PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biomol Screen. Author manuscript; available in PMC Jan 1, 2012.
Published in final edited form as:
PMCID: PMC3019245
NIHMSID: NIHMS260117
Chemical Genetic Screening for Compounds that Preferentially Inhibit Growth of Methylthioadenosine Phosphorylase (MTAP) Deficient Saccharomyces Cerevisiae
Yuwaraj Kadariya, Baiqing Tang, Cynthia B. Myers, Jami Fukui, Jeffrey R. Peterson, and Warren D. Kruger*
Cancer Biology Program, Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, Pennsylvania 19111, USA
*To whom correspondence should be addressed: warren.kruger/at/fccc.edu Tel: 215-728-3030, Fax: 215-214-1623
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP deficient cells. To determine if MTAP inactivation could be targeted, we have performed a differential chemical genetic screen in isogenic MTAP+ and MTAPS. cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP cells exhibiting a four-fold shift in IC50 compared to MTAP+ cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP yeast. These compounds were also examined for growth inhibition of isogenic MTAP+ and MTAP HT1080 fibrosarcoma cells, and four of the five compounds exhibited evidence of modest, but significant, increased potency in MTAP cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP cells.
Keywords: Methionine Salvage Pathway, Drug screening, Yeast, Genetic-chemical interaction
A quarter century ago, Toohey first recognized that certain murine malignant hematopoietic cell lines lacked methylthioadenosine phosphorylase (MTAP) activity.1 We now know that loss of MTAP activity occurs mainly through homozygous gene deletion and that it is common in many kinds of human cancers including non-small cell lung cancer, glioma, T-cell acute leukemia, bladder cancer, osteosarcoma, endometrial and pancreatic cancer.214 MTAP is expressed in all normal human tissues15 and is highly evolutionarily conserved and is found in organisms ranging from yeast to humans. In S. cerevisiae MTAP is encoded by MEU1, a gene originally identified in a screen designed to identify transcription factors.16 The high frequency of loss of MTAP expression in tumor cells combined with its apparent expression in all normal tissues suggests the idea of using MTAP-deficiency as a way to selectively target tumor cells.
MTAP functions as the first enzyme in the methionine salvage pathway that converts the byproduct of polyamine synthesis, 5'-deoxy-5'-methylthioadenosine (MTA), into adenine and methionine. This pathway is not essential at the cellular level as demonstrated by the fact that inhibition of MTAP function either by mutation or drug has no effect on cell growth under standard conditions.17,18 However, often in highly conserved pathways there are biochemical redundancies that explain why loss of a single gene does not give an obvious phenotype. In such situations it is often possible to create a novel phenotype by inhibiting two pathways at the same time. In genetics such interactions are termed synthetic lethal or synthetic sick interactions. In addition to gene-gene synthetic interactions, it is also possible to create such interactions using small molecules (chemical genetic lethality).19
In this study, we set out to determine if it was possible to identify small molecules that preferentially inhibit the growth of MTAP cells. As a test for the feasibility, we performed a chemical genetic screening in S. cerevisiae to identify compounds that can preferentially inhibit the growth of MTAP (meu1Δ) cells with a relatively simple screening methodology. Using this approach we have identified a novel class of compounds that can preferentially inhibit the growth of MTAP yeast and a MTAP human cell line.
Yeast strains
The base strain used in these experiments was RDY84 (Mata, pdr1ΔKAN, pdr3ΔHIS3, ade2, trp1, his3, leu2, ura3, can1). To create a meu1Δ version of this strain it was transformed with a MEU1Δ::LEU2 knock-out cassette using primers sense (5′- ATC AAA TCC GAT CAG CTT ACT GTG TTG ACG - 3′) and anti-sense (5′ - CAC GTT CAG TTG GTT GAA TGC AGA ACT TC - 3′) to amplify the cassette from yeast strain AS3-2a.18 The PCR product was sequenced and introduced into RDY84 using lithium acetate transformation. The transformed yeast was plated on synthetic complete media lacking leucine. The presence of the mutation was confirmed in two different ways; loss of MTAP enzyme activity and growth in adenine free medium supplemented with MTA. Basic yeast procedures were all performed as described previously.20
Yeast extract preparation and enzyme assay
Yeast extracts and MTAP enzyme assays were performed as described previously.18 One unit of MTAP activity is defined as the enzyme amount that catalyzes the formation of one μmol of adenine per minute under the conditions of the assay described.
