residues in each docked complex**

*PSI-BLAST results of the target organisms using hPGP as the query.*

Max Scores, but low query coverage. These protein sequences were also found to be unannotated. For these reasons, the proteins which had a lower Max Score in comparison to other results, but satisfied other parameters, were chosen.

#### **3.2 Template selection and multiple sequence alignment**

Certain metazoan, algal and bacterial crystal structures shown in **Table 2** were selected as potential templates for homology modeling [38].

Each target protein sequence was aligned with the set of chosen templates using ClustalX 2.1. The MSA between Leishmania major and the 4M1M and 4F4C templates showed the highest sequence identity, as shown in **Figure 1**. Additionally, the phylogenetic distances between the sequences were calculated using the NJ algorithm (**Table 3**).

#### **3.3 Homology modeling**

The chosen P-glycoprotein sequences of the organisms were used as target sequences for homology modeling using the SWISS-MODELER. Each protein was modeled using several templates, and the predetermined templates were used if they were found to have a fairly high GMQE score. Each modeled structure was saved as a PDB file. The results are summarized in **Table 4**.

Global Model Quality Estimation (GMQE) is a score that provides an estimation of the quality of the alignment. It is expressed as a value between 0 and 1, where the reliability of the model is directly proportional to the score. The GMQE of the homology models are found to be (mostly) between 0.60 and 0.70 for all organisms, with the exception of *Trypanosoma cruzi*, which gave scores in the range 0.29–0.52.

The templates 4M1M, 4F4C, and 3WME were found to be comparatively more reliable. Hence, only the protein structures modeled using these templates were used for further validation studies.


**Table 2.** *Templates chosen for multiple sequence alignment.*


**Template Phylogenetic distance**

*\**

**Table 3.**

**Table 4.**

**135**

*Homology modeling results.*

*identity with the helminthic P-glycoproteins.*

*Bold values signify the final template in the case of each agent.*

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

*Leishmania major Onchocerca volvulus Schistosoma mansoni Trypanosoma cruzi*

4M1M 0.685 0.648 0.646 **0.847\*** 4F4C **0.642 0.638 0.605** 0.861 3WME 0.653 0.679 0.649 0.867 2HYD 0.72 0.709 0.694 0.826 3B5Z 0.731 0.707 0.698 0.841

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

*The distance between* T. cruzi *and 4M1M is prioritized as the 4M1M and 4F4C templates were found to have higher sequence*

**Organism Template Sequence identity Query coverage GMQE** *Leishmania major* 4F4C 34.43 0.91 0.64

*Onchocerca volvulus* 4F4C 38.83 0.97 0.69

*Schistosoma mansoni* 4F4C 38.60 0.97 0.69

*Trypanosoma cruzi* 4F4C 15.11 0.81 0.45

4M1M 36.09 0.90 0.65 3WME 37.30 0.43 0.29 4Q9I 36.20 0.90 0.65 4KSC 38.25 0.90 0.64 4KSB 38.25 0.90 0.64 5KPJ 38.25 0.90 0.66

4M1M 37.10 0.96 0.69 3WME 31.75 0.44 0.33 3G5U 38.77 0.95 0.66 4KSB 38.77 0.95 0.67 4Q9I 36.97 0.95 0.69 4LSG 38.77 0.95 0.67

4M1M 36.09 0.90 0.65 3G5U 39.41 0.97 0.68 3G60 42.11 0.95 0.68 5KPJ 39.52 0.97 0.70 4KSC 42.11 0.95 0.69 4KSB 42.11 0.95 0.69 4LSG 39.41 0.97 0.69

4M1M 14.36 0.78 0.29 3WME 18.37 0.51 0.33 4KSC 14.23 0.79 0.44 3G5U 14.46 0.78 0.43 5TSI 23.43 0.90 0.52 4LSG 14.46 0.78 0.43

*Phylogenetic distance matrix between the target sequence of each organism and the templates.*

#### **Figure 1.**

*Multiple sequence alignment of the target sequence P-glycoprotein of* Leishmania major *(tr\_Q4Q3A6) with the templates of interest.*

#### **3.4 Structure validation**

The quality of each structure was assessed using Procheck. Criteria such as model geometry and the Ramachandran plot were used to validate the structures. The PDB file of each structure was used to run the Procheck, and the Ramachandran plot values were obtained. The Ramachandran values are summarized in **Table 5**.

#### *Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*


*\* The distance between* T. cruzi *and 4M1M is prioritized as the 4M1M and 4F4C templates were found to have higher sequence identity with the helminthic P-glycoproteins.*

*Bold values signify the final template in the case of each agent.*

#### **Table 3.**

*Phylogenetic distance matrix between the target sequence of each organism and the templates.*


**Table 4.** *Homology modeling results.*

**3.4 Structure validation**

rized in **Table 5**.

**134**

**Figure 1.**

*templates of interest.*

The quality of each structure was assessed using Procheck. Criteria such as model geometry and the Ramachandran plot were used to validate the structures.

*Multiple sequence alignment of the target sequence P-glycoprotein of* Leishmania major *(tr\_Q4Q3A6) with the*

Ramachandran plot values were obtained. The Ramachandran values are summa-

The PDB file of each structure was used to run the Procheck, and the

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

The structures were finalized by analyzing overall Ramachandran value, Phylogenetic tree distance, and taxonomy parameters. The 4F4C template was found to suitable for all the organisms excluding *Leishmania major*, for which the 4M1M template was selected (**Table 5**).

#### *3.4.1 Validation of the P-glycoprotein structure modeled using the 4M1M template for* Leishmania major

The Ramachandran plots having a core region of at least 90% are prioritized for further studies. The core, allowed, generous and disallowed regions are colored and distinguished (**Figure 2**). The red, brown, and yellow regions represent the favored, allowed, and generously allowed regions.

A more comprehensive analysis of the structure is provided by other programs that generate other data such as Phi-Psi graphs and Chi1-Chi2 plots for each residue type. Each Phi-Psi plot provides an analysis of the torsion angle of each residue type. The red, brown, and yellow regions represent the favored, allowed, and generously allowed regions (shown in **Figure 3**).

The Chi1-Chi2 plot describes the side-chain torsion angles combinations for each amino acid [28]. The darker regions indicate a more favorable angle combination (shown **Figure 4**).

#### *3.4.2 Validation of the P-glycoprotein structure modeled using the 4F4C template for* Onchocerca volvulus, Schistosoma mansoni *and* Trypansoma cruzi

For all the three P-glycoproteins, the structures were modeled using the 4F4C template and as such and showed remarkable structural similarity with respect to the Ramachandran plot (90.8% in the core region), and residue torsion angles. **Figures 5**–**7** summarize this exercise.

