Vidushi Sharma

Structural insight into selective phosphodiesterase 4B inhibitors: pharmacophore-based virtual screening, docking, and molecular dynamics simulations

Phosphodiesterase (PDE4) is the predominantly expressing family of PDE enzyme in immune and inflammatory cells. Inhibition of PDE4 has been reported to suppress a diverse spectrum of inflammatory responses both in vitro and in vivo. Many PDE4 inhibitors in development are shown to be efficacious in animal models of various inflammatory disorders, such as asthma, COPD, psoriasis, inflammatory bowel diseases, and rheumatoid arthritis, as well as in clinical trials for asthma and COPD. Therefore, PDE4 might prove as potential therapeutic targets in inflammatory and autoimmune diseases. The PDE4 enzyme family consists of four members (named PDE4A, PDE4B, PDE4C, and PDE4D) that are highly expressed in neutrophils, monocytes, central nervous system, and smooth muscles of the lung. Non-selective PDE4 inhibitors like Rolipram are associated with severe side effects like nausea and emesis. Second-generation PDE4 inhibitors like Roflumilast are reported with improved side effects, but narrow therapeutic window. The behavioral correlation of emesis in mice by deleting PDE4D encoding gene confirmed that inhibition of PDE4D, but not PDE4B is responsible for emetic effect of non-selective PDE inhibitors (Jin, Ding, & Lin, 2012). Therefore, selective PDE4B inhibitors can provide anti-inflammatory efficacy without any side effect. However, conserved active site residues of PDE4B and PDE4D make it difficult to design selective PDE4B inhibitors. In the line of identifying selective PDE4B inhibitors, a combined receptorand ligand-based approach can improve the binding affinity and selectivity of PDE4B inhibition. Thirty-four PDE4B structures are available in RCSB-PDB. Hitherto, no structural classification of available co-crystallized PDE4B inhibitors is reported. An in-depth analysis of available PDE4B-inhibitor complexes may help to find empirical parameters for selective enzyme inhibition. Chen et al. (2010) developed multiple pharmacophore models based on the 18 crystal structures of phosphodiesterase 4 (PDE4) with co-crystal ligand for discovering potential PDE4 inhibitors. Srivani, Usharani, Jemmis, and Sastry (2008) performed the crystal structure analysis of PDE4B and PDE4D which led to find significant variations in the M-loop region, which is the integral part of the active site of PDE4B and PDE4D. Ke (2004) performed the similar crystal structure analysis for PDE4 and PDE5 with respect to the metal binding site. In present study, first we retrieved the information from available PDE4B crystal structures and grouped the crystal ligands in different classes based on chemical scaffolds. We have employed structure and ligand-based dual approach for identifying potential and selective molecules for PDE4B. Structure-based approach presents advantage over ligand-based approach by incorporating spatial distribution of hotspot. At the same time ligandbased approach helps to identify key pharmacophoric features in ligands essential for bioactivity. We combined the chemical features of reported crystal ligands of PDE4B in RCSB PDB data bank and developed ligandbased pharmacophores. Using these queries, SPECS database of commercially available small molecules was screened to find new PDE4B inhibitors. To predict the selectivity in PDE4B over PDE4D, the screened molecules were docked in PDE4B (PDB ID: 3G45) and PDE4D (PDB ID: 3G4G) crystal structures using Glide. The stability of top-scoring ligand in PDE4B active site was confirmed by a 10 ns MD simulations (Figure 1). Our results provided critical insight for designing selective PDE4B inhibitors. This work is organized as follows. The crystal structure analysis, pharmacophore modeling, virtual screening (VS), docking and ADME studies, MD simulation materials, and reproducible methods are provided in next section. Further part includes is the analysis and discussion of crystal structure analysis and pharmacophore

Pharmacophore generation and atom based 3D-QSAR of quinoline derivatives as selective phosphodiesterase 4B inhibitors

