INTRODUCTION:

One of the current significant approaches for the discovery of new antibacterial drugs and thereby solving the global problem of antimicrobial resistance is called "repurposing old drugs". This approach involves the investigation of the antibacterial activities of non-antibiotic drugs (NADs) which were initially approved for non-infectious diseases but can be repurposed as new antibacterial drugs^1,2. Amlodipine {3-Ethyl 5-methyl (±)-2-[(2-aminoethoxy) methyl]-4-(o-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridinedicarboxylate}³ is an oral calcium channel blocker (CCB) clinically used for treatment of hypertension and angina⁴.

It is one of these NADs that have been the concern of many studies that focused on the relevant approach. The drug was reported to possess in vitro activity alone and/or in combination with antibiotics against a wide-spectrum of pathogenic bacteria, such as Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp, Listeria monocytogenes and multidrug-resistant Acinetobacter baumanii^5-8. The main obstacle that hinders the repurposing of amlodipine to a new systemic antibacterial drug is its expected adverse actions on cardiovascular system due to its action as CCB. Therefore, the aim of this study was to design and evaluate novel structural analogs of amlodipine that might have similar or higher antibacterial activity than amlodipine but with diminished activity as CCBs.

MATERIALS AND METHODS:

Materials:

All materials were obtained from computational and data retrieval techniques including softwares, web servers and web databases as described later. The chemoinformatics work employed in this study was performed on a laptop "Toshiba/Japan, model: Tecra C660-A236 with Intel Arc A770 Limited Edition graphics card, Core 15 processor, and 16 GB RAM.

Prediction of amlodipine bacterial target:

The prediction was performed using a machine learning web server followed by virtual molecular docking.

a) SuperPred 3:

SuperPred 3 is a web server operated by the Institute of Physiology at Charité - Universitaetsmedizin Berlin, Germany⁹. It is a machine learning model that uses logistic regression and Morgan fingerprints of length 2048. The results of the prediction calculations for each molecule tested were the probability values (P %), for each potential target, that estimate the probability that the entered molecule binds to the targets, leading to the prediction of a corresponding therapeutic indication^9, ¹⁰. In the present study, only the targets associated with the indication "bacterial infection" were selected. The probability and accuracy of model prediction for each selected target were at least 50% and 95%, respectively.

b) Molecular Docking:

Virtual molecular docking of amlodipine to the bacterial targets predicted by "SuperPred 3" was performed not only to confirm the prediction of the web server, but also to select the most potent target among the targets predicted by the web server. In addition, the results of docking amlodipine to each predicted target protein were compared with those of a reference ligand for the target. Docking was performed using Molecular Operating Environment (MOE)^11-13 (version 2022.02) s which is a software developed by Chemical Computing Group Inc. Canada. A series of procedures were performed prior to docking. First, the structures of the bacterial target proteins predicted by "SuperPred" were downloaded as "pdb" from the UniProt protein database¹⁴. Second, the structure of the ligand "amlodipine" was created in "smiles" format using ChemDraw^® version 12, which is software developed by Cambridge soft corporation, USA ¹⁵, and then converted to "mol" format using “Open Babel” version 3.1.1 software¹⁶. Third, the binding site at the target protein was determined using the "site finder" tool of the software MOE, which ranks the potential binding sites according to their propensity for ligand binding (PLB) and uses a methodology based on alpha shapes, which are a generalization of the convex hulls developed in the software. Later, the structures of the ligand "amlodipine" and the protein were corrected and 3D-protonated using the "structure preparation" tool in the software. After these pre-docking procedures, the docking parameters were set to the standard accurate "rigid receptor" protocol in MOE and docking was initiated. Post-docking analysis performed by the software provided a number of data points. However, the final score (s) was the most meaningful. The score predicted the total binding energy in kcal/mol required for the best position of the molecule to bind to the binding site residues. Another parameter provided by the software was "rmsd_ refine," which gave the squared deviation between the molecular position before and after refinement. As recommended by the software, the molecule with the best pose (i.e., the pose with the greatest binding affinity to the receptor residues) was the one with the highest negative value (S), where rmsd_ refine ≤ 2. Post-docking analysis also provided a detailed description of the modes of protein-ligand interactions (PLIs), of which only those with a maximum distance of 4.5 Å were considered^11-13. In addition, the protein-ligand complex was prepared using the software MOE and the 3D visualization of the complex was prepared using the software "UCSF Chimera, alpha version, 2023, developed by the Regents of the College of California¹⁷.