*Leishmania*

**Template**

**137**

4F4C

4M1M

3WME

**Template**

4F4C

4M1M

3WME

**Template**

4F4C

4M1M

3WME

**Template**

4F4C

4M1M

3WME

**Table 5.** *Justification*

 *of the template chosen for each organism using the* 

 89.8

 86.0

 90.8

 **Core region** 

**Additionally**

 **allowed region Generously**

8.1 11.9

8

 **allowed region Disallowed**

1.2 1.8 1.6 *Ramachandran*

 *plot values and the phylogenetic*

 *distance between the target protein and the template.*

 93.7

 91.1

 90.8

 **Core region** 

**Additionally**

 **allowed region Generously**

8.1 6.9 5.8

 **allowed region Disallowed**

1.2 1.8 0.2

 93.1

 91.1

 90.8

 **Core region** 

**Additionally**

 **allowed region Generously**

8.1 7.6 5.8

 **allowed region Disallowed**

1.2 1.2 0.6

 94.6

 92

 90.8

 **Core region** 

**Additionally**

 **allowed region Generously**

8.1 6.4 4.6

 **allowed region Disallowed**

1.2 1.4 0.4

 *major*

 **region No. of residues**

> 0

0.2 0.4

*Onchocerca*

 *volvulus*

 **region No. of residues**

> 0

0.1 0.6

*Schistosoma*

 *mansoni*

 **region No. of residues**

> 0

0.2 0.4

*Trypanosoma*

 *cruzi*

 **region No. of residues**

> 0

0.2 0.6

573

0.51

18.37

0.861

1034

0.51

17.80

0.867

1250

0.81

15.11

 **Query coverage**

 **Sequence identity** 

**Phylogenetic**

 **distance**

0.847

567

0.45

38.31

0.649

572

0.46

36.6

0.605

1250

0.97

38.6

 **Query coverage**

 **Sequence identity** 

**Phylogenetic**

 **distance**

0.646

1180

0.45

33.51

0.679

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

571

0.95

37.1

0.638

1250

0.97

38.83

 **Query coverage**

 **Sequence identity** 

**Phylogenetic**

 **distance**

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

0.648

573

0.43

36.46

0.653

1188

0.9

36.09

0.642

1250

0.43

34.43

 **Query coverage**

 **Sequence identity** 

**Phylogenetic**

 **distance**

0.685

#### **3.5 Creation of the ligand dataset**

Upon extensive survey of the literature, a comprehensive data set of the known and potential drugs was compiled (**Table 6**). The list of potential drugs comprises of both unapproved, investigational drugs that are undergoing phase trials, and FDA approved antibiotics. In this study, these known drugs have been repurposed for other helminthic diseases.

#### **3.6 Molecular docking of the helminthic efflux pumps with known and potential antibiotics**

The molecular docking was carried out using the AutoDock suite of tools. The search algorithm used was the Lamarckian Genetic Algorithm, and the docking parameters were set to 10 runs per protein-drug complex. Each docked complex yielded 10 poses, and the best pose was defined as the conformation possessing the least free binding energy.

#### *3.6.1 Molecular docking results of benznidazole with P-glycoprotein (*Leishmania major*)*

The drug benznidazole is docked with P-glycoprotein (*Leishmania major*), and their interaction is studied (**Table 7**). The best pose has a free binding energy of �5.00 kcal/mol. The clustering was performed at 2.0 *Å* r.m.s. to validate the convergence to the best pose. The clustering figure (**Figure 8**) shows closer peaks near �2.5 kcal/mol, whereas the least binding energy of the complex, that is, most


*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

> **Table 5.**

*Justification of the template chosen for each organism using the Ramachandran plot values and the phylogenetic distance between the target protein and the template.*

The structures were finalized by analyzing overall Ramachandran value, Phylogenetic tree distance, and taxonomy parameters. The 4F4C template was found to suitable for all the organisms excluding *Leishmania major*, for which the 4M1M

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

*3.4.1 Validation of the P-glycoprotein structure modeled using the 4M1M template for*

The Ramachandran plots having a core region of at least 90% are prioritized for further studies. The core, allowed, generous and disallowed regions are colored and distinguished (**Figure 2**). The red, brown, and yellow regions represent the favored,

A more comprehensive analysis of the structure is provided by other programs that generate other data such as Phi-Psi graphs and Chi1-Chi2 plots for each residue type. Each Phi-Psi plot provides an analysis of the torsion angle of each residue type. The red, brown, and yellow regions represent the favored, allowed, and generously

The Chi1-Chi2 plot describes the side-chain torsion angles combinations for each amino acid [28]. The darker regions indicate a more favorable angle combination

*3.4.2 Validation of the P-glycoprotein structure modeled using the 4F4C template for* Onchocerca volvulus, Schistosoma mansoni *and* Trypansoma cruzi

For all the three P-glycoproteins, the structures were modeled using the 4F4C template and as such and showed remarkable structural similarity with respect to the Ramachandran plot (90.8% in the core region), and residue torsion angles.

Upon extensive survey of the literature, a comprehensive data set of the known and potential drugs was compiled (**Table 6**). The list of potential drugs comprises of both unapproved, investigational drugs that are undergoing phase trials, and FDA approved antibiotics. In this study, these known drugs have been repurposed

The molecular docking was carried out using the AutoDock suite of tools. The search algorithm used was the Lamarckian Genetic Algorithm, and the docking parameters were set to 10 runs per protein-drug complex. Each docked complex yielded 10 poses, and the best pose was defined as the conformation possessing the

*3.6.1 Molecular docking results of benznidazole with P-glycoprotein (*Leishmania major*)*

The drug benznidazole is docked with P-glycoprotein (*Leishmania major*), and their interaction is studied (**Table 7**). The best pose has a free binding energy of �5.00 kcal/mol. The clustering was performed at 2.0 *Å* r.m.s. to validate the convergence to the best pose. The clustering figure (**Figure 8**) shows closer peaks near �2.5 kcal/mol, whereas the least binding energy of the complex, that is, most

**3.6 Molecular docking of the helminthic efflux pumps with known and**

template was selected (**Table 5**).

allowed, and generously allowed regions.

allowed regions (shown in **Figure 3**).

**Figures 5**–**7** summarize this exercise.

**3.5 Creation of the ligand dataset**

for other helminthic diseases.

**potential antibiotics**

least free binding energy.

**136**

(shown **Figure 4**).