Compound library
The collection of compounds used for screening was obtained from a commercial provider (Chemical Diversity, Inc., San Diego, CA). The compounds have been selected for maximum structural diversity while largely meeting Lipinski's empirically-derived “rule of 5” for drug-like properties21. The compounds were solubilized in dimethyl sulfoxide to a final concentration of 5 mM, formatted in 384-well microtiter plates, and stored at −80°C.
Screening methodology
Isogenic strains of yeast (MEU1+ and meu1Δ) were grown in SC media overnight; concentration was determined by measuring OD600, and then the strains were diluted to OD600 0.003 and 0.0015, respectively. The difference in starting cell number is to compensate for the slightly faster doubling time of the meu1Δ strain. Using an automated dispenser, 40 μl of culture was added to a 384 well plates (Corning Inc, NY). For each plate of the compound library tested, four yeast plates were made (two MEU1 and two meu1Δ). To each plate compound was added from the library using a 384 pin transfer apparatus (Scinomix lab, Earth City, MO). These pins are designed to transfer 20 nl compounds in each transfer resulting in a final compound concentration of 2.5 μM. Plates were sealed with Thermowell sealing tape (Fisher Scientific) and incubated at 30°C without shaking. Each plate had a column of untreated wells that were used to normalize between plates. A total of 30,080 compounds were tested for each strain in duplicate. After 18 hours, cells were resuspended by vortexing, plates were read at 570 nm by a plate reader, and statistical computation was done.
Data processing
The OD570 for compound-treated well was normalized by dividing with the average of the untreated wells on the same plate. The average of the normalized meu1Δ values were divided by normalized MEU1 values to get meu1Δ / MEU1 ratio. Hits were selected based on this ratio. A meu1Δ / MEU1 ratio of less than 0.5 (growth inhibition more than 50%) was set as a cutoff point. There were 118 compounds that showed at least two-fold increased growth inhibition in the meu1Δ strain compared with MEU1. In addition, we excluded those compounds which exhibited more than a 50% reduction in growth in MEU1 cells or exhibited a marked difference in OD between duplicate wells (SD >30%).
Dose response analysis
To determine IC50 concentrations meu1Δ and MEU1 yeast were plated in 100 μl of SC complete media in 96 well plates. Drug was added at concentrations ranging from 0 to 300 μM in 25 μM increments for compounds to each well in triplicate. For some compounds (3A and 7C) 10 μM increments were used. Growth was measured by OD570 after 18 hours. Dose response curve and IC50 were calculated using non-linear regression analysis using GraphPad Prism 4.0 software (GraphPad Software, Inc).
HT1080 studies
HT1080 cells are a human fibrosarcoma derived cell line that is known to lack MTAP expression.22 Stable MTAP expressing HT1080 cells were created by stable transformation with pTRE2:MTAP. This plasmid was created by inserting the hMTAP containing BamHI/EcoRV fragment from pCR:sMTAP22 into pTRE2hyg (Clontech, Palo Alto, CA). HT1080 cells (Clontech) were cultured in DMEM medium supplemented with 2 mM glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin, 10% fetal bovine serum (FBS), and 250 μg/ml G418. Transformations were performed using Fugene6 reagent (Roche, Indianapolis, IN), a nonliposomal transformation agent according to manufacturer's instructions. Four micrograms of pTRE2:MTAP were used for each transformation and pTRE2hyg was used as vector control. Clones were selected using 250 μg/ml Hygromycin from a 50 mg/ml stock solution in PBS (Sigma). Twenty-four individual clones transfected with pTRE2:MTAP were isolated and examined for MTAP expression by Western blot and all were found to express MTAP at similar levels. The pTRE2:MTAP and pTRE2 transfected cells were then pooled together in equal numbers to create two new pooled cell lines (called HT1080M+ and HT1080M−, respectively) in order to control for non-MTAP related clonal variation.
