In silico Analysis of Novel Azetidinone substituted benzotriazole and benzimidazole derivatives as Plasmodium falciparum Glutamate Dehydrogenase Inhibitors

Sandip N. Badeliya¹*, Pankaj P. Kapupara², Ankit B. Chaudhary³

¹Research Scholar, Faculty of Pharmacy, RK University, Rajkot, Gujarat, India.

²Department of Pharmaceutical Chemistry, School of Pharmacy, R K University, Rajkot, Gujarat, India.

³Departmet of QA and Chemistry, Saraswati Institute of Pharmaceutical Sciences, Dhanap,

Di. Gandhinagar, Gujarat, India.

*Corresponding Author E-mail: snb.success@gmail.com, pankaj.kapupara@rku.ac.in, ankitbchaudhary@gmail.com

ABSTRACT:

NADP-dependent enzyme Glutamate dehydrogenase is responsible for the maintenance of reduced state in plasmodia. Chloroquine and Mefloquine inhibit glutamate dehydrogenase enzyme and also glutathione reductase like antioxidative enzyme and thioredoxin, inducing oxidative stress. Plasmodia can't survive in the highly oxidized medium. From a detailed study on the SAR of quinolines, a series of compounds were designed and developed using molecular docking, In silico analysis was done using SWISSADME online tool, and bioactivity prediction was performed using Molinspiration online tool. Among the all designed compounds, in the benzotriazole series, compound code 1(d) (-103.22kcal/mol), 1(e) (-102.05kcal/mol), and 1(b) (-100.78 kcal/mol) show good binding affinity. Whereas, in the benzimidazole series, compound code 2(f) (-104.98 kcal/mol), 2(b) (-104.86kcal/mol) and 2(g) (-104.08kcal/mol) shows good binding affinity. The performed research reveals that benzimidazole derivatives offer an advantage over benzotriazole moiety for binding affinity with the enzyme Plasmodium Falciparum glutamate dehydrogenase.

KEYWORDS: Glutamate dehydrogenase, in silico, Chloroquine, Mefloquine, NADP.

INTRODUCTION:

Glutamate dehydrogenase, an NADP-dependent enzyme can be found in a large number of microbes and mitochondria of eukaryotes. Glutamate dehydrogenase, present in Plasmodium Falciparum is responsible for the synthesis of α-ketoglutarate as well as ammonia and reduces NADP in NADPH.^1,2 Reduced NADP provides electrons to glutathione reductase and thioredoxin-like antioxidative enzymes. Enzyme glutathione reductase carries out the reduction of glutathione disulfide and converts into glutathione.^3,4 Formed glutathione reductase prevents oxidative stress and provides an antioxidative (reduced) environment to the Plasmodium Falciparum.

Thioredoxin, an oxidoreductase enzyme, possesses a molecular weight around 12 kiloDalton. This possesses radical scavenging activity and performs a major role to prevent oxidative stress. This will result in a reduced environment that is the primary requirement for the survival of Plasmodium Falciparum.^5,6,7

Glutamate dehydrogenase inhibitors:

Glutamate dehydrogenase inhibitors compete with NADP and at the active site, it binds with the enzyme glutamate dehydrogenase. They inhibit the conversion of NADP into NADPH and inhibit the catalysis. As a result, glutathione reductase and thioredoxin-like antioxidative enzymes cannot get electron for reduction. Plasmodium Falciparum fails to survive due to the induction of oxidative stress. Due to the absence of glutamate dehydrogenase in the host erythrocyte, this is a target of interest for the design of newer antimalarial agents.^8,9