Leishmania major

**Figure 2.**

*(a) The Ramachandran plot generated for P-glycoprotein (*Leishmania major*), modeled using the 4M1M template and (b) plot statistics of the P-glycoprotein (*Leishmania major*), modeled using the 4M1M template.*

**Figure 5.**

*template.*

**139**

*(a) The Ramachandran plot generated for P-glycoprotein (*Onchocerca volvulus*), modeled using the 4F4C template and (b) plot statistics of the P-glycoprotein (*Onchocerca volvulus*), modeled using the 4F4C*

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

**S. no. Drug PubChem CID 3-D Structure**

1. Albendazole 2082

2. Amphotericin B 5280965

3. Artesunate 65664

4. Benznidazole 5798

5. Cladosporin 13990016

6. Dapsone 2955

7. Diethylcarbamazine 15432

8. Emodepside 6918632

#### **Figure 3.**

*Phi-psi plot of residues of the P-glycoprotein structure of* Leishmania major*, modeled using the 4M1M template (a) Ala, (b) Arg, and (c) Asn.*

#### **Figure 4.**

*Chi1-Chi2 plot of residues of the P-glycoprotein structure of* Leishmania major*, modeled using the 4M1M template (a) Arg, (b) Asn and (c) Asp.*

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

**Figure 5.**

**Figure 2.**

**Figure 3.**

**Figure 4.**

**138**

*template (a) Arg, (b) Asn and (c) Asp.*

*(a) Ala, (b) Arg, and (c) Asn.*

*(a) The Ramachandran plot generated for P-glycoprotein (*Leishmania major*), modeled using the 4M1M template and (b) plot statistics of the P-glycoprotein (*Leishmania major*), modeled using the 4M1M template.*

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

*Phi-psi plot of residues of the P-glycoprotein structure of* Leishmania major*, modeled using the 4M1M template*

*Chi1-Chi2 plot of residues of the P-glycoprotein structure of* Leishmania major*, modeled using the 4M1M*

*(a) The Ramachandran plot generated for P-glycoprotein (*Onchocerca volvulus*), modeled using the 4F4C template and (b) plot statistics of the P-glycoprotein (*Onchocerca volvulus*), modeled using the 4F4C template.*


**S. no. Drug PubChem CID 3-D Structure**

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

21. Nifurtimox 6842999

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

22. Oxamniquine 4612

23. Paromomycin 165580

24. Pentamidine 4735

25. Posaconazole 468595

26. Praziquantel 4891

27. Ravuconazole 467825

28. Sodium stibogluconate 76968133

29. Suramin 5361

30. Terbinafine 1549008

31. Thiabendazole 5430

32. Tipifarnib 159324

**Table 6.**

**141**

*3D structures of the drugs are visualized using python molecular viewer (PMV-1.5.6).*

*PubChem compound ID and 3D structure of the ligands used for docking studies.*



*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

#### **Table 6.**

**S. no. Drug PubChem CID 3-D Structure**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

9. Fexinidazole 68792

10. Flubendazole 35802

11. Fluconazole 3365

12. Furozan 67517

13. Imatinib 5291

14. Ivermectin 6321424

15. Jaspamide 9831636

16. Mebendazole 4030

17. Metrifonate 5853

18. Miltefosine 3599

19. Moxidectin 9832912

20. Niclosamide 4477

**140**

*PubChem compound ID and 3D structure of the ligands used for docking studies.*

#### **Figure 6.**

*Phi-psi plot of residues of the P-glycoprotein structure of* Onchocerca volvulus*, modeled using the 4F4C template (a) Ala, (b) Arg, and (c) Asn.*

**Figure 8.**

**Table 8.**

**Figure 9.**

**143**

*(a) Clustering analysis of the benznidazole- P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Leishmania major*]). (c) The interacting residues in the benznidazole-*

**Rank of complex Free binding energy (kcal/mol)**

*(a) Clustering analysis of the niclosamide- P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Onchocerca volvulus*]). (c) The interacting residues in the niclosamide- P-*

1 �5.29 2 �5.01 3 �4.78 4 �5.14 5 �5.08 6 �5.02 7 �4.59 8 �4.53 9 �4.42 10 34.78

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

*P-glycoprotein (*Leishmania major*) docked complex is viewed using RasMol 2.1.*

*Interaction of the drug niclosamide with P-glycoprotein (*Onchocerca volvulus*).*

*glycoprotein (*Onchocerca volvulus*) docked complex is viewed using RasMol 2.1.*


#### **Table 7.**

*Interaction of the drug benznidazole with P-glycoprotein (*Leishmania major*).*

#### **Figure 7.**

*Chi1-Chi2 plot of residues of the P-glycoprotein structure of* Onchocerca volvulus*, modeled using the 4F4C template. (a) Arg, (b) Asn, and (c) Asp.*

clustering is at �5.66 kcal/mol. This shows that convergence to the best pose can be achieved through consecutive dockings with more iterations. **Figure 8(b)** depicts the binding site on the receptor, and **Figure 8(c)** shows the interacting residues in the benznidazole-P-glycoprotein (*Leishmania major*) docked complex viewed through RasMol 2.1.

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

#### **Figure 8.**

*(a) Clustering analysis of the benznidazole- P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Leishmania major*]). (c) The interacting residues in the benznidazole-P-glycoprotein (*Leishmania major*) docked complex is viewed using RasMol 2.1.*


#### **Table 8.**

*Interaction of the drug niclosamide with P-glycoprotein (*Onchocerca volvulus*).*

#### **Figure 9.**

clustering is at �5.66 kcal/mol. This shows that convergence to the best pose can be achieved through consecutive dockings with more iterations. **Figure 8(b)** depicts the binding site on the receptor, and **Figure 8(c)** shows the interacting residues in the benznidazole-P-glycoprotein (*Leishmania major*) docked complex viewed

*Chi1-Chi2 plot of residues of the P-glycoprotein structure of* Onchocerca volvulus*, modeled using the 4F4C*

*Phi-psi plot of residues of the P-glycoprotein structure of* Onchocerca volvulus*, modeled using the 4F4C*

**Rank of complex Free binding energy (kcal/mol)**

1 �5.00 2 �4.84 3 �4.2 4 �4.41 5 �3.77 6 �3.48 7 �2.96 8 �2.64 9 �2.54 10 �2.48

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

*Interaction of the drug benznidazole with P-glycoprotein (*Leishmania major*).*

through RasMol 2.1.

*template. (a) Arg, (b) Asn, and (c) Asp.*

**Figure 6.**

**Table 7.**

**Figure 7.**

**142**

*template (a) Ala, (b) Arg, and (c) Asn.*

*(a) Clustering analysis of the niclosamide- P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Onchocerca volvulus*]). (c) The interacting residues in the niclosamide- Pglycoprotein (*Onchocerca volvulus*) docked complex is viewed using RasMol 2.1.*

#### E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*


*3.6.2 Molecular docking results of niclosamide with P-glycoprotein*

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

*3.6.3 Molecular docking results of praziquantel with P-glycoprotein*

(*Schistosoma mansoni*) docked complex viewed through RasMol 2.1.

*3.6.4 Molecular docking results of cladosporin with P-glycoprotein*

*vulus*) docked complex viewed through RasMol 2.1.

