Indian Journal of Pharmaceutical and Biological Research (IJPBR)

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1 Indian J. Pharm. Biol. Res. 2015; 3(2):58-65 CODEN (USA): IJPB07 ISSN: Indian Journal of Pharmaceutical and Biological Research (IJPBR) Journal homepage: Original Research Article Small-scale screening program for the identification of cytotoxic Oxazolo[5,4-d]pyrimidine derivatives based on Whole Cell Viability Assay Lawaly Maman Manzo 1,3, Yijuan Cheng 2,3, Ling yang 1,3, Jiping liu 1, Ya-Hui Deng 1, Li-Ping Sun 2 * and Yu liu 1 * 1 Department of Biochemistry, School of Life Science and Technology, China Pharmaceutical University, Nanjing , P. R. China 2 Jiangsu Key Laboratory of Drug Design & Optimization, Nanjing, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing , P. R. China 3 China Pharmaceutical University, Nanjing , P. R. China ARTICLE INFO: Article history: Received: 6 April 2015 Received in revised form: 10 May 2015 Accepted: 5 June 2015 Available online: 30 June 2015 Keywords: Cytotoxic ativity; small scale screening; MTT assay; clonogenic assay ABSTRACT For over last couple of decades, there has been a robust activity aimed towards the discovery of novel anti-cancer therapeutics. An approach to identify starting points for new drug candidates is high throughput screening of compound library collection. In this work, we describe the application of a Tetrazolium-based, 96-well small scale screening assay to screen a mini library of 19 compounds bearing Oxazolo[5,4-d]pyrimidine structures against human umbilical vein endothelial cells. Primary actives identified against HUVEC were retested and the IC 50 value compounds were estimated for HUVEC. The screening program (Primary screening) identified 4 compounds with inhibition rate percentage 70% each. Retest screening of these compounds, taking into account criteria required for high cytotoxic compounds, afforded a panel of 1 compound for further biological analysis. This compound had IC 50 value of 12.19µM, 12.16µM, 10.24µM, 20.43µM for HUVECs, SGC7901, MCF7, and HeLa respectively. Furthermore, a clonogenic assay was performed in order to confirm the cytotoxic activity of the selected compound on the survival and proliferation of MCF7. This compound was found to significantly effect the survival and proliferation of MCF7. Taken together, the selected compound, namely SCYJ32, was found to be highly cytotoxic against the numerous cell lines. Further studies are ongoing in order to unravel various mechanisms of action of this novel small compound. Introduction Cancer is a leading cause of death worldwide. In 2012, 8.2 million people died of cancer, and it is expected that annual cancer cases will rise to 22 million within the next 2 decades [1]. Although chemotherapy is one of the most frequently used forms of cancer therapy, the use of available chemotherapeutics is often limited mainly due to the adverse side effects and resistance of available anticancer agents. Therefore, searching for new anticancer agents with better activity remains important [2]. Oxazolo[5,4-d]pyrimidine derivatives are of great interest due to their broad spectrum of biochemical effects and pharmaceutical functions. In particular, there have been intense research efforts in recent years in the design and development of Oxazolo[5,4-d]pyrimidine derivatives as a class of antitumor agents [3-6]. In the present study, we investigated for novel cytotoxic compound by using a whole cell based screening approach to identify yet unknown compounds bearing Oxazolo[5,4- d]pyrimidine structures with putative anti-proliferative properties. One method for the identification of active compounds against cancer cells is the application of high throughput screening methods. Whole cell-based assays are becoming increasingly popular, and are often used for screening collections of compounds to determine if the test molecules have effects on cell proliferation or show direct cytotoxic effects that eventually lead to cell death. We * Corresponding Author: Yu liu, Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing , P. R. China. 58