Design and novelty of amlodipine structural analogs:

Eighty-five structural analogs of amlodipine were designed by substituting one or more of the pharmacophore moieties on the 1, 4-dihydropyridine nucleus of the drug. These chemical moieties, designated G1, G2, G3, G4, G5, and G6 as shown in Figure 1, were selected because they are associated with the drug's activity as a calcium channel blocker (CCB)¹⁸. Based on the numbering on the dihydropyridine nucleus, the relevant moieties included: H atom in conjunction with the N atom of the dihydropyridine ring, 2-[(2- aminoethoxy) methyl], 3-(ethyl carboxylate), 4-(o-chlorophenyl), 5-(methyl carboxylate), and 6-methyl. The groups on the designed structural analogs that substituted these moieties are demonstrated in (Table 1). The chemical structures of the analogs were drawn, checked for errors, and converted to "smiles" using ChemDraw® software as described earlier for amlodipine. The novelty of each designed analog was confirmed using search tools from three reliable chemical web databases, including the similarity search at “ChemSpider” of the Royal Society of Chemistry, UK¹⁹, SciFinder at the Chemical Abstract Service (CIS) of the American Chemical Society, USA²⁰, and the search service at “PubChem” of the National Institutes of Health (NIH), USA²¹.

Table 1: Chemical moieties, at the structures of amlodipine structural analogs, substituted the pharmacophore moieties (G1-G6) of amlodipine.

Code	G1	G2	G3	G4	G5	G6
D001	- H	-Me-O-Et-NH₂	-(C=O)H	o-Cl-Phe-	H(O=C)-	Me-
D002	- H	-Me-O-Et-NH₂	-COOH	o-Cl-Phe-	HOOC-	Me-
D003	- H	-Me-O-Et-NH₂	-(C=O)-Me	o-Cl-Phe-	Me-O(O=C)-	Me-
D004	- H	-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-	Pr-O(O=C)-	Me-
D005	-cycloPr	-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-	Me-O(O=C)-	Me-
D006	-Phe	-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-	Me-O(O=C)-	Me-
D007	-Pr	-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-	Me-O(O=C)-	Me-
D008	- Me	-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-	Me-O(O=C)-	Me-
D009	- H	-Me-O-Et-NH₂	-(C=O)-(CH-SH) –Et	o-Cl-Phe-	SH-Me(O=C)-	Me-
D010	- H	-Me-O-Et-NH₂	-(C=O)-S-Et	o-Cl-Phe-	Me-S(O=C)-	Me-
D011	-cycloPr	-Me-O-Et-NH₂	-COOH	o-Cl-Phe-	HOOC-	Me-
D012	-cycloPr	-Me-O-Et-NH₂	-(C=O)H	o-Cl-Phe-	H(O=C)-	Me-
D013	-cycloPr	-Me-O-Et-NH₂	-(C=O)-Me	o-Cl-Phe-	Me-O(O=C)-	Me-
D014	-cycloPr	-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-	Et(O=C)-	Me-
D015	-Me	-Me-O-Et-NH₂	-(C=O)H	o-Cl-Phe-	H(O=C)-	Me-
D016	-Me	-Me-O-Et-NH₂	-COOH	o-Cl-Phe-	HOOC-	Me-
D017	-Me	-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-	Et(O=C)-	Me-
D018	-Me	-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-	Me(O=C)-	Me-
D019	-cycloPr	-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	o-Cl-Phe-	SH-Me(O=C)-	Me-
D020	-Me	-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	o-Cl-Phe-	SH-Me(O=C)-	Me-
D021	-cycloPr	-Me-O-Et-NH₂	-(C=O) –S –Et	o-Cl-Phe-	Me-S(O=C)-	Me-
D022	-Me	-Me-O-Et-NH₂	-(C=O) –S –Et	o-Cl-Phe-	Me-S(O=C)-	Me-
D023	-Pr	-Me-O-Et-NH₂	-(C=O)-O-Me	o-Cl-Phe-	- Me-O(O=C)-	Me-