The best pose has a free binding energy of �5.29 kcal/mol (**Table 8**). The clustering figure shows the most number of conformations at �1.30 kcal/mol (**Figure 9**). **Figure 9(b)** depicts the binding site on the receptor, and **Figure 9(c)** shows the interacting residues in the niclosamide-P-glycoprotein (*Onchocerca vol-*

The best pose has a free binding energy of �5.83 kcal/mol (**Table 9**). The clustering figure (**Figure 10**) shows the most number of conformations at �5.0 kcal/mol. **Figure 10(b)** depicts the binding site on the receptor, and **Figure 10(c)** shows the interacting residues in the Praziquantel-P-glycoprotein

The best pose has a free binding energy of �6.23 kcal/mol (**Table 10**). The clustering figure (**Figure 11**) shows the most number of conformations at �5.0 kcal/ mol. **Figure 11(b)** depicts the binding site on the receptor, and **Figure 11(c)** shows the interacting residues in the the cladosporin-P-glycoprotein (*Trypanosoma cruzi*)

These steps were carried out for each receptor-ligand complex, and the least free binding energy of each docked complex was determined. These results are summa-

The differential affinity of the potential drug for a given efflux pump protein relative to the known drug is estimated as the difference between the binding

*(a) Clustering analysis of the cladosporin-P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Trypanosoma cruzi*]). (c) The interacting residues in the cladosporin-*

*P-glycoprotein [*Trypanosoma cruzi*] docked complex are viewed using RasMol 2.1.*

*(*Onchocerca volvulus*)*

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

*(*Schistosoma mansoni*)*

*(*Trypanosoma cruzi*)*

rized in **Table 11**.

**Figure 11.**

**145**

docked complex viewed through RasMol 2.1.

energies of the known and potential drugs.

**3.7 Calculation of differential ligand binding affinity**

#### **Table 9.**

*Interaction of the drug Praziquantel with P-glycoprotein (*Schistosoma mansoni*).*

#### **Figure 10.**

*(a) Clustering analysis of the Praziquantel-P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Schistosoma mansoni*]). (c) The interacting residues in the Praziquantel-P-glycoprotein [*Schistosoma mansoni*] docked complex are viewed using RasMol 2.1.*


#### **Table 10.**

*Interaction of the drug cladosporin with P-glycoprotein (*Trypanosoma cruzi*).*

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

*3.6.2 Molecular docking results of niclosamide with P-glycoprotein (*Onchocerca volvulus*)*

**Rank of complex Free binding energy (kcal/mol)**

1 �5.83 2 �5.51 3 �5.21 4 �4.71 5 �4.47 6 �4.15 7 �3.57 8 9.34 9 29.83 10 36.47

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

*Interaction of the drug Praziquantel with P-glycoprotein (*Schistosoma mansoni*).*

*(a) Clustering analysis of the Praziquantel-P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Schistosoma mansoni*]). (c) The interacting residues in the Praziquantel-*

**Rank of complex Free binding energy (kcal/mol)**

*P-glycoprotein [*Schistosoma mansoni*] docked complex are viewed using RasMol 2.1.*

1 �6.23 2 �6.04 3 �5.67 4 �4.81 5 �5.92 6 �5.25 7 �4.89 8 �4.83 9 �4.15 10 �3.34

*Interaction of the drug cladosporin with P-glycoprotein (*Trypanosoma cruzi*).*

**Table 9.**

**Figure 10.**

**Table 10.**

**144**

The best pose has a free binding energy of �5.29 kcal/mol (**Table 8**). The clustering figure shows the most number of conformations at �1.30 kcal/mol (**Figure 9**). **Figure 9(b)** depicts the binding site on the receptor, and **Figure 9(c)** shows the interacting residues in the niclosamide-P-glycoprotein (*Onchocerca volvulus*) docked complex viewed through RasMol 2.1.

#### *3.6.3 Molecular docking results of praziquantel with P-glycoprotein (*Schistosoma mansoni*)*

The best pose has a free binding energy of �5.83 kcal/mol (**Table 9**). The clustering figure (**Figure 10**) shows the most number of conformations at �5.0 kcal/mol. **Figure 10(b)** depicts the binding site on the receptor, and **Figure 10(c)** shows the interacting residues in the Praziquantel-P-glycoprotein (*Schistosoma mansoni*) docked complex viewed through RasMol 2.1.

#### *3.6.4 Molecular docking results of cladosporin with P-glycoprotein (*Trypanosoma cruzi*)*

The best pose has a free binding energy of �6.23 kcal/mol (**Table 10**). The clustering figure (**Figure 11**) shows the most number of conformations at �5.0 kcal/ mol. **Figure 11(b)** depicts the binding site on the receptor, and **Figure 11(c)** shows the interacting residues in the the cladosporin-P-glycoprotein (*Trypanosoma cruzi*) docked complex viewed through RasMol 2.1.

These steps were carried out for each receptor-ligand complex, and the least free binding energy of each docked complex was determined. These results are summarized in **Table 11**.

#### **3.7 Calculation of differential ligand binding affinity**

The differential affinity of the potential drug for a given efflux pump protein relative to the known drug is estimated as the difference between the binding energies of the known and potential drugs.

#### **Figure 11.**

*(a) Clustering analysis of the cladosporin-P-glycoprotein docked complex. (b) Location of the binding site on the receptor (P-glycoprotein [*Trypanosoma cruzi*]). (c) The interacting residues in the cladosporin-P-glycoprotein [*Trypanosoma cruzi*] docked complex are viewed using RasMol 2.1.*



ΔΔGinvest*:* ¼ ΔGbind,potential � ΔGbind,known

**Receptor Drug Interacting residues**

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

Arg867

Gln383, Gly384

Asn960, Lys964

Ile1058, Lys1060

*Interacting residues between the P-glycoprotein of* Onchocerca volvulus *and the chosen drugs.*

4F4C Nifurtimox Asn733, Asn734, Gln849, Asn850, Arg1056, Lys1057, Ile1058

Arg830, Ala860, Thr861, Arg867

*Interacting residues between the P-glycoprotein of* Schistosoma mansoni *and the chosen drugs.*

**Receptor Drug Interacting residues**

Ala385

Suramin ,Lys727, Lys923, Val925

Ser1036, Pro1039

**Table 14.**

**Table 15.**

**149**

4F4C Mebendazole Glu267, Thr268, Tyr271, Ala272, Gly275, Lys276, Lys315, Arg830, Ala860, Thr861, Pro864, Arg867

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

Suramin Lys720, Leu723, Ser724, Lys727, Lys923, Val925, Lys936 Diethylcarbamazine Arg918, Arg919, Phe920, Gly922, Lys923, Asn924, Gln979