2 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2):58-65 have developed a whole cell viability screening assay based on MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) tetrazolium reduction. MTT assay was the first homogeneous cell viability assay developed for a 96-well format that was suitable for HTS [7-9]. The assay can identify compounds that directly or indirectly affect HUVEC cell proliferation. Here we describe the small scale screening method of a compound mini library using a 96-well whole cell HUVEC assay, and the retesting of the identified active compounds against HUVEC cell line, in order to assess cell cytotoxicity. The selected best active compound was further tested against numerous tumor cell lines. Materials and methods Human cell line and cell culture The normal human umbilical vein endothelial cell line HUVEC, was used for cytotoxic screening of the candidate chemical compounds. HUVEC cell line were kindly provided by a collaborative laboratory of China pharmaceutical university (Benbu campus) and was maintained in log phase growth in T-75 culture flasks (Corning, NY, USA) by subculturing at either 24 or 48 hour intervals. HUVEC was grown in Dulbecco s modified Eagle s medium (DMEM) medium, supplemented with 10% fetal bovine serum (FBS). Cells were maintained at 37 0 C in a 5% CO 2 atmosphere with 95% humidity. Test chemical compounds A list of candidate compounds from Medicinal Chemistry Laboratory (China Pharmaceutical University, Nanjing, China, PRC) was provided under cooperation. Compounds were dissolved in 100% DMSO (Shanghai Titanchem Co.Ltd, CAS- China) based on the solubility of each compound, and stock solution of 20mM was prepared for each chemical compound, stored at C and diluted to the final concentration in fresh media before each experiment. In all experiments, the final DMSO concentration was < 0.8% v/v. MTT assay The MTT assay was used for screening 19 compounds and for testing cancer cell lines. HUVEC cell line was seeded in a 96- well plates at a density of 5 x 10 3 cells /well/100µl and treated with various concentrations of chemical compound. After 2 days of treatment, 20µl of MTT was added, and the incubation continued for 4 h at 37 0 C. The precipitated formazan was solubilized with 100µl of dimethyl sulfoxide (DMSO, Sigma) and the absorbance was determined at 490 nm by imark TM Microplate Reader (Bio-Rad Labs, USA). The percentage of cell growth inhibition was determined as the ratio of absorbance of the sample versus that without compound as control. IC50 values were calculated as those concentrations of compound yielding 50% cell survival, taking the values obtained for the control to be 100%. Clonogenic assay Clonogenic assay serves as a useful tool to test whether a given cancer therapy can reduce the clonogenic survival of tumor cells. Clonogenic assay is the method to determine cell reproductive death after treatment with cytotoxic agents. MCF7 cells in exponential phase of growth were exposed to the selected compound (2.5µM, 5µM, and 10µM) and or sunitinib (2.5µM, 5µM). Following drug exposure, the cells are disaggregated to form a single-cell suspension and are plated out at low density to allow colony formation. The colonies are fixed, stained, and counted. Each colony is assumed to be derived from a single cell and thus the colony count is an estimate of the number of cells that survived the drug treatment. Primary screening campaign Primary screening of the chemical compounds obtained from a medicinal chemistry laboratory (China pharmaceutical university), consisting of 19 compounds each marked with a distinct symbol code, was undertaken in single point against HUVEC cell lines. Stock solutions consisted of test compound at a concentration of 20mM in 100% DMSO. A defined volume of each compound stock solution was diluted by the addition of adequate volume of dilution medium (high glucose DMEM without FBS) by a multidrop liquid handler. A 100 µl sample of this diluted solution was then added to each well of the precultured 96-microplate for MTT assay. The final various concentrations of each test compound in the assay was 100µM, 10µM, 1µM, 0.1µM, 0.01µM and that of DMSO was < 0.8% v/v. Compounds were screened over a total of 5 days, at an average of two plates per day, taking into consideration that the MTT assay incubation from cells seeding up to drug exposure was 3 days total. Test compounds were added to plates in batches of 2 at two hour intervals, to maintain the timing of additions and reads. Compound activity was calculated as the percentage inhibition in relation to positive and negative controls. The positive controls, sunitinib and sorafenib, were