Table 1: continue

Code	G1	G2		G3		G4	G5		G6
D024	-Phe		-Me-O-Et-NH₂	-(C=O)H	o-Cl-Phe-			H(O=C)-	Me-
D025	-Phe		-Me-O-Et-NH₂	-COOH	o-Cl-Phe-			HOOC-	Me-
D026	-Phe		-Me-O-Et-NH₂	-(C=O)-O-Me	o-Cl-Phe-			- Me-O(O=C)-	Me-
D027	-Pr		-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-			Et(O=C)-	Me-
D028	-Pr		-Me-O-Et-NH₂	-(C=O)H	o-Cl-Phe-			H(O=C)-	Me-
D029	-Pr		-Me-O-Et-NH₂	-COOH	o-Cl-Phe-			HOOC-	Me-
D030	-Pr		-Me-O-Et-NH₂	-(C=O)-Pr	o-Cl-Phe-			Et(O=C)-	Me-
D031	-Phe		-Me-O-Et-NH₂	-(C=O)-S-Et	o-Cl-Phe-			Me-S(O=C)-	Me-
D032	-Pr		-Me-O-Et-NH₂	-(C=O) –(CH-SH)-Et	o-Cl-Phe-			SH-Me(O=C)-	Me-
D033	-Pr		-Me-O-Et-NH₂	-(C=O)-S-Et	o-Cl-Phe-			Me-S(O=C)-	Me-
D034	-Phe		-Me-O-Et-NH₂	-(C=O) –(CH-SH)-Et	o-Cl-Phe-			SH-Me(O=C)-	Me-
D035	-H		-Me-O-Et-NH₂	-(C=O)H	H-			H(O=C)-	Me-
D036	-H		-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	H-			SH-Me(O=C)-	Me-
D037	-H		-Me-O-Et-NH₂	-(C=O)-S-Et	H-			Me-S(O=C)-	Me-
D038	-H		-Me-O-Et-NH₂	-(C=O)-O-Et	H-			Me-O(O=C)-	Me-
D039	-Phe		-Me-O-Et-NH₂	-(C=O)-O-Et	H-			Me-O(O=C)-	Me-
D040	-Me		-Me-O-Et-NH₂	-(C=O)-O-Et	H-			Me-O(O=C)-	Me-
D041	-cycloPr		-Me-O-Et-NH₂	-(C=O)-O-Et	H-			Me-O(O=C)-	Me-
D042	-H		-Me-O-Et-NH₂	-(C=O)-Pr	H-			Et(O=C)-	Me-
D043	-H		-Me-O-Et-NH₂	-(C=O)-Me	H-			Me(O=C)-	Me-
D044	-H		-Me-O-Et-NH₂	-COOH	H-			HOOC-	Me-
D045	-Me		-Me-O-Et-NH₂	-(C=O)-S-Et	H-			Me-S(O=C)-	Me-
D046	-Me		-Me-O-Et-NH₂	-COOH	H-			HOOC-	Me-
D047	-Me		-Me-O-Et-NH₂	-(C=O)H	H-			H(O=C)-	Me-
D048	-Me		-Me-O-Et-NH₂	-(C=O)-Me	H-			Me(O=C)-	Me-
D049	-H		-Me-O-Et-NH₂	-(C=O)-Me	H-			H(O=C)-	H-
D050	-Me		-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	H-			SH-Me(O=C)-	Me-
D051	-cycloPr		-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	H-			SH-Me(O=C)-	Me-
D052	-cycloPr		-Me-O-Et-NH₂	-(C=O)-Pr	H-			Et(O=C)-	Me-
D053	-cycloPr		-Me-O-Et-NH₂	-COOH	H-			HOOC-	Me-
D054	-cycloPr		-Me-O-Et-NH₂	-(C=O)-S-Et	H-			Me-S(O=C)-	Me-
D055	-cycloPr		-Me-O-Et-NH₂	-(C=O)H	H-			H(O=C)-	Me-
D056	-Me		-Me-O-Et-NH₂	-(C=O)-Pr	H-			Et(O=C)-	Me-
D057	-cycloPr		-Me-O-Et-NH₂	-(C=O)-Me	H-			Me(O=C)-	Me-
D058	-Phe		-Me-O-Et-NH₂	-(C=O)H	H-			H(O=C)-	Me-
D059	-Phe		-Me-O-Et-NH₂	-COOH	H-			HOOC-	Me-
D060	-Phe		-Me-O-Et-NH₂	-(C=O)-Me	H-			Me(O=C)-	Me-
D061	-Phe		-Me-O-Et-NH₂	-(C=O)-Pr	H-			Et(O=C)-	Me-
D062	-Pr		-Me-O-Et-NH₂	-(C=O)H	H-			H(O=C)-	Me-
D063	-Pr		-Me-O-Et-NH₂	-COOH	H-			HOOC-	Me-
D064	-Pr		-Me-O-Et-NH₂	-(C=O)-Me	H-			Me(O=C)-	Me-
D065	-Pr		-Me-O-Et-NH₂	-(C=O)-Pr	H-			Et(O=C)-	Me-
D066	-Phe		-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	H-			SH-Me(O=C)-	Me-
D067	-Phe		-Me-O-Et-NH₂	-(C=O)-S-Et	H-			Me-S(O=C)-	Me-
D068	-Pr		-Me-O-Et-NH₂	-(C=O)-(CH-SH)-Et	H-			SH-Me(O=C)-	Me-
D069	-Pr		-Me-O-Et-NH₂	-(C=O)-S-Et	H-			Me-S( O=C)-	Me-
D070	-H		-Me-O-Et-NH₂	-(C=O)-O-Et	m-Cl-Phe-			Me-O(O=C)-	Me-
D071	-H		-Me-O-Et-NH₂	-(C=O)-O-Et	p-Cl-Phe-			-Me-O(O=C)-	Me-
D072	-Pr		-Me-O-Et-NH₂	-(C=O)-O-Et	m-Cl-Phe-			Me-OO=C)-	Me-
D073	-Pr		-Me-O-Et-NH₂	-(C=O)-O-Et	p-Cl-Phe-			Me-O( O=C)-	Me-
D074	-Bu		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D075	-Bu		-Me-O-Et-NH₂	-(C=O)-O-IsoPr	o-Cl-Phe-			Et-O-(C=O)-	Me-
D076	-Bu		-Me-O-Et-NH₂	-COOH	o-Cl-Phe-			HOOC-	Me-
D077	-Bu		-Me-O-Et-NH₂	-(C=O)-O-IsoPr	o-Cl-Phe-			Et-O-(C=O)-	H-
D078	-IsoPr		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D079	-IsoBu		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D080	-(C=O)H		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D081	-NH2		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D082	-CH2-COOH		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D083	-CH2-(C=O) CH3		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D084	-IsoPe		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-
D085	-cycloHe		-Me-O-Et-NH₂	-(C=O)-O-Et	o-Cl-Phe-			Me-O(O=C)-	Me-