Albendazole Glu36, Gly37, Asp38, Ile40, Glu267, Thr268, Tyr271, Val305, Ala308,

Ivermectin Arg172, Thr197, Phe200, Asp201, Glu204, Lys720, Lys923, Asn924, Val925, Ala928, Phe931, Ala932, Gly935, Lys936, Ile939

Praziquantel Leu11, Glu165, Lys207, Asp212, Arg918, Phe920, Lys923, Asn924, Ser927, Phe931, Ala972, Glu975, Gln979 Moxidectin Leu11, Arg12, Asp15, Lys26, Lys30, Glu33, Pro374, Gln913, Tyr914,

Niclosamide Asn4, Gly5, Ser6, Leu7, Ile48, Thr49, Val56, Lys59, Gly380, Thr381,

Ala860, Thr861, Pro864, Asn865, Arg867, Lys1043 Thiabendazole Phe504, Asn505, Cys506, Asp933, Lys936, Ile937, Glu940, Phe957,

Metrifonate Gln840, His841, Gly843, Phe844, Ser847, Gln849, Asn850, Lys1057,

Emodepside Arg172, Thr197, Phe200, Asp201, Glu204, Asp550, Val925, Ser929, Phe931, Ala932, Gly935, Lys936, Ile939

Benznidazole Asn4, Gly5, Ser6, Leu7, Thr49, Glu55, Val56, Arg205,Thr381,Gly384,

Cladosporin Tyr35, Glu36, Ile40, Glu267, Thr268, Tyr271, Ala272, Gly275, Lys315,

Tipifarnib Gly373, Asp38, Ile40, Asp41, Ser42, Asn43, Glu267, Thr268, Tyr271,

Jaspamide Gln913, Tyr914, Arg916, Gly917, Arg918, Arg919, Lys923, Gly1032, Ph1033, Thr1035, Ser1036, Phe1038, pro1039

Fexinidazole Gly37, Asp38, Ile40, Asp41, Ser42, Asn43, Thr268, Tyr271, Ala272, Gly275, Arg830, Ser856, Ala860, Thr861, Pro864, Arg867

Ravuconazole Ala910, Gln913, Tyr914, Gly917, Arg919, Gly1032, Phe1033, Thr1035,

Posaconazole Glu33, Leu161, Gly917, Arg918, Arg919, Phe920, Gly922, Lys923, Asn924, Glu975, Ala976, Gln979, Phe1033, Thr1035, Ser1036, Pro1039

Ala272, Gly275, Arg830, Ser856, Thr857, Ala860, Thr861, Arg867

Flubendazole Glu36, Gly37, Ser42, Thr268, Tyr271, Ala272, Gly275, Arg830,

Lys309, Glu823, Thr826, Arg827, Arg830, Ala860, Thr861, Pro864,

Arg916, Gly917, Gly1032, Phe1033, Thr1035, Ser1036, Pro1039

where ΔΔGinvest. = differential ligand affinity, kcal/mol;ΔGbind = free energy of binding, kcal/mol.

For each disease, the differential ligand binding affinity is calculated for every known-potential drug pair. The ΔΔGinvestiational values are given in **Table 12**. All values are expressed in kcal/mol. The drugs having ΔΔGinvest values greater than the ΔGinvest. values may have better antihelminthic activity.

All values are expressed in kcal/mol. It can be inferred from these results that many of the repurposed antiparasitic drugs show promise for treatment against other helminths. The results shown in **Table 12** serve as an indicator of which drugs may be promising antihelminthics:



#### **Table 13.**

*Interacting residues between the P-glycoprotein of* Leishmania major *and the chosen drugs.*

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*


#### **Table 14.**

ΔΔGinvest*:* ¼ ΔGbind,potential � ΔGbind,known

binding, kcal/mol.

mol)

may be promising antihelminthics:

and Nifurtimox (�6.87 kcal/mol).

**Receptor Drug Interacting residues**

where ΔΔGinvest. = differential ligand affinity, kcal/mol;ΔGbind = free energy of

For each disease, the differential ligand binding affinity is calculated for every known-potential drug pair. The ΔΔGinvestiational values are given in **Table 12**. All values are expressed in kcal/mol. The drugs having ΔΔGinvest values greater than the

All values are expressed in kcal/mol. It can be inferred from these results that many of the repurposed antiparasitic drugs show promise for treatment against other helminths. The results shown in **Table 12** serve as an indicator of which drugs

1.Leishmaniasis: Cladosporin (�7.63 kcal/mol), Jaspamide (�7.19 kcal/mol),

2.Trypanosomiasis: Cladosporin (�1.21 kcal/mol) and Tipifarnib (�0.85 kcal/

4M1M Amphotericin B Thr172, Asp173, Ser176, Ala683, Asp687, Ser876, Ala879, Leu880, Lys883,

Fluconazole Val129, Cys133, Ala136, Asn179, Glu180, Gly181, Gly183, Asp184, Lys185, Met188, Leu875, Asp882, Lys930, Phe934 Pentamidine Glu239, Leu240, Ala242, Tyr243, Ala244, Gly247, Ala248, Glu251, Arg785, Thr811, Ala815, Asn816, Ala819, Gln820 Miltefosine Asp173, Ser176, Lys177, Glu180, Lys185, Leu875, Ala879, Leu880, Lys883 Cladosporin Gln434, Leu437,Leu439, Val468, Ser470, Glu472, Val474,Asn899,

Jaspamide Leu254, Ala255, Ala256, Ile257,Arg258, Thr259,Phe800, Asn805, Thr806,

Thr807, Gly808, Leu810, Glu1115, Ile1117 Nifurtimox Ala288, Asn292, Gln769, Gly770, Phe773, Gly774, Glu778, Ala819, Gln820, Lys822, Gly823, Ser827, Phe990, Pro992 Praziquantel Leu254, Ala255, Ala256, Ile257,Arg258, Thr259, Phe800, Asn805, Thr806,

Dapsone Val474, Leu475, Phe476, Ala477, Gly521, Glu522, Lys523, Lys891,

Benznidazole Asp685, Val688, Pro689, Trp799, Asp802, Lys804, Asn805, Arg813,

Tipifarnib Ala244, Gly247, Ala248, Val249, Glu251, Glu252, Asp1120, Gly1166,

Flubendazole Phe159, Asp160, His162, Asp163, Val164, Ser470, Glu472, Val474, Ile897,

Val474, Ile897, Glu898, Asn899, Phe900, Arg901, Thr902, Ser905

Ser470, Glu472, Pro473, Val474, Leu475, Ala477, Glu522, Lys532, Glu895,

His1003, Arg1006, Ile1007, Lys1010

Gly898, Asn899, Phe900, Arg901, Thr902 Terbinafine Phe159, Val164, Gln434, Gln437, Leu439, Val468, Ser470, Glu472,

Glu898, Asn899, Arg901, Thr902 Paromomycin Val164, Glu472, Pro473, Val474, Glu522, Glu898, Asn899

*Interacting residues between the P-glycoprotein of* Leishmania major *and the chosen drugs.*

Thr894, Glu895, Glu898, Asn899, His1003, Arg1006, Ile1007, Lys1010

Lys884, Glu887, Lys996

Arg901, Thr902, Ser905

Thr807, Leu810, Ser1113

Asp1167, Lys1168

Sodium stibogluconate

**Table 13.**

**148**

ΔGinvest. values may have better antihelminthic activity.