Chloroquine and chloroquine phosphate are 4-amino quinoline contains 4-amino quinoline nucleus that can be useful in prophylaxis and treatment of malaria. Chloroquine forms complex with heme (Ferriprotoporphyrin IX), which is toxic to the parasite. The formed complex is referred to as chloroquine-heme complex. Hence, heme cannot be converted into hemozoin (nontoxic to the parasite) and malarial parasite cannot survive. Chloroquine has many side effects like nausea, vomiting, abdominal cramp, headache, loss of appetite, loss of hearing, blurred vision, diarrhea, skin color change, hair loss, weight loss, seizures. Also, this drug cannot be given during pregnancy. Mefloquine, 4-aminoquinoline derivative, is available in the market as R,S-enantiomer. It contains trifluoromethyl moieties. It enhances pH in vesicles of malarial parasite and interferes with the heme formation. Mefloquine has side effects like exacerbate mental disorders hence it cannot be given to the patient with active depression, psychosis, anxiety, schizophrenia, and seizures.^10,11

Figure 1. Design of novel Azetidinone substituted benzimidazole and benzotriazole derivatives as Plasmodium Falciparum Glutamate Dehydrogenase Inhibitors

A detailed quinoline structure-activity relationship shows that the quinoline ring gives activity. At the 4^th position of quinoline, a side chain having 2 to 5 carbon atoms should be attached between two nitrogen atoms, and at the terminal end, tertiary nitrogen must be present for the antimalarial activity. In the side chain, the aromatic ring enhances the activity and the heterocyclic ring reduces toxicity. All these parameters were kept in mind for the designing of new molecules. Figure 1 reveals a general designed newer 1-Azetidine substituted benzotriazole and benzimidazole derivatives that are having all the requirements. It was one attempt made, replacement of quinoline moiety with benzotriazole and benzimidazole nucleus. The designed compounds contain one to two carbon atom side chain between two nitrogen atoms, one to two phenyl ring (one inside the chain and one on azetidine ring), and terminal tertiary nitrogen as azetidine ring.^12,13,14

To date, no compound has been invented, that can inhibit the glutamate dehydrogenase and glutathione reductase. Also glutamate dehydrogenase and glutathione reductase both have different folds than that present in the mammals. Hence, these enzymes can be a key target for the design and development of novel antimalarial agents.

MATERIAL AND METHODS:

Protein Preparation:

Crystallographic structure from Protein Data Bank, of the target protein (PDB ID: 2BMA) was prepared and saved in the standard 3D coordinate format.¹⁵  The energy states and conformations of added hydrogens were fixed and corrected by energy minimization using YASARA energy minimization server.

Molecular Docking:

Docking is a computational technique that evaluates the preferred orientation of one molecule to the target protein binding site. It can be predicted when they bound with each other to form a stable complex. Molecular docking was performed using iGEMDOCK version 2.1-1 between protein (enzyme) and inhibitor (ligand), that confers binding orientation in kcal/mol. Population size of 200 and the number of generations 2 were taken to generate accurate settings in docking. To refine the proteins, water molecules were removed and polar hydrogen, as well as Kollmann charges, was added. The protein-ligand complex was subjected to 2.5 million evaluations. The binding energies were analyzed with the docking score of the reference ligand structure chloroquine and mefloquine. Table 1 reveals the binding affinity of designed newer Plasmodium Falciparum glutamate dehydrogenase inhibitors.^{16,17,18,19,20,21}

Table 1. The binding affinity of designed novel Plasmodium Falciparum glutamate dehydrogenase inhibitors [^#Chq= Chloroquine (Standard 1), ^#Mfq= Mefloquine (Standard 2)].