For dose response curves, HT1080M+ and HT1080M− cells were seeded onto 96-well cell culture plate in the medium mentioned above, at 3000 cells/0.2mL per well. After seeding for 24 h, testing agents were added at final concentration range from 0 (vehicle only) to 200 μM for an additional 48 hours. Cell viability was determined by CellTiter 96 Aqueous One Solution (MTS assay, Promega, Madison, WI) following manufacturer's instructions. Each treatment was carried out in triplicate. At the end of treatment, media was replaced by 100 μl regular media containing 15% Cell Titer 96 Aqueous One Solution and plate was incubated at 37°C for 3 h. The optical absorbance of the MTS metabolite formazin at 492 nm wavelength was recorded immediately by using an automated plate reader, Multiskan Ascent (Thermo Bioanalysis, Santa Fe, NM). Dose response analysis was performed identically to the yeast analysis described above.
Synthesis of compounds
Each of the compounds was generated using a two-step procedure in which the first step is the creation of the appropriate bromopropiophenone, and the second step reacts to this compound with thiosemicarbazide to produce the 1,3,4-thadiazine ring.23 Details are described below.
Materials
4-Chloropropiophenone, 3′,4′-dichloropropiophenone, thiosemicarbazide hydrochloride and anhydrous DMF were purchased from Acros Organics (available from VWR International). 2-Bromopropiophenone and 3′-chloropropiophenone were purchased from Sigma-Aldrich, St. Louis, MO. Chloroform was dried by storage over CaCl2. Ethanol was distilled from calcium hydride and stored over molecular sieves. All other reagents were from stock and were dried in vacuo for several hours prior to use. 1H NMR analysis was performed on a Bruker Avance 300 MHz wide-bore instrument.
Step 1 preparation of a-bromopropiophenone
(1) 2-Bromo-4′-chloropropiophenone
Exactly, 11.1 grams (0.0658 moles) of 4′-chloropropiophenone and 60 mg (0.00045 moles) of aluminium chloride were dissolved with stirring in 30 ml of chloroform under a nitrogen atmosphere at room temperature. Bromine (10.5 ml; 0.0658 moles) was added dropwise over 45 minutes. Then, the solution was stirred for 60 minutes and analyzed by TLC (10% toluene and 90% hexanes). HBr gas was removed with a nitrogen stream. The product was dried under reduced pressure (16.073 grams; 98% yield). 1H NMR (CDCl3): d 1.90 (d, 3H, 6.3 Hz); 5.22 (q, 1H, 6.3 Hz); 7.46 (d, 2H, 8.1 Hz); 7.97 (d, 2H, 8.1 Hz).
(2) 2-Bromo-3′-chloropropiophenone
Exactly, 5.53 grams of 3′-chloropropiophenone and 30 mg of aluminium chloride were dissolved with stirring in 20 ml of chloroform under a nitrogen atmosphere. Bromine (1.68 ml; 3.366g) was added dropwise over 30 minutes at room temperature. Then, the solution was stirred for 2 hours and analyzed by TLC (10% toluene and 90% hexanes). HBr gas was removed with a nitrogen stream. The product was dried under reduced pressure to give an orange liquid (7.73 grams, 95% yield). 1H NMR (CDCl3): d 1.90 (d, 3H, 6.6 Hz); 5.23 (q, 1H, 6.6 Hz); 7.41 (t, 1H, 8.1 Hz); 7.54 (d, 1H, 8.1 Hz); 7.88 (d, 1H, 8.1 Hz); 7.98 (s, 1H).