o-Cl-Phe: o-chlorophenyl; m-Cl-Phe: m-chlorophenyl; p-Cl-Phe: p-chlorophenyl; Bu: butyl; cycloHe: Cyclohexyl; cycloPr: cyclopropyl; Et: ethyl; IsoPe: Isopentyl; IsoPr: isopropyl; Me: methyl; Phe: phenyl; Pr: propyl

Figure 2: 3D structures of proteins downloaded from the protein databases (I: Bacterial Acyl CoA desaturase¹⁴ ^(a); II:^{Bacterial DNA topoisomerase I 14(b); III: Human DNA
topoisomerase I 14(c); IV: Ca}V^{Ab: Calcium ion transporter protein24).}

Table 2: SuperPred 3 probability and molecular docking results for predicting the bacterial target of amlodipine

Ligand	SuperPred (P %)			Molecular Docking; s (kcal/mol)
Ligand	BAD	BDT1	HDT1	BAD	BDT1	HDT1
Amlodipine	53.54	51.59	51.59	- 5.480	- 6.366	- 4.532
Acyl CoA ^▲	65.95	52	52	-8.607	n.t	n.t
N,N-dimethyl(phenanthro]3,4-d][dioxol-5-yl) ethanamine ^	n.r	66.86	66.86	n.t	-4.739	-5.522

(P %): prediction probability percentage; BAD: Bacterial acyl CoA desaturase; BDT1: Bacterial DNA topoisomerase I; HDT1: Human DNA topoisomerase I; n.t: not tested; n.r: no results provided; s: final docking score; ^▲: a reference ligand of Acyl CoA desaturase; ^: a reference ligand of DNA topoisomerase I;