contained in each test plate, containing test compounds (2 per plate), and the negative control (no effect) comprised of %DMSO < 0.8%, in column 11 of each test compound assay plate. These in-plate negative controls were used in an effort to normalize compound activity in relation to any plate to plate variation in the assay signal. The in-plate positive controls were used to calculate compound activity for batches of 2 compound plates. Retest screening campaign Compounds identified from primary screening were retested against HUVEC cells in duplicate and at varying concentrations to obtain a dose-response curve. Thus, a defined volume of fresh compound stock solution was picked and serially diluted to 20mM, 10mM, 1mM, 0.1mM in dilution medium (DMSO). Then, 20mM, 10mM, 1mM, Original Research Article 59

3 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2): mM concentrations were respectively been prepared in a second dilution medium (high glucose DMEM without FBS). This resulted in a total of 4 doses per sample with 100µM as the highest concentration of test compound followed by 50µM, 25µM, 10µM, 1µM, and 0.1µM as the lowest one.the DMSO final working concentration for the MTT assay was maintained within 0.5% ~ 1.5%. Compound activity in the retest campaign was calculated as percentage inhibition in relation to positive and negative controls, in the same manner as the primary screening campaign. Data analysis Data were analyzed with GraphPad Prism and or Minitab software. Non-linear regression analyses were performed to generate dose-response curves and calculate IC 50 values. Data are presented as mean and SD. P<0.05 Results Screening steps statistic To discover new compounds with anti-proliferative activity against human umbilical vein endothelial cell, a list of 19 compounds was screened. The screening steps are schematically illustrated in figure 1. Four primary actives were obtained out of the 19 compounds, as displayed in figure 2, which represents the distribution of the normalized absorbance readings of the 19 compounds. After retesting in exactly the same conditions, 3 actives were confirmed (15.78% of the total). Figure 1: Schematic screening steps with number of compounds and percentage of total compound at each step Primary screen (100µM) (Table 1). However, in an effort to reduce false positives during the primary screening assay, several repeats This campaign was concerned with the screening of 19 were made for each dose response. Compounds with more compounds and for preliminary evaluation for possible than 70% activity were therefore considered active. From the cytotoxicity activity against HUVEC cells proliferation. The primary screening campaign, four compounds inhibited whole preliminary results were tabulated as percentage HUVEC growth by 70% (figure 2). inhibition generated per compound at high dose response Table 1: Summary of IR% per compound (100µM) Series No Compound Mean IR% SCYJ4 33±7.1 SCYJ5 35±5.1 SCYJ6-13 SCYJ7-29 SCYJ8 11±3.3 SCYJ15-10 SCYJ16 15±1.3 Original Research Article 60

4 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2): SCYJ17 3.3±4 9 SCYJ SCYJ19 19± SCYJ20 12± SCYJ21 50± SCYJ ± SCYJ26 72± SCYJ27 82± SCYJ28 81± SCYJ29 5± SCYJ31 3± SCYJ32 90±2.8 Figure 2: Small-scale screening of 19 compounds. Result of the primary screening of 19 compounds in duplicate setup. Compounds with more than 70% inhibition of the HUVEC cells proliferation were further tested. Retest screen Of the 4 primary actives that were retested, 4 compounds (75%) reconfirmed in duplicate to be more than 50% inhibitory in the HUVEC assay (SCYJ26:76±3.21; SCYJ27: 82±3.51; SCYJ28: 82±5.13; SCYJ32: 90±3.01). A doseresponse plateau is necessary for IC 50 values to be determined for these compounds. Hence, a compound needed to display about or more than 80% inhibitory in duplicate. There were only 3 compounds among the 4 reconfirmed actives that passed these criteria. For all these compounds, titration data were imported into GraphPad Prism and the IC 50 values estimated. All the 3 compounds produced a sigmoidal curve in the HUVEC assay and therefore could have an IC 50 value (figure 3A,B). Control plates, used as a measure of reproducibility, showed that the HUVEC assay had an average Z of 0.84 for sunitinib and 0.62 for sorafenib both the two as reference compounds (data not shown). Original Research Article 61

