journal of pharmacy research 6 (2013) 173e178

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Original Article Cross evaluation of different classes of alpha- receptor antagonists to identify overlapping pharmacophoric requirements

Neetesh Pandey a, Mukesh Yadav b,*, Anuraj Nayarisseri c, Meenakshi Ojha a, Jyoti Prajapati a, Saurabh Gupta c a School of Computer Science and Information Technology, DAVV, Indore, Madhya Pradesh, India b Department of Pharmaceutical Chemistry, Softvision College, Indore, Madhya Pradesh, India c Bioinformatics Research Laboratory, Eminent Biosciences, Indore, Madhya Pradesh, India article info abstract

Article history: Structurally dispersive classes of drugs targeting identical receptor binding site can serve Received 6 August 2012 as a source of information in the design of novel drug candidates. Their comparative cross Accepted 1 November 2012 structural features can be utilized toward optimization of receptoreligand interactions. Five established alpha-adrenergic receptors antagonists were selected as representative Keywords: compounds of their respective classes. The selected antagonists are Phenoxybenzamine, Alpha , , Mesylate and . A small library of 1000 molecules, antagonist 200 from each class, were submitted to molecular docking in the antagonist binding site of Pharmacophores alpha-adrenergic receptor. The present work includes homology modeling of alpha- Homology modeling adrenergic receptor using SPDBV and its structure validation from Procheck. The molecule Flexible molecular docking library was developed using drug likeness filters (Lipinski’s rules). Flexible molecular docking was performed using MVD (Molegro Virtual Docker) after receptor and ligand preparation. The conclusive outcome of the present work is identification of antagonist binding sites of the alpha-1 (a1)-adrenergic receptor exclusively as hydrophobic due to presence of the amino acids Val 107, Val 157, Asp 106, Ile 157, Ser 158, Ser 192, Ala 189, Phe 288 and Phe 289.The amino acids identified were found crucial to identify pharmacophoric features for alpha-1 (a1)-adrenergic receptor and antagonists. Results also include identi- fication of new molecule (PubChem ID 10289950) similar to Ergoloid Mesylate with re-rank score 113.571. The present work explains successive workflow of homology modeling, flexible molecular docking, and pharmacophoric features identification. Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

* Corresponding author. Tel.: þ91 975420031. E-mail address: [email protected] (M. Yadav). 0974-6943/$ e see front matter Copyright ª 2012, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2012.11.036 174 journal of pharmacy research 6 (2013) 173e178

1. Introduction

A physiological condition when blood pressure stands consistently higher than normal magnitudes is referred to as .1 This physiological event implies extra perfor- mance and also poses serious health risks. Hypertension has been identified and proven to be a major cause of strokes and heart attacks. In addition, higher blood pressure also results into the devastation of coronary arteries, kidneys, brain and eyes.2,3 Target identification events have confirmed the cardinal role in regulation of a variety of physiological events, mark- edly within the cardiovascular system. Recent advances encompass the concerned studies related to physiological Fig. 2 e Modeled structure of alpha-1A-adrenergic receptor a events and messenger systems in which the -adrenergic showing antagonist binding site with identified cavity in 4,5 receptors are involved. Literature survey reveals develop- green color. ment of agonists and antagonists, highly selective for the various subtypes of a-adrenergic receptors and with possible e therapeutic values and lesser side effects.6 9 The target site selection in alpha-adrenergic receptor was hypertension resulted from surgical events. Labetalol identified from the literature survey pertaining to current (DB00598) particularly antagonizes alpha-adrenergic receptor work. The active site residues were found to be Phe 312 and Phe in hypertension and compatible in angina pectoris. Ergoloid 308, which is the major site of antagonist affinity. This active Mesylate (DB01049) has been found significant in dementia site is present on the transmembrane domain 7 of the alpha causing slow down of the heart rate. Prazosin (DB00457) with (1a)-adrenergic receptor.10 Mutation of either Phe 312 or Phe even larger profile is employed in symptomatic benign pros- 308 results into a significant loss of affinity for the antagonists tatic hyperplasia and severe congestive heart failure along Prazosin, Phentolamine, Labetalol, Phenoxybenzamine, with with hypertension. no changes in affinity for agonists compounds such as Phen- Molecular docking is a computational technique used in ylephrine, Epinephrine and .10 measuring the receptoreligand interactions on the basis of Information retrieved from drug bank (http://www. physicoechemical interactions pertaining to force-field drugbank.ca/) affirmed that drugs like Phenoxybenzamine, (molecular mechanics). Molecular docking helps to identify Phentolamine, Labetalol, Ergoloid Mesylate and Prazosin are pharmacophores, particularly in structure-based drug implied in cardiovascular diseases after binding alpha- design.12 Pharmacophoric atoms, groups and substructures adrenergic receptor as antagonists. Phenoxybenzamine controlling H-bond, electrostatic, hydrophobic, hydrophilic, (DB00925) is employed to dilate blood vessels leading muscle van der Waals interactions are to be identified as the objective repose.11 Phentolamine (DB00692) is prescribed during pheo- of present investigations. Present work is an overlapping chromocytomectomy to guard patients from paroxysmal information extraction from structure based drug design and