Phosphodiesterase 4B (PDE4B) hydrolyses cyclic adenosine monophosphate (cAMP) and thus regulates its intracellular levels. The enzyme has been proposed as a potential drug target against diseases like inflammation and chronic obstructive pulmonary disease. But use of current PDE4B inhibitors is limited due to dose-dependent nausea and vomiting. Adverse effects associated with current PDE4B inhibitors are possibly results of PDE4D inhibition, a highly similar homolog of PDE4B. Here we considered quinoline analogs and applied ligand-based pharmacophore and atom based 3D-QSAR modeling with structure-based docking and ADME approach. A 5-point pharmacophore model was developed and used to derive a predictive 3D-QSAR model for the studied dataset. The obtained r2 and q2 values were 0.96 and 0.91, respectively. The result suggested that the generated 3D-QSAR model is reliable and can be considered for PDE4B activity prediction. Further, a pharmacophore model was employed for virtual screening to identify potent PDE4B inhibitors. The selective ligands for PDE4B were identified through docking and prime binding energy analysis of ligands in both PDE4B and PDE4D. ADME analysis was performed to confirm the drug ability of the selective ligand. To validate docking results, a molecular dynamics simulation was performed for PDE4B complexed with a top scoring ligand, AQ-390/42425549. AQ-390/42425549-PDE4B interactions reported in MD analysis were consistent with the docking results. All the hit molecules were procured and biologically evaluated for percentage inhibition of PDE4B and PDE4D in in vitro enzymatic assays. Among the total of thirteen molecules that were active against PDE4B, ten were selective with little PDE4D inhibition.

Design, synthesis and evaluation of newer 5,6-dihydropyrimidine-2(1H)-thiones as GABA-AT inhibitors for anticonvulsant potential

Several new 5,6-dihydropyrimidine-2(1H)-thione derivatives have been prepared and investigated for their potencies for anticonvulsant activity against maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) test in mice. The acute neurotoxicity was measured by rotarod test. Compounds 3c and 3l were found active in both of the animal models. Further, in vitro GABA-AT enzyme activity assay was carried out to investigate the possible mechanism of action through GABA-AT inhibition. The most potent compounds 3c and 3l showed inhibitory potency (IC50) of 18.42μM and 19.23μM, respectively. The molecular modeling was performed for all the synthesized compounds. The docking results were found in concordant with the observed animal studies.

Design, Synthesis, and Docking Study of Pyrimidine-Triazine Hybrids for GABA Estimation in Animal Epilepsy Models: Pyrimidine-Triazine Hybrids as Anticonvulsants

A series of new pyrimidine–triazine hybrids (4a–t) was designed and synthesized, from which potent anticonvulsant agents were identified. Most of the compounds exhibited promising anticonvulsant activity against the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) tests, along with minimal motor impairment with higher safety compared to the standard drugs, phenytoin and carbamazepine. In the series, 5‐(4‐(4‐fluorophenyl)‐6‐(4‐hydroxyphenyl)‐2‐thioxo‐5,6‐dihydropyrimidin‐1(2H)‐yl)‐1,2‐dihydro‐1,2,4‐triazin‐3(6H)‐one (4o) and 5‐(6‐(4‐hydroxy‐3‐methoxyphenyl)‐4‐(4‐hydroxyphenyl)‐2‐thioxo‐5,6‐dihydropyrimidin‐1(2H)‐yl)‐1,2‐dihydro‐1,2,4‐triazin‐3(6H)‐one (4s) emerged as most potent anticonvulsant agents with median doses of 22.54 and 29.40 mg/kg (MES ED50), 285.02 and 293.42 mg/kg (scPTZ ED50), and 389.11 and 412.16 mg/kg (TD50), respectively. Docking studies were also performed for all synthesized compounds to get insight into the binding pattern toward the GABAA receptor as a possible mechanism of their anticonvulsant action, and in silico ADME studies were carried out to predict the safety and stability of the molecules. The increased GABA level in the experimental animals in the neurochemical estimation assay confirmed their GABAergic modulating activity. The most potent compounds were also evaluated for their neurotoxic and hepatotoxic effects. Fortunately, they did not show any sign of neurotoxicity or hepatotoxicity, suggesting that they have a broad spectrum of anticonvulsant activity with a large safety margin. Together, this research suggested that 4o and 4s may serve as leads in the discovery and development of new anticonvulsant drugs.