5 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2): (A) Inhibition % SCYJ28 Log[µM] (B) Inhibition % SCYJ32 Log[µM] pound IC 50 (µm) Com SCYJ27 SCYJ28 SCYJ ± ± ±0.9 Figure 3 (A): Dose-dependent effect of active compounds on the in vitro growth of human umbilical vein endothelial cells (HUVEC). The compound concentrations required to inhibit cell proliferation were measured by a spectrophotometer at OD 490 and the inhibitory rate was calculated according to the formula: Inhibition (%) = 1 - [a-b / c-b] x 100; where a is OD 490 value derived from a well added with a test chemical compound, b is mean OD 490 value derived from blank wells, c is mean OD 490 value derived from control wells (i.e., added culture medium as a test chemical). Each point represents the mean of quadruplicates; (B) half-maximal inhibitory concentration (IC 50 ) values calculated from dose-response curves as the concentration of compound yielding 50% of cell inhibition. They are expressed as means ± SD of three independent experiments. Selection of active (s) Our next goal was to select amongst the 3 confirmed active compounds the best candidates for further biological profiling. To this aim, compound with high cytotoxic activity was selected. The threshold for selecting best active was set as the average of different inhibition rate percentages across different (A) rounds of the retest screen plus three times the standard deviation [mean ODtest + 3 x SD]. All over, the scoring aimed to statistically identify the best active amongst the three, the one after threshold introduction has shown activity (IR%) 85% (figure 4). After scoring, 1 active compound was selected (5.26% of the total). (B) 90 Standard Threshold Mean IR% SCYJ27 SCYJ28 SCYJ32 Figure 4 (A): Selection of active (s) The line in dashes shows the active compound selection cutoff. Amongst the three compounds that have been subjected for scoring, one appeared to fulfill the criteria earlier introduced and the compound was SCYJ32. Notes: Original Research Article 62

6 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2):58-65 Standard refers to the initial IR% before the introduction of the threshold formula while Threshold refers to the new IR% after threshold formula introduction. (B) Chemical structure of SCYJ32[3-bromo-5-methyl-N-(4-((5-methyl-2-(4-(3-(4-methylpiperazin- 1-yl)propoxy)phenyl)oxazolo[5,4-d]pyrimidin-7-yl)amino)phenyl)picolinamide]. Cytotoxic activity of the selected active (SCYJ32) on tumor cells Three cancer cell lines were used in this experiment. These three cell lines were human breast adenocarcinoma cell line MCF7, human gastric cancer cell line SGC7901, cervix adenocarcinoma cell line Hela. These cells were used to test the efficacy of the selected active in vitro. Different dose responses (100, 10, 0.1 and 0.01µM) of the active compound were applied to different cancer cell line following the MTT assay protocol for cytotoxicity evaluation. After 48 h of exposure, data obtained spectrophotometrically were evaluated, means and standard deviations of relative cell viability are calculated (figure 5A). Data obtained after spectrophotometric reading were used to evaluate the inhibition activity. SCYJ32 was found to inhibit cancer cell line proliferation in a dose-response manner. SCYJ32 was found to produce a sigmoidal curve in the cancer cells assay and therefore could have an IC 50 value. Mean and standard deviations of relative IC 50 are calculated (figure 5B). The values were not significantly different from one another, as determined by a one way ANOVA in GraphPad Prism, with a significant difference of P<0.05. (A) (B) Cancer cell lines MCF7 SGC7901 HeLa IC50 (µm) 10.24± ± ±0.93 Figure 5:Identical number (5,000/well) of cells was plated onto 96-well plates and exposed to various doses of the active compound concentration (0.1, 1, 10, and 100µM) for 48 hours. (A) Dose-dependent effect of SCYJ32 on the in Vitro growth of MCF7, SGC7901, and HeLa. Cell survival is presented as a percentage of control-cell growth in cultures containing no drug. Each point represents the mean of triplicates; (B) Half-maximal inhibitory concentration (IC 50 ) values calculated from dose-response curves as the concentration of compound yielding 50% of control cell survival. They are expressed as means ± SD of three and two independent experiments for cancer cell lines. Cytotoxic activity of the selected active (SCYJ32) on MCF7 survival: clonogenic