Fig. 1 e a- 3D structure of alpha-1A-adrenergic receptor. b- Quality assessment of modeled 3D structure of alpha-1A- adrenergic receptor using Ramachandran plot. journal of pharmacy research 6 (2013) 173e178 175

Table 1 e Molecular docking results of established (leads) antagonists of alpha-1A-adrenergic receptor taken as lead in compound library formation. Compound name Ligand name Mol dock Mol dock Re-rank score H-bond Torsion (PubChem CID) score (Grid) score interaction

Ergoloid Mesylate 592735 124.154 121.716 56.59 3.10142 5 Labetalol 3869 99.5906 104.037 66.8474 3.41745 8 Phenoxybenzamine 4768 88.339 92.7128 68.3864 0.25 8 Phentolamine 5755 104.342 105.932 1.5526 4.69197 4 Prazosin 4893 95.5977 96.4742 78.9892 0.52842 4

ligand based drug design. The current work explain successful that the compound library consists of structural features stepwise application of computational techniques like derived from five different classes which cover overlapping homology modeling, small molecule library formation, flex- features and thereafter holds good chances of identification of ible molecular docking, structure superimposition and phar- pharmacophoric requirements. macophoric features identification. Primary limiting factors in this approach are the availability of different classes of 2.2. Molecular modeling of alpha-adrenergic receptor antagonists having identical mode of action at the common active site region of receptor. After retrieving sequence of alpha-1 (a1)-adrenergic receptor from uniprot (P35348), BLAST15 has resulted in 36% identity and core conserved similarity 71 % with similar template of 2. Methodology chain A beta2 adreno receptor (PDB ID 2R4R_A) having sequence length of 365 in Homo sapiens from Protein Database 16 2.1. Compound library selection Bank (PDB). Protein modeling has been performed using Deep View/Swiss PDB Viewer and Swiss Model server.17 The a Five established drugs (Phenoxybenzamine, Phentolamine, primary polypeptide chain of alpha-1 ( 1)-adrenergic receptor Prazosin, Ergoloid Mesylate, and Labetalol), structurally was aligned on the backbone of template (chain A beta2 dispersive and acceptable pharmacokinetics and pharmaco- adreno receptor, PDB ID 2R4R_A) which then was followed by dynamics profile were chosen as the leads of their respective side chain optimization using the simultaneous global classes. All (five) available antagonists found suitable to create optimization of the energy for all non-identical residues. a a library of antagonists targeting alpha-1 (a1)-adrenergic Structural validation of the modeled 3D alpha-1 ( 1)-adren- receptor. Chemical and structure information resource “Pub- ergic receptor was assessed using most popular structure 18 19 chem” (http://pubchem.ncbi.nlm.nih.gov/search/) has been validation tool Procheck and Ramchandranplot. used in the filtration of the structurally similar compounds to Phenoxybenzamine, Phentolamine, Prazosin, Ergoloid Mesy- 2.3. Antagonists binding site identification and late, and Labetalol. On the basis of the structural similarities molecular docking parameters and drug likeness13 between the chosen compounds and utilization of the filters including Lipinski’s rule14 of five, 200 Molecular docking program Molegro Virtual Docker (MVD) compounds similar to each candidate have been used to based on PLP score and PLANTS Score provided a flexible create a library of 1000 molecules. It is worthy to mention here platform for docking of the compound library of all 1000

Fig. 3 e Electrostatic, H-bond and secondary structure docking view of Prazosin with alpha-1A-adrenergic receptor. 176 journal of pharmacy research 6 (2013) 173e178

Table 2 e Molecular docking results of two best new candidates from library showing better antagonistic effect on alpha- 1A-adrenergic receptor than those five established antagonists. PubChem CID Mol dock score (Grid) Mol dock score Re-rank score H-bond interaction Torsion

10289950 (Similar to 188.327 183.386 113.571 4.24591 11 Ergoloid Mesylate ) 16191408 (Similar to Prazosin) 150.23 150.702 112.604 3.73026 8

Fig. 4 e Electrostatic, H-bond and secondary structure docking view of Compound similar to Ergoloid Mesylate, PubChem CID 1028995 with alpha-1A-adrenergic receptor.

candidates. The PLP scoring functions was first reported by Interaction), internal hydrogen bond interactions and Gehlhaar et al20,21 and its advanced form was introduced by sp2esp2 torsions. With reference to literature reported and Yang and Chen22 Similarly PLANTS scoring function was discussed above,10 the center of binding site was set on the recently incorporated in MVD developed and reported by Korb coordinates values X ¼ 11.49, Y ¼ 57.28, and Z ¼ 43.36. Default et al.23 GRID resolution was set to 0.30 A0. Antagonists were parameters were used including maximum iteration of 1500 evaluated on the basis of the internal ES (Internal electrostatic and a maximum population size of 50.

