Virtual screening, molecular simulations and bioassays: Discovering novel microsomal prostaglandin E Synthase-1 (mPGES-1) inhibitors.

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase expressed following exposure to pro-inflammatory stimuli. The mPGES-1 enzyme represents a new target for the therapeutic treatment of acute and chronic inflammatory disorders and cancer. In the present study, compounds from the ZINC15 database with an indole scaffold were docked at the mPGES-1 binding site using Glide (high-throughput virtual screening [HTVS], standard precision [SP] and extra precision [XP]), and the stabilities of the complexes were determined by molecular simulation studies. Following HTVS, the top 10% compounds were retained and further screened by SP. Again, the top 10% of these compounds were retained. Finally, the Glide XP scores of the compounds were determined, 20% were analyzed, and the Prime MM-GBSA total free binding energies of the compounds were calculated. The molecular simulations (100 ns) of the reference ligand, LVJ, and the two best-scoring compounds were performed with the Desmond program to analyze the dynamics of the target protein-ligand complexes. In human lung cells treated with the hit compounds, cell viability by colorimetric method and PGE2 levels by immunoassay method were determined. These in vitro experiments demonstrated that the two indole-containing hit compounds are potential novel inhibitors of mPGES-1 and are, therefore, potential therapeutic agents for cancer/inflammation therapies. Moreover, the compounds are promising lead mPGES-1 inhibitors for novel molecule design.

Design, synthesis and antiproliferative activity evaluation of fluorine-containing chalcone derivatives.

A series of new chalcones containing fluoro atom at B ring have been designed, synthesized, and evaluated to be antiproliferative activity against a panel of human tumor cell lines. Some of the analogs (8, 9, 12, 45, 46 and 48) displayed powerful antiproliferative effects to certain human tumor cells, but all of them were devoid of any cytotoxicity towards the normal HEK 293. Acridine orange staining data supported that the cytotoxic and antiproliferative effects of the synthesized analogs on tumor cells are mediated through apoptosis. The compounds 12 and 46 manifested concentration-dependent antiproliferative activity in human hepatocellular carcinoma cell lines using an xCELLigence assay. The structures and antiproliferative activity relationship were further supported by in silico molecular docking study of the compounds against tubulin protein which suggests our compounds interference to cell division. Communicated by Ramaswamy H. Sarma.

Discovery of novel and selective inhibitors targeting protein tyrosine phosphatase 1B (PTP1B): Virtual screening and molecular dynamic simulation.

Protein tyrosine phosphatase 1B (PTP1B) is a promising target for Type II diabetes, obesity, and cancer therapeutics. However, capturing selectivity over T cell protein tyrosine phosphatase (TCPTP) is key to PTP1B inhibitor discovery. Current studies demonstrate that the phosphotyrosine (pTyr) binding site confers selectivity to inhibitors. To identify novel selective inhibitors of PTP1B, drugs in the DrugBank were docked into the active and pTyr site using virtual docking tools. The most suitable drugs were selected based on their docking scores, similarity, and visual results before molecular dynamic simulations were performed. A combination of virtual screening and molecular dynamic simulation approaches indicated that five drugs (DB03558, DB05123, DB03310, DB05446, DB03530) targeting the active and second pTyr binding site of PTP1B could be potential selective inhibitors. This study showed that the hit drugs (experimental, research, and approved) could serve as potential selectivity PTP1B inhibitors and as useful treatments for diabetes and cancer. The hit drugs can be experimentally validated via in vitro molecular testing and in vivo animal testing; alternatively, they can be included in ongoing clinical trials. In addition, more effective molecules can be designed by derivatizing these drugs.

Synthesis, molecular modeling studies, ADME prediction of arachidonic acid carbamate derivatives, and evaluation of their acetylcholinesterase activity.