Sr. No.	Compound Code	Chemical name of designed compounds	Binding energy score (Kcal/mol)
1	^#Chq	7-Chloro-N-(5-(diethylamino)pentan-2-yl)quinolin-4-amine	-86.26
2	^#Mfq	(2,8-Bis(trifluromethyl)quinolin-4-yl)(piperidin-2-yl)methanol	-93.19
3	1(a)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-oxo-4-phenylazetidin-1-yl)acetamide	-92.8
4	1(b)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(4-methoxyphenyl)-4-oxoazetidin-1-yl)acetamide	-100.78
5	1(c)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(2-chlorophenyl)-4-oxoazetidin-1-yl)acetamide	-98.43
6	1(d)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(3-nitrophenyl)-4-oxoazetidin-1-yl)acetamide	-103.22
7	1(e)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(2-hydroxyphenyl)-4-oxoazetidin-1-yl)acetamide	-102.05
8	1(f)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(2-nitrophenyl)-4-oxoazetidin-1-yl)acetamide	-94.8
9	1(g)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(2-methoxyphenyl)-4-oxoazetidin-1-yl)acetamide	-98.56
10	1(h)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(4-chlorophenyl)-4-oxoazetidin-1-yl)acetamide	-95.34
11	1(i)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(2-bromophenyl)-4-oxoazetidin-1-yl)acetamide	-92.81
12	1(j)	2-(1-H-Benzo[d][1,2,3]triazol-1-yl)-N-(3-chloro-2-(4-bromophenyl)-4-oxoazetidin-1-yl)acetamide	-90.51
13	2(a)	1-((1-H-Benzo[d]imidazol-1yl)(phenyl)methyleneamino)-3-chloro-4-phenylazetidin-2-one	-99.86
14	2(b)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(4-methoxyphenyl)azetidin-2-one	-104.86
15	2(c)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(2-chlorophenyl)azetidin-2-one	-94.79
16	2(d)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(3-nitrophenyl)azetidin-2-one	-100.67
17	2(e)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(2-hydroxyphenyl)azetidin-2-one	-103.59
18	2(f)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(2-nitrophenyl)azetidin-2-one	-104.98
19	2(g)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(2-methoxyphenyl)azetidin-2-one	-104.08
20	2(h)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(4-chlorophenyl)azetidin-2-one	-87.45
21	2(i)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(2-bromophenyl)azetidin-2-one	-103.5
22	2(j)	1-((1-H-Benzo[d]imidazol-1-yl)(phenyl)methyleneamino)-3-chloro-4-(4-bromophenyl)azetidin-2-one	-100.56

Table 2. In silico toxicity studies of designed compounds [^#Chq= Chloroquine (Standard 1), ^#Mfq= Mefloquine (Standard 2)]

Sr.No	Sample Code	H-Bond Donor	H-Bond Acceptor	Log Po/w (iLOGP)	MW (g/mol)	CYP1A2 inhibitor	CYP2C19 inhibitor	CYP2C9 inhibitor	CYP2D6 inhibitor	CYP3A4 inhibitor	BBB Penetration
1	^#Chq	1	2	3.95	319.87	Yes	No	No	Yes	Yes	Yes
2	^#Mfq	2	9	2.78	378.31	No	No	No	Yes	Yes	No
3	1(a)	1	4	2.19	355.78	No	Yes	No	No	No	No
4	1(b)	1	5	2.51	385.80	No	Yes	No	No	No	No
5	1(c)	1	4	1.96	390.22	No	Yes	Yes	No	No	No
6	1(d)	2	6	-4.03	401.78	Yes	Yes	No	No	No	No
7	1(e)	2	5	1.83	371.78	No	Yes	No	No	No	No
8	1(f)	2	6	-3.55	401.78	Yes	Yes	No	No	No	No
9	1(g)	1	5	2.18	385.80	No	Yes	No	No	No	No
10	1(h)	1	4	2.51	390.22	No	Yes	Yes	No	No	No
11	1(i)	1	4	2.31	434.67	No	Yes	Yes	No	No	No
12	1(j)	1	4	2.01	434.67	No	Yes	No	No	No	No
13	2(a)	0	3	2.74	400.86	No	Yes	Yes	No	No	Yes
14	2(b)	0	4	3.08	430.89	No	Yes	Yes	No	No	Yes
15	2(c)	0	3	2.94	435.31	No	Yes	Yes	No	No	Yes
16	2(d)	1	5	-3.14	446.87	No	Yes	No	No	No	No
17	2(e)	1	4	2.43	416.86	No	Yes	Yes	No	No	Yes
18	2(f)	1	5	-4.03	446.87	No	Yes	No	No	No	No
19	2(g)	0	4	2.94	430.89	No	Yes	Yes	No	No	Yes
20	2(h)	0	3	2.85	435.31	No	Yes	Yes	No	No	Yes
21	2(i)	0	3	2.81	479.76	No	Yes	Yes	No	No	Yes
22	2(j)	0	3	3.01	479.76	No	Yes	Yes	No	No	Yes