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

*Interacting residues between the P-glycoprotein of* Onchocerca volvulus *and the chosen drugs.*


#### **Table 15.**

*Interacting residues between the P-glycoprotein of* Schistosoma mansoni *and the chosen drugs.*

3. Schistosomiasis: Cladosporin (�2.24 kcal/mol) and Jaspamide (�2.23 kcal/ mol)

1.P-glycoprotein (*Leishmania major*): **(Ser470, Glu472, Val474, Ile897,**

2.P-glycoprotein (*Onchocerca volvulus*): **(Arg830, Ala860, Thr861, Pro864,**

3.P-glycoprotein (*Schistosoma mansoni*): **(Glu267, Thr268, Tyr271, Ala272,**

4.P-glycoprotein (*Trypanosoma cruzi*): **(Arg830, Ala860, Thr861, Arg867); (Gly917, Arg918, Arg919, Phe920, Gly922, Lys923); (Phe1033, Thr1035,**

A PSI-BLAST was performed to search for P-glycoprotein homologs in *E. coli* using hPGP as the query. The top BLAST hits showed low percentage identity (< 30%) and low score and were not annotated as bacterial P-glycoprotein. Though we could not reliably ascertain P-glycoprotein homologs in *E. coli*, there exist other mechanisms that could potentially lead to multidrug resistance phenotypes in *E. coli*. Multidrug efflux systems are of five types, namely the super-families ATP Binding Cassete (ABC) and Major Facilitador Super-family (MFS), Small Multidrug Resistance (SMR), Resistance, Nodulation, Division (RND) and Multidrug and Toxic Compound Extrusion (MATE). In *E.coli*, the examples for various systems include: MFS system Bcr, EmrB and EmrD; SMR family EmrE; RND family AcrB; and Mate family YdhE9 [39]. *E. coli* contains five putative ABC-type MDR-like transporters. These systems were all cloned and expressed in a drug-sensitive *E. coli* strain, and the drug resistance phenotypes were investigated. None of these systems provided an appreciable drug resistance to *E. coli*, except for YbjYZ, which conferred resistance to erythromycin [40]. The AcrAB-TolC system of *E. coli* is one of the best-characterized MDR transporters that is responsible for the acquisition of multiple antimicrobial resistance of the *mar* mutants, including resistance to tetracycline, chloramphenicol, ampicillin, nalidixic, and rifampicin [41, 42]. *E. coli* infections could modulate the pharmacokinetics of the drug enrofloxacin by alter-

The study of the human P-glycoprotein homologs, namely the P-glycoproteins of *Leishmania major*, *Onchocerca volvulus*, *Schistosoma mansoni*, and *Trypanosoma cruzi* has provided an insight into their drug resistance mechanisms. The investigational drugs such as cladosporin, jaspamide, nifurtimox, and tipifarnib are strong contenders for novel antihelminthic treatment. Known drugs such as praziquantel and moxidectin have shown great promise for use as treatment against other

**Glu898, Asn899, Phe900, Arg901, Thr902, Ser905)**

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated…*

ing the expression of intestinal P-glycoprotein in broilers [43].

**Arg867)**

**Gly275, Lys276)**

*DOI: http://dx.doi.org/10.5772/intechopen.93175*

**Ser1036, Pro1039)**

**3.9 P-glycoprotein in** *E. coli*

**4. Conclusions**

helminthic diseases.

**151**

4.Onchocerciasis: Praziquantel (�4.81 kcal/mol) and Moxidectin (�4.18 kcal/ mol)

#### **3.8 Analysis of interacting residues in each docked complex**

The best pose of each docked complex was viewed using RasMol 2.1, and all interacting residues within a radius of 4.5 Å of the ligand were restricted and analyzed. The results are summarized in **Tables 13–16**.

The interacting residues are shown and the binding pockets found in each protein sequence with respect to different drugs are highlighted. Analysis of the interacting residues showed certain binding pockets in each efflux pump protein studied. Certain residues were found to be preferred over others, for drug binding. These preferred binding pockets are:


#### **Table 16.**

*Interacting residues between the P-glycoprotein of* Trypanosoma cruzi *and the chosen drugs.*


#### **3.9 P-glycoprotein in** *E. coli*

3. Schistosomiasis: Cladosporin (�2.24 kcal/mol) and Jaspamide (�2.23 kcal/

4.Onchocerciasis: Praziquantel (�4.81 kcal/mol) and Moxidectin (�4.18 kcal/

The best pose of each docked complex was viewed using RasMol 2.1, and all interacting residues within a radius of 4.5 Å of the ligand were restricted and

The interacting residues are shown and the binding pockets found in each protein sequence with respect to different drugs are highlighted. Analysis of the interacting residues showed certain binding pockets in each efflux pump protein studied. Certain residues were found to be preferred over others, for drug binding.

4F4C Praziquantel Asn505, Arg551, Asp933, Lys936, Ile937, Ile939, Glu940, Glu943, Asn944,

Mebendazole Arg172, Thr197, Phe200, Asp201, Glu204, Lys207, Asn924, Ala928, Phe931,

Oxamniquine Ile40, Asp41, Ser42, Thr268, Tyr271, Ala272,Gly275, Arg830, Ser856,

Albendazole Ser42, Glu267, Thr268, Tyr271, Ala272, Gly275, Lys276, Lys315, Arg830,

Cladosporin Tyr35, Glu36, Gly37, Ile40, Phe263, Ala264, Ile265, Glu267, Thr268, Tyr271, Lys315, Arg830, Thr861, Asn865, Arg867, Thr868, Glu1040, Lys1043

Jaspamide Leu161, Lys207, Glu208, Gly211, Asp212, Lys213, Gly917, Arg918, Arg919,

Niclosamide Lys26 Lys30, Ala910, Gln913, Tyr914, Arg916, Gly917, Leu1031, Gly1032,

Tipifarnib Leu161, Glu204, Lys207, Glu208, Gly211, Asp212, Lys213, Val378, Asn924 Imatinib Ser42, Asn43, Glu267, Tr268, Tyr271, Ala272, Gly275, Lys276, Arg830,

Furozan Ala910, Gln913, Tyr914, Arg916, Gly917, Arg919, Lys923, Gly1032,

Suramin Asn4, Arg8, Asp51, Glu55, Thr194, Asp201, Asn202, Arg205, Glu716,

Metrifonate Ile40, Phe263, Ala264, Glu267, Thr268, Tyr271, Ala308, Lys315, Arg830,

Nifurtimox Tyr35, Glu36, Gly37, Ile40, Phe263, Ala264, Glu267, Thr268, Lys315, Pro864,Asn865, Arg867, Thr868, Glu1040, Lys1043 Artesunate Arg8, Leu11, Arg12,Asp15, Lys26, Lys30, Leu371, Pro374, Arg916, Phe1033,

Phe920, Gly922, Lys923, Asn924, Gln979

Benznidazole Asn505, Arg551, Asp933, Lys936, Ile937, Glu940, Lys964

Thr1035, Ser1036, Phe1038, Pro1039

**3.8 Analysis of interacting residues in each docked complex**

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

analyzed. The results are summarized in **Tables 13–16**.