In this work, a series of novel anandamide units containing carbamate were designed and synthesized. All the derivatives were evaluated in vitro for their inhibitory potential against the electric eel acetylcholinesterase enzyme (AChE) and showed reversible inhibitions. The compounds 7a, 7d, 7e, and 7f are mixed inhibitors of AChE, while the compounds 7b, 7c, and 7g are uncompetitive (Ki in the range 0.93-8.86 µM). The kinetic studies revealed that compounds 7b, 7c, 7f, and 7g inhibit considerably AChE activity. Molecular docking analyses were made to evaluate the binding type and interactions of the synthesized compounds to the ligand-binding site of hAChE. It was observed that the docking results were in parallel with the in vitro results. The adsorption, distribution, metabolism, and excretion properties were computed for the compounds, and were found within the acceptable range. This study suggests the compounds 7b, 7c, 7f, and 7g identified as novel reversible AChE inhibitors may be useful lead compounds for the treatment of Alzheimer's disease.

Some indazoles reduced the activity of human serum paraoxonase 1, an antioxidant enzyme: in vitro inhibition and molecular modeling studies.

Background: Paraoxonase 1 (PON1: EC 3.1.8.1) is a vital antioxidant enzyme against mainly atherosclerosis and many other diseases associated with oxidative stress. Thus, studies related to PON1 have an important place in the pharmacology. In this study, we aimed to evaluate the in vitro inhibition effects of some indazoles on the activity of human PON1. Methods: PON1 was purified from human serum with a specific activity of 5000 U/mg and 13.50% yield by using simple chromatographic methods. Results: The indazoles showed Ki values in a range of 26.0 ± 3.00-111 ± 31.0 µM against hPON1. All these indazoles exhibited competitive inhibition. In addition, molecular docking studies were performed in order to assess the probable binding mechanisms into the active site of hPON1. Molecular modeling studies confirmed our results. Conclusions: Inhibition of PON1 by indazoles supplies a verification to further consideration of limitation dosage of indazole molecule groups as drug.

A Comparison of the Inhibitory Effects of Anti-Cancer Drugs on Thioredoxin Reductase and Glutathione S-Transferase in Rat Liver.

BACKGROUND: While Thioredoxin Reductase (TrxR) plays an important role in regulation of the intracellular redox balance and various signalling pathways, Glutathione S-Transferase (GSTs) enzymes belong to the detoxification family that catalyse the conjugation of glutathione with various endogenous and xenobiotic electrophiles. Since TrxR and GSTs are overexpressed in many cancer cells, they have been identified as potential targets to develop chemotherapeutic strategies. METHOD: The mitochondrial TrxR (TrxR2) enzyme and the cytosolic GST enzyme was purified from rat liver via affinity chromatography. After the purification, the in vitro inhibition effects of some anticancer drugs (cisplatin, calcium folinate, carboplatin, epirubicin hydrochloride, doxorubicin hydrochloride, paclitaxel, etoposide, fluorouracil, and methotrexate) were investigated on both enzymes. Since only methotrexate inhibits both enzymes among all the anticancer drugs, a molecular docking study was performed to determine the binding site and the binding affinity of methotrexate to the enzymes. RESULTS: Firstly, TrxR2 and GST were found to have a specific activity of 0.436, 1765 EU/mg proteins with a yield of 39.20%, 31.28% and 207.6, 3516.6 of purification fold, respectively. While TrxR2 was strongly inhibited by all of the anticancer drugs, GST was not inhibited by any of the anticancer drugs except methotrexate. CONCLUSION: Both enzymes were inhibited by only methotrexate in rat liver, and methotrexate was well placed in the active sites of both proteins. Therefore, it may be argued that methotrexate may be a more effective anticancer drug than all other drugs used in this study against the multi drug resistance that will occur during chemotherapy.

Efficient expression of recombinant human telomerase inhibitor 1 (hPinX1) in Pichia pastoris.