Table 3: Binding interaction modes of amlodipine and reference ligands with the target residues

ligand	Target	Bonded amino acid residue	Number, type, (energy kcal/mol) of interaction
Amlodipine	BDT1	Glu86	2, ionic bonds, (- 1, - 5.1)
		Arg139	1, H bond ( - 0.2)
		Arg458	1, H bond (- 0.6)
	HDT1	Asp483	1, H bond (- 2.6); 1, ionic bond (- 3.9)
	HDT1	Gln600	1, H bond (- 0.2)
	BAD	Thr13	1, H bond (- 0.2)
		Asn90	1, H bond (- 0.4)
		Glu136	1, H bond (- 0.5); 2, ionic bonds ( - 4.3, - 0.7)
		Tyr158	1, Polar -π bond (- 0.2)
N,N-dimethyl-2- (phenanthro]3,4- d][dioxol-5-yl) ethanamine	BDT1	Arg140	3, Polar -π bond (- 0.2, - 1.7, -1.5)
N,N-dimethyl-2- (phenanthro]3,4- d][dioxol-5-yl) ethanamine	HDT1	Ala625	2, Polar -π bond ( - 0.5, - 0.6)
Acyl CoA	BAD	Ala14	1, H bond (- 0.4)
		Tyr114	1, Polar -π bond (- 0.3)
		Met128	1 H bond (- 0.7)
		Glu136	1 H bond ( - 5.4)
		Tyr158	1 H bond (- 0.6)

BAD: Bacterial acyl CoA desaturase; BDT1: Bacterial DNA topoisomerase I; HDT1: Human DNA topoisomerase I; Ala: Alanine; Arg: Arginine;; Asp: Aspartic acid;Asn: Asparagine; Gln: Glutamine; Glu: Glutamic acid; Met: Methionine; Thr: Threonine; Tyr: Tyrosine;

Figure: 3 3D diagrams of amlodipine and reference ligands interactions with targets (I: amlodipine with bacterial DNA topoisomerase I, II: amlodipine with human DNA topoisomerase I, III: amlodipine with bacterial Acyl CoA desaturase, IV: a ligand with bacterial DNA topoisomerase I; V: a reference ligand with human DNA topoisomerase I, VI: Acyl CoA with bacterial Acyl CoA desaturase); Magenta color: a part of the binding site pocket), Cyan color: bonded residues, gold color: ligand, blue color at ligand: N atom, red color at ligand:O atom, green color: Cl atom, black line between ligand and residue: an interaction bond)

Table 4: Prediction of antibacterial and CCB activities of amlodipine structural analogs

Ligand code	Antibacterial activity		Molecular Docking with HDT1; s^▲ (kcal/mol)	Activity as CCB Molecular Docking with ^CaV^{Ab; s▲ (kcal/mol)}
Ligand code	SuperPred 3 (P %^) with BDT1	Molecular Docking with BDT1; s^▲ (kcal/mol)	Molecular Docking with HDT1; s^▲ (kcal/mol)
AMLO	51.59	-6.366	- 4.532	- 6.393
D004	52.55	- 6.115	n.t	n.t
D006	52.18	- 5.451	n.t	n.t
D018	53.04	- 4.981	n.t	n.t
D027	55.86	- 5.347	n.t	n.t
D029	51.59	- 5.300	n.t	n.t
D030	53.78	- 5.800	n.t	n.t
D038	54.27	- 4.889	n.t	n.t
D067	59.88	- 5.934	n.t	n.t
D074	61.30	- 7.051	- 4.381	- 6.068
D075	60.05	- 6.808	- 4.029	- 3.138
D076	61.39	- 5.258	n.t	n.t
D077	57.2	-5.812	n.t	n.t
D082	55.41	- 6.392	- 4. 183	- 5.011

BDT1: Bacterial DNA topoisomerase I; HDT1: Human DNA topoisomerase I; CCB: calcium channel blocker; CaVAb: human calcium channel; AMLO: amlodipine; ^: Probability of prediction; ^▲: Final docking score representing the energy of binding; n.t: not tested