Table 3. Bioavailability score of designed compounds [^#Chq= Chloroquine (Standard 1), ^#Mfq= Mefloquine (Standard 2)]

Sr. No.	Compound Code	GPCR Ligand	Ion Channel Modulator	Kinase Inhibitor	Nuclear Receptor Ligand	Protease Inhibitor	Glutamate Dehydrogenase Inhibitor
1	^#Chq	0.32	0.32	0.38	-0.19	0.05	0.11
2	^#Mfq	0.45	0.21	-0.05	0.30	0.36	0.21
3	1(a)	-0.26	-0.70	-0.40	-0.69	-0.23	-0.36
4	1(b)	-0.28	-0.72	-0.41	-0.66	-0.28	-0.39
5	1(c)	-0.27	-0.71	-0.51	-0.66	-0.32	-0.42
6	1(d)	-0.32	-0.41	-0.40	-0.42	-0.36	-0.30
7	1(e)	-0.26	-0.66	-0.40	-0.58	-0.24	-0.32
8	1(f)	-0.29	-0.37	-0.48	-0.38	-0.38	-0.28
9	1(g)	-0.29	-0.71	-0.45	-0.66	-0.30	-0.39
10	1(h)	-0.25	-0.68	-0.41	-0.69	-0.26	-0.38
11	1(i)	-0.36	-0.81	-0.51	-0.74	-0.41	-0.43
12	1(j)	-0.34	-0.74	-0.43	-0.78	-0.34	-0.42
13	2(a)	-0.05	-0.15	-0.10	-0.22	-0.08	-0.07
14	2(b)	-0.09	-0.20	-0.13	-0.22	-0.13	-0.11
15	2(c)	-0.07	-0.17	-0.20	-0.21	-0.17	-0.13
16	2(d)	-0.18	-0.19	-0.20	-0.29	-0.20	-0.17
17	2(e)	-0.06	-0.13	-0.11	-0.14	-0.09	-0.04
18	2(f)	-0.16	-0.14	-0.29	-0.26	-0.23	-0.16
19	2(g)	-0.10	-0.19	-0.16	-0.22	-0.15	-0.12
20	2(h)	-0.05	-0.15	-0.11	-0.24	-0.11	-0.10
21	2(i)	-0.14	-0.27	-0.20	-0.28	-0.24	-0.14
22	2(j)	-0.13	-0.20	-0.14	-0.31	-0.18	-0.13

In silico ADME/T properties:

The designed compounds were analyzed for in silico toxicity study by the SWISSADME program. A drug molecule, to be effective, it must reach with appropriate concentration at the target in the body. Likewise, it should remain in the body for enough time so that regular biological events can be proceeding. Drug design and development particularly involve assessment of absorption, distribution, metabolism, and excretion (ADME) at the earliest but the compounds are numerous approaches to the physical samples that are limited. In such a situation, the computer models and software perform a dominant role as an option to this approach. Table 2 reveals in silico toxicity studies of novel Plasmodium Falciparum glutamate dehydrogenase inhibitors.

Bioactivity Prediction:

Bioactivity prediction via computational approach to predict biological activity of designed compounds was performed by the Molinspiration online software tool. If the bioactivity score is more than 0 then the drug molecule is active, if in between -5 to 0 then moderately active and if less than -5 then the drug molecule is inactive. Table 3 reveals the bioactivity scores of the designed compounds.^22-26

RESULTS AND DISCUSSION:

In oxidative stress, Plasmodia cannot survive. NADP-dependent Plasmodium Falciparum glutamate dehydrogenase enzyme is responsible for the production of NADPH in the parasite. The formed NADPH serves as an electron source for glutathione reductase and thioredoxin reductase like antioxidative enzymes. This will lead to the suppression of the oxidized state in plasmodia and make it difficult to survive. Furthermore, glutamate dehydrogenase is not present in the host erythrocyte so it can be a key target for the design of some potential antimalarial drug candidates. After the docking studies, it was observed that novel 1-azetidinone substituted benzotriazole and benzimidazole derivatives scaffold performed a dominant role on Plasmodium Falciparum glutamate dehydrogenase enzyme. Among all designed compounds, in benzotriazole series, compound code 1(f) containing nitro group at the ortho position of the benzene ring on azetidine nucleus is having enzyme inhibitor activity -0.28 shows the most significant activity. It binds with SER-391, HIS-392, GLU-404, ASN-408, HIS-392, TRP-393, TRP-393, LEU-119, GLU-404, ILE-405. Whereas, compound code 1(i) containing bromo group at the ortho position of a benzene ring on azetidine nucleus is having enzyme inhibitor activity -0.43shows less significant activity. In the case of benzimidazole series, compound code 2(e) containing hydroxy group at the ortho position of a benzene ring on the azetidine nucleus is having enzyme inhibitor activity -0.04 shows the most significant activity. It binds with SER-260, GLY-237, SER-260, ASP-261, SER-262, GLU-307, ALA-321. Whereas, compound code 2(d) containing nitro group at meta position of a benzene ring on azetidine nucleus is having enzyme inhibitor activity -0.17 shows less significant activity. Figure 2 indicates the binding of compound 2(d) and 1(i) with the enzyme glutamate dehydrogenase enzyme. After the bioactivity prediction, it can be concluded that all the designed compounds are moderately active (activity between -5.0 to 0). Bioactivity of designed compounds in decreasing order is as under:

Mefloquine (Std 2) > Chloroquine (Std 1)> 2(e) > 2(a) > 2(h) > 2(b) > 2(g)> 2(c), 2(j) > 2(i) > 2(f) > 2(d)> 1(f) > 1(d) > 1(e) > 1(a) > 1(h) > 1(b), 1(g) > 1(c) > 1(j) > 1(i)

Figure 2. Binding of compound 2(d) with enzyme glutamate dehydrogenase

Glutamate dehydrogenase enzyme is found to be a key target for the design and development of newer antimalarial candidates. Molecular modeling, molecular docking, and virtual screening play a dominant role in drug discovery and drug development. 1-Azetidinone substituted benzotriazole and benzimidazole scaffold is a putative pharmacophore needed for inhibition of NADP-dependent Plasmodium Falciparum glutamate dehydrogenase enzyme. All the designed compounds show moderate activity. All the compounds of the benzotriazole series follow the Lipinski rule of five without any violation whereas all the compounds of the benzimidazole series follow the Lipinski rule of five with one violation (MLOGP> 4.15). So, in the future, compounds of both benzotriazole and benzimidazole series can be utilized to prepare orally bioavailable drugs.

List of Abbreviations:

NADP     = Nicotinamide adenine nucleotide phosphate

NADPH                 = Reduced Nicotinamide adenine nucleotide phosphate

PDB                        = Protein Drug Binding

ADME/T               =Absorption, Distribution, Metabolism, Excretion, Toxicity

BBB                       = Blood brain barrier

MW                        = Molecular weight

GPCR                     = G-Protein couple receptor

RMSD                    = Root mean square deviation

ARG                       = Arginine

ASN                        = Asparagine

ALA                       = Alanine

SER                        = Serine

LYS                        = Lysine

PHE                        = Phenylalanine

LEU                        = Leucine

PRO                        = Proline

GLU                       = Glutamine

GLN                       = Glycine

ASP                        = Aspartic acid

ILE                         = Isoleucine

HIS                         = Histidine

TRP                        = Tryptophan

ACKNOWLEDGEMENT:

We are thankful to Dr Pankaj Kapupara (Professor, School of Pharmacy, R K University, Rajkot) and Dr Ankit B. Chaudhary (Professor and Head, Saraswati Institute of Pharmaceutical Sciences) for providing his bizarre proficiency and expertise towards this research work.

CONFLICT OF INTEREST:

There is no any conflict of interest.

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Received on 18.02.2021            Modified on 10.04.2021

Accepted on 16.06.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2022; 15(4):1431-1436.

DOI: 10.52711/0974-360X.2022.00237