PinX1 encoded by a remarkable tumor suppressor gene and located in human chromosome 8p23 is known as telomerase inhibitor. In recent years, this protein has been of interest as clinically tumor suppressor. Pichia pastoris expression system is preferred to produce heterologous proteins and is suitable for industrial and research purposes. In the present study, human PinX1 gene (hPinX1) was cloned in E. coli One Shot TOP10 cells and overexpressed in P. pastoris strain X-33 intracellularly, using a strong AOX (alcohol oxidase) promoter. The recombinant cells were grown in shaking flask. Induction time, methanol concentration and initial pH were optimized for obtaining high levels of hPinX1 protein production. Recombinant protein production was confirmed by Western blot analysis and the relative expression levels of rhPinX1 were quantified. According to Western blot analysis, molecular mass of produced hPinX1 was determined as 47.5 kDa. At the end of optimization studies, the best fermentation conditions were determined as induction time 48 h, methanol concentration 3% and initial culture pH 5.0. This process would be an applicable way for obtaining recombinant hPinX1 using P. pastoris expression system. This is the first report on recombinant production of hPinX1 in P. pastoris.

Synthesis and biological evaluation of phloroglucinol derivatives possessing α-glycosidase, acetylcholinesterase, butyrylcholinesterase, carbonic anhydrase inhibitory activity.

A series of novel phloroglucinol derivatives were designed, synthesized, characterized spectroscopically and tested for their inhibitory activity against selected metabolic enzymes, including α-glycosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and human carbonic anhydrase I and II (hCA I and II). These compounds displayed nanomolar inhibition levels and showed Ki values of 1.14-3.92 nM against AChE, 0.24-1.64 nM against BChE, 6.73-51.10 nM against α-glycosidase, 1.80-5.10 nM against hCA I, and 1.14-5.45 nM against hCA II.

Synthesis and Carbonic Anhydrase Inhibition of Tetrabromo Chalcone Derivatives.

In the present study, human carbonic anhydrase (hCA) enzyme was purified and characterized from fresh blood human red cells by Sepharose-4B-l-tyrosine-sulfanilamide affinity gel chromatography. Secondly, a series of new tetrabromo chalcone derivatives containing 4,7-methanoisoindol-1,3-dione (2a-i) were synthesized from the addition of Br2 to related chalcone derivatives (1a-i). The structures of the new molecules (2a-i) were confirmed by means of 1 H NMR, 13 C NMR and elemental analysis. Finally, the inhibitory effects of 2a-i on CA activities were investigated using the esterase method under in vitro conditions. The compounds 2a-i exhibited excellent inhibitory effects, in the low nanomolar range, with Ki values in the range of 11.30-21.22 nM against hCA I and in the range of 8.21-12.86 nM against hCA II. Our findings suggest that the new compounds 2a-i have superior inhibitory effect over acetazolamide (AZA), which is used as clinical CA inhibitor, with obtained Ki values of 34.50 and 28.93 nM against the hCA I and II isozymes, respectively. In addition to the inhibition assays, molecular modeling approaches were implemented for prediction of the binding affinities of compounds 2a and 2c, which had the highest inhibition effects, against the hCA I and II isozymes.

Assessment of the inhibitory effects and molecular docking of some sulfonamides on human serum paraoxonase 1.

Paraoxonase-1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. Due to the PON1's crucial functions, inhibitors and activators of PON1 must be known for pharmacological applications. In this study, we investigated the in vitro effects of some sulfonamides compounds on human serum PON1 (hPON1). For this aim, we purified the hPON1 from human serum with high specific activity by using simple chromatographic methods, and after the purification processes, we investigated in vitro interactions between the enzyme and some sulfonamides (2-amino-5-methyl-1,3-benzenedisulfonamide, 2-chloro-4-sülfamoilaniline, 4-amino-3-methylbenzenesulfanilamide, sulfisoxazole, sulfisomidine, and 5-amino-2-methylbenzenesulfonamide). IC50 , Ki values, and inhibition types were calculated for each sulfonamide. 2-amino-5-methyl-1,3-benzenedisulfonamide and 2-chloro-4-sülfamoilaniline exhibited noncompetitive inhibition effect, whereas 4-amino-3-methylbenzenesulfanilamide, sulfisoxazole, and sulfisomidine exhibited mixed type inhibition. On the other hand, 5-amino-2-methylbenzenesulfonamide showed competitive inhibition and so molecular docking studies were performed for this compound in order to assess the probable binding mechanism into the active site of hPON1.