Table 5: Prediction of the toxicity profiles of amlodipine structural analogs

Toxicity Parameter	AMLO	D074	D075	D082
^LD50 ^mg/kg	37	37	37	37
Toxicity Class	II	II	II	II
Endpoints	Probability confidence for Inactive toxicity
Hepatotoxicity	0.84	0.55	0.57	0.82
Carcinogenicity	0.60	0.96	0.95	0.61
Immunotoxicity	0.96	0.75	0.79	0.98
Mutagenicity	0.76	0.68	0.67	0.78
Cytotoxicity	0.64	0.91	0.88	0.65
Aryl hydrocarbon Receptor	0.91	0.95	0.95	0.92
Androgen Receptor	0.96	0.84	0.84	0.96
Androgen Receptor Ligand Binding Domain	0.66	0.79	0.79	0.77
Aromatase	0.61	0.91	0.86	0.74
Estrogen Receptor	0.93	0.92	0.92	0.91
Estrogen Receptor Ligand Binding Domain	0.90	0.94	0.92	0.94
Peroxisome Proliferator Activated Receptor Gamma	0.79	0.87	0.86	0.93
Nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element	0.80	0.87	0.86	0.85
Heat shock factor response element	0.80	0.84	0.81	0.85
Mitochondrial Membrane Potential	0.80	0.73	0.76	0.87
Phosphoprotein (Tumor Supressor) p53	0.54	0.98	0.99	0.71
ATPase family AAA domain containing protein 5	0.97	0.55	0.57	0.98

DISCUSSION:

As reported in the literature, the assumption suggesting that amlodipine antibacterial action is due to blocking the bacterial calcium channel may be doubtful³⁵. It is well known that mammalian voltage- dependent calcium channels (Ca_Vs) are phylogenetically related to the bacterial voltage-gated sodium channels (BacNa_Vs) with very similar amino acid sequences present in both types, and so they are used as a model for gating and ion permeation³⁶. Recently, two bacterial voltage-dependent calcium channels (Ca_VMr and Na_VPp) have been recognized, which are selective for Ca²⁺, and for Na⁺ with Ca²⁺ dependent inhibition, respectively³⁷. When amlodipine is administered to humans at a standard daily dose of 10 mg, it produces a maximal serum concentration of 5.9 x10^-3 µg/ml which is adequate for the drug’s action as CCB³⁵. Therefore, owing to the similarity between mammalian and bacterial calcium channels, it is supposed that the same concentration is sufficient to block the ion channels in bacteria as well, resulting in bacterial killing. However, in vitro investigations of amlodipine antibacterial activity revealed that the minimum inhibitory concentration (MIC) of the drug against all tested bacteria (e.g. S. aureus ~10-400 µg/ml) which is extremely greater than its maximum serum concentration³⁵. This may indicate that the drug may have a bactericidal mechanism of action that is unrelated to its activity as CCB. In another theory, it was found that amlodipine enhances the activity of imipenem against multidrug-resistant Acinetobacter baumanii by inhibiting expression of the resistance nodulation cell division efflux pump AdeABC⁶. However, this theory was not in agreement with other studies that reported the presence of factors other than blocking the efflux pump that influenced the antibacterial action of the drug, such as its ionization as well as the environmental pH that surrounds the infection pathway^38-40.

Bacterial Acyl CoA desaturase was one of the two bacterial targets initially predicted in this study as bacterial target for amlodipine. This enzyme is a membrane enzyme belonging to the superfamily of "fatty acid desaturases (FADs)," which catalyze the introduction of double bonds into fatty acids and are therefore necessary components of cell membrane anabolic long-chain polyunsaturated fatty acids^31, ³². Although there are studies which have shown that it is problematic to consider the fatty acid biosynthetic pathway as a potential bacterial target because exogenous fatty acids from the host serum can overcome inhibition of the bacterial pathway³², this enzyme was investigated in this study for the purpose of verification by molecular docking. The other initial predicted target "bacterial DNA topoisomerase I" is an enzyme that belongs to the family of DNA topoisomerases. The latters are known to play important roles in DNA replication, transcription, repair, and recombination. The results of the present study predicted not only higher binding affinity of amlodipine than that of a reference ligand of that enzyme, but also stronger bond interactions between amlodipine and this enzyme. Indeed, the results supported the theory that DNA topoisomerase I could be a potential bacterial target for new antibiotics as reported in the litearature³³.