These preferred binding pockets are:

**Receptor Drug Interacting residues**

Lys964

Ala932, Gly935, Ile939

Ala860, Pro864, Arg867

Ala860, Arg867

Phe1033,Thr1035

Ala860, Thr861, Arg867,

Gly719, Lys720, Asp721

Ala860, Pro864, Arg867

*Interacting residues between the P-glycoprotein of* Trypanosoma cruzi *and the chosen drugs.*

**Table 16.**

**150**

Thr1035

mol)

mol)

A PSI-BLAST was performed to search for P-glycoprotein homologs in *E. coli* using hPGP as the query. The top BLAST hits showed low percentage identity (< 30%) and low score and were not annotated as bacterial P-glycoprotein. Though we could not reliably ascertain P-glycoprotein homologs in *E. coli*, there exist other mechanisms that could potentially lead to multidrug resistance phenotypes in *E. coli*. Multidrug efflux systems are of five types, namely the super-families ATP Binding Cassete (ABC) and Major Facilitador Super-family (MFS), Small Multidrug Resistance (SMR), Resistance, Nodulation, Division (RND) and Multidrug and Toxic Compound Extrusion (MATE). In *E.coli*, the examples for various systems include: MFS system Bcr, EmrB and EmrD; SMR family EmrE; RND family AcrB; and Mate family YdhE9 [39]. *E. coli* contains five putative ABC-type MDR-like transporters. These systems were all cloned and expressed in a drug-sensitive *E. coli* strain, and the drug resistance phenotypes were investigated. None of these systems provided an appreciable drug resistance to *E. coli*, except for YbjYZ, which conferred resistance to erythromycin [40]. The AcrAB-TolC system of *E. coli* is one of the best-characterized MDR transporters that is responsible for the acquisition of multiple antimicrobial resistance of the *mar* mutants, including resistance to tetracycline, chloramphenicol, ampicillin, nalidixic, and rifampicin [41, 42]. *E. coli* infections could modulate the pharmacokinetics of the drug enrofloxacin by altering the expression of intestinal P-glycoprotein in broilers [43].

#### **4. Conclusions**

The study of the human P-glycoprotein homologs, namely the P-glycoproteins of *Leishmania major*, *Onchocerca volvulus*, *Schistosoma mansoni*, and *Trypanosoma cruzi* has provided an insight into their drug resistance mechanisms. The investigational drugs such as cladosporin, jaspamide, nifurtimox, and tipifarnib are strong contenders for novel antihelminthic treatment. Known drugs such as praziquantel and moxidectin have shown great promise for use as treatment against other helminthic diseases.

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#### **Author details**

Nivedita Jaishankar<sup>1</sup> , Sangeetha Muthamilselvan<sup>2</sup> and Ashok Palaniappan<sup>2</sup> \*

1 Department of Biotechnology, Sri Venkateswara College of Engineering, Sriperumbudur, India

2 Department of Bioinformatics, School of Chemical and BioTechnology, SASTRA Deemed University, Thanjavur, India

\*Address all correspondence to: apalania@scbt.sastra.edu

© 2020 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, provided the original work is properly cited.

*Computational Studies of Drug Repurposing Targeting P-Glycoprotein-Mediated… DOI: http://dx.doi.org/10.5772/intechopen.93175*

#### **References**

[1] Nikaido H. Multidrug resistance in bacteria. Annual Review of Biochemistry. 2009;**78**:119-146. DOI: 10.1146/annurev.biochem.78.082907. 145923

[2] Handzlik J, Matys A, Kieć-Kononowicz K. Recent advances in multi-drug resistance (MDR) efflux pump inhibitors of gram-positive bacteria S. aureus. Antibiotics. 2013; **2**(1):28-45. DOI: 10.3390/ antibiotics2010028

[3] Sharma A, Gupta VK, Pathania R. Efflux pump inhibitors for bacterial pathogens: From bench to bedside. The Indian Journal of Medical Research. 2019;**149**(2):129-145. DOI: 10.4103/ ijmr.IJMR\_2079\_17

[4] Higgins CF, Callaghan R, Linton KJ, Rosenberg MF, Ford RC. Seminars in cancer biology. Structure of the multidrug resistance P-glycoprotein. 1997;**8**:135-142

[5] Amin ML. P-glycoprotein inhibition for optimal drug delivery. Drug Target Insights. 2013;**7**:27-34. DOI: 10.4137/ DTI.S12519

[6] Jin MS, Oldham ML, Zhang Q, Chen J. Crystal structure of the multidrug transporter P-glycoprotein from *C. elegans*. Nature. 2012; **490**(7421):566-569. DOI: 10.1038/ nature11448

[7] Sauna ZE, Muller MM, Kerr KM, Ambudkar SV. The mechanism of action of multidrug-resistance- linked P-glycoprotein. Journal of Bioenergetics and Biomembranes. 2001;**33**(6): 481-491. DOI: 10.1023/A: 1012875105006

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**Author details**

Nivedita Jaishankar<sup>1</sup>

Sriperumbudur, India

**152**

Deemed University, Thanjavur, India

provided the original work is properly cited.