Fig. 5 e Electrostatic, H-bond and secondary structure docking view of Compound similar to Prazosin, PubChem CID 16191408 with alpha-1A-adrenergic receptor. journal of pharmacy research 6 (2013) 173e178 177

3. Results and discussion

The 3D structure of alpha-1A-adrenergic receptor model 592735 Ergoloid Mesylate (Fig. 1a) qualified all the structure protein quality parameters. Results of homology modeling of alpha-1A-adrenergic receptor and its structure validation using Ramachandran plot confirm

the structural quality by allocating only 0.6% of total residues in 3869 Leu 155 Ile 157 Val 156 Val 107 Val 107 disallowed region. The remaining 71.4 % of the amino acids are Labetalol found in the core region, 25.1 % of them are distributed in the allowed region, while 2.9% are found in the generously allowed region (Fig. 1b). The energy minimization tool for modeled 4768 Phe 289 Phe 289 Phe 289 Phe 288 Phe 288 Phe 288 Ser 192 Ser 192 Ser 192 Ala 189 Ala 189 Ala 189 structures calculated that thermodynamical free energy of the Ser 158 Phenoxy- modeled structure to 835.042 KJ/mol. benzamine Newly modeled 3D Structure of alpha-1A-adrenergic receptor was chosen for carrying out docking studies. Five established molecules have a common mode of action therefore it was conclusive to dock them in the same binding pocket. The 5755 Ala 189 antagonist binding pocket identified from literature for alpha- Ile 157

1A-adrenergic receptor is shown in the figure below (Fig. 2). Phentolamine Outcome of molecular docking of five established (lead) molecules showing appreciable interactions in terms of re- rank scores are provided in Table 1. Perusal of tables 4893 Phe 289 Phe 289 Phe 288 Phe 288 Ser 192 Ser 192 Ser 158 Ser 158 Ile 157 Val 156 concludes that among five chosen lead compounds (Phenox- Val 107 Prazosin ybenzamine, Phentolamine, Prazosin, Ergoloid Mesylate, and Labetalol), Prazosin binds strongly and efficiently to alpha-1A- adrenergicas an antagonist (Fig. 3). Surprisingly, when all similar compounds from library were analyzed based on re- rank score, two new chemical compounds (Table 2) were Phe 289 Phe 288 Ser 192 Ser 158 Ile 157 Val 156 Val 107 (similar

identified which are structurally similar to Ergoloid Mesylate 10289950 ( CID 10289950) and Prazosin (pubchem CID compound) 16191408). Hydrogen bond and electrostatic interactions are Ergoloid Mesylate shown in diagrams separately (Figs. 3e5). Best candidate ob- tained in present studies is a compound similar to Ergoloid Mesylate (pubchem CID 10289950). Chemically this compound Phe 289 Phe 288 Leu 155 Ala 189 Ser 158 Ile 157 Val 156 Val 107 is N-(4,6-dimethoxy-2-{[3-(piperidin-1-yl)propyl]amino}pyr- 644725 (similar Labetalol imidin-5-yl)-5-[(1,1,3,3,6-pentamethyl-1,3-dihydro-2- compound) benzofuran-5-yl)methyl]furan-2-carboxamide with Molecular docking score 183.386 and re-rank score 113.571 (Table 2). The next molecule with accepted antagonist effect is a compound similar to Prazosin with pubchem CID 16191408, which is chemically 3-[5-(2H-1,3-benzodioxol-5-yl)-1,3,4- ligand docking of established and similar compounds in the antagonists site of alpha-2-adrenergic receptor. Phe 289 Phe 288 Ser 192 Leu 155 Ala 189 Ser 158 Ile 157 Val 156 Val 107 4717082

oxadiazol-2-yl]-N-ethyl-N-[2-(1H-pyrazol-1-yl)ethyl]prop- Phenoxy- benzamine anamide with Molecular docking score 150.702 and re-rank score is 112.604 (Table 2). Both the molecules mentioned (similar compound) above are newer and have never been tested for their alpha- 1A-adrenergic effects.

3.1. Pharmacophoric feature identification Phe 289 Phe 288 Ser 192 Leu 155 Ala 189 Ser 158 Ile 157 Val 156 Val 107 760357 (similar compound) Identification of the pharmacophores was achieved from the Phentolamine study of presence of amino acids around docked molecules with the best scores, as illustrated in Table 3. A comparative perusal of Table 3 proves that amino acids which play crucial Phe 289 Phe 288 Ser 192 Ala 189 Ser 158 Ile 157 Val 156 role are Val 107, Val 157, Asp 106, Ile 157, Ser 158, Ala 189, and Val 107 (similar Prazosin 16191408 compound) Ser 192, with most important and repetitive Phe 288 and Phe List of amino acids identified ine receptor

289. Based on the chemical nature of amino acids, it was e significantly concluded that the antagonist binding site of alpha-1A-adrenergic receptor is extensively and completely 9 8 7 5 6 4 3 2 Table 3 1 Amino acid hydrophobic in nature. The above analysis is obtained from 178 journal of pharmacy research 6 (2013) 173e178

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