Synthesis, cyclooxygenase-2 inhibition, anti-inflammatory evaluation and docking study of substituted-N-(3,4,5-trimethoxyphenyl)-benzo[d]oxazole derivatives

BACKGROUND Non-steroidal anti-inflammatory drugs are widely used for many years, but the chronic use of NSAIDs leads to gastric side effects, ulceration and kidney problems. These side effects are due to non-selective inhibition of COX-2 along with COX-1. Therefore, it is imperative to develop novel and selective COX-2 inhibitors. OBJECTIVE In this paper wehave synthesized a series of novel hybrids comprising of substituted-N- (3,4,5-trimethoxyphenyl)-benzo[d]oxazole derivatives and screened for the treatment of inflammation. METHODS The structures of the obtained compounds were elucidated by elemental and spectral analysis (ATR-FTIR, 1H NMR, 13C NMR, Mass spectroscopy). All of the compounds were evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activity by in vitro enzymatic assay. The compound which showed COX-2 activity (3a - 3e, 3g - 3h, 3k, 3m and 3o) was further screened for in vivo anti-inflammatory activity and ulcerogenic liability. Molecular docking study was also performed with resolved crystal structure of COX-2 to understand the binding mechanism of newly synthesized inhibitors in the active site of COX-2enzyme. RESULTS The in vitro COX-1 and COX-2 inhibitory studies showed that the synthesized compounds potentially inhibited COX-2 (IC50 = 0.04 - 26.41 µM range) over COX-1 (IC50 = 0.98 - 33.33 µM range). The in vivo studies predicted that compounds 3c (70.9%, 0.6±0.22), 3m (68.1%, 1.9±0.41) and 3o (70.4%, 1.7±0.27) produced more efficacy against carrageenan induced paw edema and less ulcerogenic effect, as compared to standard ibuprofen (65.9%, 2.2±0.44). The results of docking studies were found to be concordant with the biological evaluation studies of the prepared compound. CONCLUSION Among all the tested compounds, 2-Chloro-N-(2-(3,4,5-trimethoxyphenyl)- benzo[d]oxazol-5-yl)-benzamide (3c) was the most potent anti-inflammatory agent and has less ulcerogenic potential. This series of compound can be explored more for development of safer and more active anti-inflammatory agents.

5,6-Dihydropyrimidine-1(2H)-carbothioamides: Synthesis, in vitro GABA-AT screening, anticonvulsant activity and molecular modelling study

Even after considerable advances in the field of epilepsy treatment, convulsions are inefficiently controlled by standard drug therapy. Herein, a series of pyrimidine-carbothioamide derivatives 4(a-t) was designed as anticonvulsant agents by doing some important structural modifications in well-known anticonvulsant drugs. Two classical animal models were used for the in vivo anticonvulsant screening, maximum electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) models; followed by motor impairment study by rotarod method. The most active compound 4g effectively suppressed seizure effect in both the animal models with median doses of 15.6 mg/kg (MES ED50), 278.4 mg/kg (scPTZ ED50) and 534.4 mg/kg (TD50) with no sign of neurotoxicity. Furthermore, in vitro GABA-AT enzyme activity assay of 4g showed inhibitory potency (IC50) of 12.23 μM. The docking study also favored the animal studies.

Synthesis, biological evaluation and docking study of N-(2-(3,4,5-trimethoxybenzyl)benzoxazole-5-yl) benzamide derivatives as selective COX-2 inhibitor and anti-inflammatory agents

A series of N-(2-(3,4,5-trimethoxybenzyl)-benzoxazole-5-yl)benzamide derivatives (3a-3n) was synthesized and evaluated for its in vitro inhibitory activity against COX-1 and COX-2. The compounds with considerable in vitro activity (IC50 < 1 µM), were evaluated in vivo for their anti-inflammatory and ulcerogenic potential. Out of the fourteen newly synthesized compounds; 3b, 3d, 3e, 3h, 3l and 3m were found to be most potent COX-2 inhibitors in in vitro enzymatic assay with IC50 in the range of 0.14-0.69 µM. In vivo anti-inflammatory activity of these six compounds (3b, 3d, 3e, 3h, 3l and 3m) was assessed by carrageenan induced rat paw edema method. The compound 3b (79.54%), 3l (75.00%), 3m (72.72%) and 3d (68.18%) exhibited significant anti-inflammatory activity than standard drug ibuprofen (65.90%). Ulcerogenic activity with histopathological studies was performed, and the screened compounds demonstrated significant gastric tolerance than ibuprofen. Molecular Docking study was also performed with resolved crystal structure of COX-2 to understand the interacting mechanisms of newly synthesized inhibitors with the active site of COX-2 enzyme and the results were found to be in line with the biological evaluation studies of the compounds.