\*Address all correspondence to: apalania@scbt.sastra.edu

, Sangeetha Muthamilselvan<sup>2</sup> and Ashok Palaniappan<sup>2</sup>

1 Department of Biotechnology, Sri Venkateswara College of Engineering,

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

2 Department of Bioinformatics, School of Chemical and BioTechnology, SASTRA

© 2020 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,

\*

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The Journal of Biological Chemistry. 2004;**279**(38):39925-39932. DOI: 10.1074/jbc.M405635200

E. Coli *Infections - Importance of Early Diagnosis and Efficient Treatment*

disease. PLoS Neglected Tropical Diseases. 2012;**6**(11):e1870. DOI: 10.1371/journal.pntd.0001870

[23] Liu J, Hajibeigi A, Ren G, Lin M, Siyambalapitiyage W, Liu Z, et al. Retention of the radiotracers 64Cu-ATSM and 64Cu-PTSM in human and murine tumors is influenced by MDR1 protein expression. Journal of Nuclear Medicine. 2009;**50**(8):1332-1339. DOI:

10.2967/jnumed.109.061879

[24] Rappa G, Lorico A, Liu MC, Kruh GD, Cory AH, Cory JG, et al. Overexpression of the multidrug

L1210 leukemia cells resistant to inhibitors of ribonucleotide reductase. Biochemical Pharmacology. 1997;**54**: 649-655. DOI: 10.1016/s0006-2952(97)

[25] Campos MCO, Castro-Pinto DB, Ribeiro GA, Berredo-Pinho MM, Gomes LHF, Bellieny MS, et al. Pglycoprotein efflux pump plays an important role in *Trypanosoma cruzi* drug resistance. Parasitology Research. 2010;**112**:2341-2351. DOI: 10.1007/

[26] Kumar A, Muthamilselvan S, Palaniappan A. Computational studies of drug repurposing targeting Pglycoprotein-mediated multidrug resistance phenotypes in priority infectious agents. In: Creatinine—a Comprehensive Update. Rijeka: Intechopen; 2020. DOI: 10.5772/

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[28] Chenna R, Sugawara H, Koike T, Lopez R, Gibson TJ, Higgins DG, et al. Multiple sequence alignment with the Clustal series of programs. Nucleic

00210-4

s00436-010-1988-6

intechopen.90745

resistance genes mdr1, mdr3 and mrp in

Bellieny MSS, Pinho RT, de Leo RMM, Seguins WS, et al. Evaluation of

thiosemicarbazones and semicarbazones as potential agents anti-*Trypanosoma cruzi*. Experimental Parasitology. 2011; **129**(4):381-387. DOI: 110.1016/j.

[17] de Silva NR, Brooker S, Hotez PJ, Montresor A, Engels D, Savioli L. Soiltransmitted helminth infections: Updating the global picture. Trends in Parasitology. 2003;**19**(12):547-551. DOI:

[16] Soares ROA, Echevarria A,

exppara.2011.08.019

10.1016/j.pt.2003.10.002

S0140-6736(13)61949-2

9781908818737.137

cbpa.2006.03.004

**154**

[18] Colley DG, Bustinduy AL, Secor WE, King CH. Human schistosomiasis. Lancet. 2014; **383**(9936):2253-2264. DOI: 10.1016/

[19] Pinto-Almeida A, Mendes T, Armada A, et al. The role of efflux pumps in *Schistosoma mansoni*

Praziquantel resistant phenotype. PLoS One. 2015;**10**(10):e0140147. DOI: 10.1371/journal.pone.0140147

[20] Cobo F. 10—Trypanosomiasis. In: Cobo F, editor. Imported Infectious Diseases. Cambridge: Woodhead Publishing; 2014. pp. 137-153. ISBN: 9781907568572. DOI: 10.1533/

[21] Maya JD, Cassels BK, Iturriaga-Vásquez P. Mode of action of natural

*Trypanosoma cruzi* and their interaction with the mammalian host. Comparative

and synthetic drugs against

Biochemistry and Physiology A Molecular and Integrative Physiology. 2007;**146**(4):601-620. DOI: 10.1016/j.

[22] Bahia MT, de Andrade IM, Martins TA, et al. Fexinidazole: A potential new drug candidate for Chagas [29] Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, et al. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Research. 2014;**42**(Web Server Issue): W252-W258. DOI: 10.1093/nar/gku340

[30] Schwede T, Kopp J, Guex N, Peitsch MC. SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Research. 2003; **31**(13):3381-3385. DOI: 10.1093/nar/ gkg520

[31] Weininger D, Weininger A, Weininger JL. SMILES 2. Algorithm for generation of unique SMILES notation. Journal of Chemical Information and Computer Sciences. 1989;**29**(2):97-101. DOI: 10.1021/ci00062a008

[32] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. Autodock4 and AutoDockTools4: Automated docking with selective receptor flexiblity. Journal of Computational Chemistry. 2009;**30**(16): 2785-2791. DOI: 10.1002/jcc.21256

[33] Sayle RA, Milner-White EJ. RASMOL: Biomolecular graphics for all. Trends in Biochemical Sciences. 1995; **20**(9):374. DOI: 10.1016/s0968-0004 (00)89080-5

[34] Messerli SM, Kasinathan RS, Morgan W, Spranger S, Greenberg RM. *Schistosoma mansoni* P-glycoprotein levels increase in response to praziquantel exposure and correlate with reduced praziquantel susceptibility. Molecular And Biochemical Parasitology. 2009;**167**(1): 54-59. DOI: 10.1016/j.molbiopara. 2009.04.007

[35] Buckner FS, Waters NC, Avery VM. Recent highlights in anti-protozoan drug development and resistance research. International Journal of

Parasitology Drugs and Drug Resistance. 2012;**2**:230-235. DOI: 10.1016/j. ijpddr.2012.05.002

[36] García MT, Lara-Corrales I, Kovarik CL, Pope E, Arenas R. Tropical skin diseases in children: A review-part II. Pediatric Dermatology. 2016;**33**: 264-274. DOI: 10.1111/pde.12778

[37] Kappagoda S, Singh SMU, Blackburn BG. Antiparasitic therapy. Mayo Clinic Proceedings. 2011;**86**(6): 561-583. DOI: 10.4065/mcp.2011.0203

[38] Varghese S, Palaniappan A. Computational pharmacogenetics of Pglycoprotein mediated antiepileptic drug resistance. The Open Bioinformatics Journal. 2018;**11**:197-207. DOI: 10.2174/1875036201811010197

[39] Moreira MAS, de Souza EC, de Moraes CA. Multidrug efflux systems in gram-negative bacteria. Brazilian Journal of Microbiology. 2004;**35**(1):2. DOI: 10.1590/S1517-8382200400 0100003

[40] Nishino K, Yamaguchi A. Analysis of a complete library of putative drug transporter genes in *Escherichia coli*. Journal of Bacteriology. 2001;**183**: 5803-5812

[41] Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE. Molecular cloning and characterization of acrA and acrE genes of *Escherichia coli*. Journal of Bacteriology. 1993;**175**: 6299-6313

[42] Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE. Genes acrA and acrB encode a stress-induced efflux system of *Escherichia coli*. Molecular Microbiology. 1995;**16**:45-55

[43] Lubelski J, Konings WN, Driessen AJM. Distribution and physiology of ABC-type transporters contributing to multidrug resistance in bacteria. Microbiology and Molecular Biology Reviews. 2007;**71**(3):463-476. DOI: 10.1128/MMBR.00001-07

Section 3

Disinfection and Antibiotic

Resistance

**157**

Section 3