analysis One of the hallmarks of tumor cells is the ability to form colonies. MCF7 cells in exponential phase of growth were exposed to SCYJ32 (2.5µM, 5µM, and 10µM) and or sunitinib. Following drug exposure, the cells are disaggregated to form a single-cell suspension and are plated out at low density to allow colony formation. After 14 Days of treatment, the colonies formed are fixed with methanol, stained with crystal violet, and counted. Each colony is assumed to be derived from a single cell and thus the colony count is an estimate of the number of cells that survived the drug treatment. As shown in Figures below, there was a decrease in colony formation in MCF7 cells when treated with SCYJ32 and or Sunitinib in a concentration-dependent manner. The colonies formed by MCF7 were observed and photographed using a solid state TV camera (Motic AE21) attached to an inverted phase-contrast microscope. Images of the colonies were captured using a Motic Image Multi-Focus system (release by Motic china Group Co.,Ltd), a multi-focal-plane Original Research Article 63

7 Liu et al. / Indian J. Pharm. Biol. Res., 2015; 3(2):58-65 image combination and manipulation software for Windows. The photos below show the inhibitory actions of SCYJ32 and or Sunitinib in reducing colonies formation by MCF7 in a concentration-dependent manner (Figure 6A). Inhibition of colony formation in MCF7 by SCYJ32 and sunitinib was quantitated from photographs by counting the number of colonies contained in two random fields from each well. Data are expressed as a percentage of the number of colonies in untreated wells (Figure 6B). One way ANOVA analysis of the data was performed with Graphpad prism software under a comparison test approach. Figure 6: Photomicrographs of colony formation in MCF7 (A). The photos show the inhibitory actions of SCYJ32 and or Sunitinib in reducing colonies formation by MCF7 in a concentration-dependent manner. Quantitation of colony formation in MCF7 (B). All the data were analyzed with a statistical software, Graphpad prism. One-way ANOVA test was performed; ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05. Discussion and Conclusion New compounds are desperately needed to fight efficiently cancer cells proliferation. To this aim, we optimized a simple and straight-forward assay that allows the screening of compounds against human umbilical vein endothelial cells. We tested a total of 19 compounds provided by a Medicinal Chemistry Laboratory of the China Pharmaceutical University under cooperation. After screening the 19 compounds, 4 confirmed actives (21%) were obtained. The rate of actives was relatively high, probably due to one main reason: the highh concentration of compounds used for primary screening (100µM). After a secondary screening to eliminate these false positive actives and select the most effective compounds, three potential candidates (15.78% of all compounds) were identified as active. The three actives we selected had IC 50 values in the micromolar range. From the three actives been identified, one was selected as the best active compound after threshold formula introduction as the criteria for selection. Both the primary and retest screening campaigns were reproducible as exemplified by the statistical of the ANOVA. In conclusion, Whole cell viability screening assays are a good tool to identify new compounds with cytotoxic activity. In this study, we found one new compound, oxazolo[5,4- d]pyrimidine scaffold with 2-substituting aromatic ring and 7- substituting aromatic amine. It is apparent that the most important feature of this highly effective compound is the presence of pyrimidine and fused heterocyclic pyrimidine. We have also confirmed its cytotoxic efficacy in inhibiting the proliferation of the selected cancer cell line by MTT assay and clonogenic assay. All the above results demonstrated that this compounds could be promising lead compound of novel antitumor drugs. Further studies about the cellular mechanism of the potent compound are in progress. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgements Original Research Article This work was supported by the National Natural Science Foundation of China (NO , NO ), the National Science and Technology Major Project (No.: 2013ZX ), the National High Technology Research and Development Program 863 (No.: 64

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