Designing novel inhibitors against falcipain-2 of Plasmodium falciparum

Coumarin containing pyrazoline derivatives have been synthesized and tested as inhibitors of in vitro development of a chloroquine-sensitive (MRC-02) and chloroquine-resistant (RKL-2) strain of Plasmodium falciparum and in vivo Plasmodium berghei malaria. Docking study was also done on cysteine protease falcipain-2 which showed that the binding pose of C-14 molecule and epoxysuccinate, inhibitor of falcipain-2, binds in the similar pattern. The most active antimalarial compound was 3-(1-benzoyl-5-(4-flurophenyl)-4,5-dihydro-1H-pyrazol-3yl)-7-(diethyamino)-2H-chromen-2-one C-14, with an IC50 of 4.21 µg/ml provided complete protection to the infected mice at 24 mg/kg X 4 days respectively.

Synthesis, biological evaluation and docking study of a new series of di-substituted benzoxazole derivatives as selective COX-2 inhibitors and anti-inflammatory agents

A new series of substituted-N-(3,4-dimethoxyphenyl)-benzoxazole derivatives 13a-13p was synthesized and evaluated in vitro for their COX (I and II) inhibitory activity, in vivo anti-inflammatory and ulcerogenic potential. Compounds 13d, 13h, 13k, 13l and 13n exhibited significant COX-2 inhibitory activity and selectivity towards COX-2 over COX-1. These selected compounds were screened for their in vivo anti-inflammatory activity by carrageenan induced rat paw edema method. Among these compounds, 13d was the most promising analogs of the series with percent inhibition of 84.09 and IC50 value of 0.04 µM and 1.02 µM (COX-2 and COX-1) respectively. Furthermore, ulcerogenic study was performed and tested compounds (13d, 13h, 13k, 13l) demonstrated a significant gastric tolerance than ibuprofen. Molecular docking study was also performed with resolved crystal structure of COX-2 to understand the binding mechanisms of newly synthesized inhibitors in the active site of COX-2 enzyme and the results were found to be concordant with the biological evaluation studies of the compounds. These newly synthesized inhibitors also showed acceptable pharmacokinetic profile in the in silico ADME/T analyses.

Synthesis, molecular docking, and pharmacological evaluation of N-(2-(3,5-dimethoxyphenyl)benzoxazole-5-yl)benzamide derivatives as selective COX-2 inhibitors and anti-inflammatory agents

A series of N‐(2‐(3,5‐dimethoxyphenyl)benzoxazole‐5‐yl)benzamide derivatives (3am) was synthesized and evaluated for their in vitro inhibitory activity against COX‐1 and COX‐2. The compounds with considerable in vitro activity (IC50 < 1 μM) were evaluated in vivo for their anti‐inflammatory potential by the carrageenan‐induced rat paw edema method. Out of 13 newly synthesized compounds, 3a, 3b, 3d, 3g, 3j, and 3k were found to be the most potent COX‐2 inhibitors in the in vitro enzymatic assay, with IC50 values in the range of 0.06–0.71 μM. The in vivo anti‐inflammatory activity of these six compounds (3a, 3b, 3d, 3g, 3j, and 3k) was assessed by the carrageenan‐induced rat paw edema method. Compounds 3d (84.09%), 3g (79.54%), and 3a (70.45%) demonstrated significant anti‐inflammatory activity compared to the standard drug ibuprofen (65.90%) and were also found to be safer than ibuprofen, by ulcerogenic studies. A docking study was done using the crystal structure of human COX‐2, to understand the binding mechanism of these inhibitors to the active site of COX‐2.