All amlodipine analogs that had considerable SuperPred predictions to have antibacterial activity by acting on the same bacterial target of amlodipine were observed to possess 4-Hydropyridine nucleus instead of 1,4-dihydropyridine nucleus of amlodipine. However, of these analogs, molecular docking investigation showed that only three analogs (D074, D075 ) and (D085) with structures bearing the nuclei of 1-butyl 4-hydropyridine and 4-hydropyridine 1-acetic acid, respectively, as demonstrated in (Figure 4), would have more binding affinity and stronger bond interactions to the target enzyme than that of amlodipine. The three analogs were also predicted to have low affinity to human DNA topoisomerase I, diminished activity as CCB, accepted toxicity profiles and drug-likeness. However, the pharmacokinetics profiles predicted for (D074) and (D075) showed greater oral and dermal absorption, wider drug distribution, similar metabolism profile and close excretion rate to that of amlodipine. Although, similar to amlodipine the two analogs had low prediction to pass blood-brain barrier and hence may be not suitable for CNS infections. This property is not not be a problem that could cease the development of these compounds as antibacterials since many antibiotics have the same drawback e.g. piperacillin, cefuroxime, cefixime and imipenem though the penetration of these antibiotics increases in case of strong meningeal inflammation⁴¹.

CONCLUSION:

Based on results of this study, it could be concluded that the structural analogs of amlodipine (D074 and D075) which bears the 1-butyl 4-hydropyridine nucleus are promising candidates to be lead compounds for the discovery of new antibacterial drugs.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

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Received on 14.08.2023 Modified on 06.11.2023

Research J. Pharm. and Tech 2024; 17(5):2271-2281.

DOI: 10.52711/0974-360X.2024.00357

Rule	Criterion	AMLO	D074	D075	D082
Lipinski's	No. of H-bond (donor) ≤ 5	2	1	1	2
	No. of H-bond (acceptor) ≤ 10	6	8	8	8
	Mwt ≤ 500	408.88	464.98	493.04	466.91
	MLOGP ≤ 4.15	1.33	2.18	2.59	0.93
Egan`s	WLOGP ≤ 5.88	1.89	3.4	4.18	1.68
Egan`s	TPSA ≤ 131.6 ^oA	99.88	91.09	91.09	128.39
Muegg`s	Mwt: 200 -600	408.88	464.98	493.04	466.91
	XLOGP3: -2 to 5	3.00	4.39	5	0.45
	TPSA ≤ 150 ^oA	99.88	91.09	91.09	128.39
	No. of rings ≤ 7	2	2	2	2
	No. of (C) atoms > 4	20	24	26	22
	No. of heteroatoms > 1	8	8	8	10
	No. of rotatable bonds ≤ 15	10	13	14	12
	No. of H-bond (donor) ≤ 5	2	1	1	2
	No. of H-bond (acceptor) ≤ 10	6	8	8	8

Abdulrahman A. Humaid¹, Maher A. Al-Maqtari², Abdulkarim K. Alzomor³,

Anes A.M. Thabit¹*

MATERIALS AND METHODS:

Prediction of amlodipine bacterial target:

a) SuperPred 3:

b) Molecular Docking:

Design and novelty of amlodipine structural analogs:

Prediction of the structural analog properties:

b) Activity as CCB:

c) Toxicity profile:

d) Drug-likeness:

e) Pharmacokinetics:

RESULTS:

Bacterial target of amlodipine:

Predicted Properties of Structural analogs:

b) Toxicity profiles:

c) Drug-likeness:

d) Pharmacokinetics:

DISCUSSION:

CONCLUSION:

Abdulrahman A. Humaid1, Maher A. Al-Maqtari2, Abdulkarim K. Alzomor3,

Anes A.M. Thabit1*

MATERIALS AND METHODS:

Prediction of amlodipine bacterial target:

a) SuperPred 3:

b) Molecular Docking:

Design and novelty of amlodipine structural analogs:

Prediction of the structural analog properties:

b) Activity as CCB:

c) Toxicity profile:

d) Drug-likeness:

e) Pharmacokinetics:

RESULTS:

Bacterial target of amlodipine:

Predicted Properties of Structural analogs:

b) Toxicity profiles:

c) Drug-likeness:

d) Pharmacokinetics:

DISCUSSION:

CONCLUSION:

Abdulrahman A. Humaid¹, Maher A. Al-Maqtari², Abdulkarim K. Alzomor³,

Anes A.M. Thabit¹*