(3) 2-Bromo-3′,4′-dichloropropiophenone
Exactly, 1.125 grams of 3′,4′-dichloropropiophenone and 6 mg of aluminium chloride were dissolved with stirring in 20 ml of chloroform under a nitrogen atmosphere. Bromine (0.284 ml) was added dropwise over 30 minutes at room temperature. Then, the solution was stirred for 2 hours and analyzed by TLC (10% toluene and 90% hexanes). HBr gas was removed with a nitrogen stream. The product was dried under reduced pressure (1.654 grams; 100% yield). 1H NMR (CDCl3): d 1.91 (d, 3H, 6.6 Hz); 5.23 (q, 1H, 6.6 Hz); 7.59 (d 1H, 8.4 Hz); 7.86 (dd, 1H, 2.1 and 8.4 Hz); 8.12 (d, 1H, 8.4 Hz).
(4) 2-Bromo-4′-chlorovalerophenone
Exactly, 4.84 grams of 4′-chlorovalerophenone and 30 mg of aluminium chloride were dissolved with stirring in 20 ml of chloroform under a nitrogen atmosphere. Bromine (3.934g; 1.262 ml; 0.024622moles) was added dropwise over 30 minutes at room temperature. The product obtained was 7.004 grams (100% yield) after the usual work up. 1H NMR (CDCl3): d 0.99 (t, 3H, 7.2 Hz); 1.50 (m, 2H); 2.14 (m, 2H); 5.09 (t, 1H, 7.2 Hz); 7.46 (d, 2H, 8.7 Hz); 7.97 (d, 2H, 8.7 Hz).
Step 2 preparation of 1,3,4-thiadiazine hydrochlorides
(1) 2-Amino-5-(4-chlorophenyl)-6-methyl-1,3,4-thiadiazine hydrochloride (65 1A)
Exactly 500 mg (0.00548 moles) of thiosemicarbazide and 976 mg (0.00394 moles) of 2-bromo-4′-chloropropiophenone were stirred in 20 ml of dry ethanol under a nitrogen stream. The solution was stirred for 1 hour at room temperature followed by 2 hour of reflux. It was allowed to cool to room temperature overnight. The solid was filtered giving 988 mg (85% yield). 1H NMR (DMSO): d 2.39 (s, 3H); 7.50 (d, 2H, 8.1 Hz); 7.64 (d, 2H, 8.1 Hz).
(2) 2-Amino-5-(3-chlorophenyl)-6-methyl-1,3,4-thiadiazine hydrochloride (91A)
Exactly 500 mg of thiosemicarbazide and 976 mg of 2-bromo-3′-chloropropiophenone were stirred in about 20 ml of dry ethanol under a nitrogen stream. The solution was stirred for 1 hour at room temperature followed by 2 hour of reflux. It was allowed to cool to room temperature overnight. The slightly yellow solid was filtered (863 mg, yield 91%). 1H NMR (DMSO): d 2.46 (s, 3H); 7.45 (m, 2H); 7.62 (d, 2H, 7.8 Hz); 7.74 (s, 1H).
(3) 2-Amino-5-(3,4-dichlorophenyl)-6-methyl-1,3,4-thiadiazine hydrochloride (3A)
Exactly 500 mg of thiosemicarbazide and 1.111 g of 2-bromo-3′,4′-dichloropropiophenone were stirred in 20 ml of dry ethanol under a nitrogen stream. The solution was stirred for 1 hour at room temperature followed by 2 hour of reflux. It was allowed to cool to room temperature overnight. The resulting solid was filtered (334 mg; 31%; NMR). 1H NMR (DMSO): d 2.47 (s, 3H); 7.62 (dd, 1H, 1.8 and 8.4 Hz); 7.71 (d, 1H, 8.4 Hz); 7.90 (d, 1H, 1.8 Hz).
(4) 2-Amino-5-phenyl-6-methyl-1,3,4-thiadiazine hydrochloride (93A)
Exactly 500 mg of thiosemicarbazide and 0.589 ml (840 mg) of 2-bromopropiophenone were stirred in about 20 ml of dry ethanol under a nitrogen stream. The solution was stirred for 1 hour at room temperature followed by 2 hour of reflux. It was allowed to cool to room temperature overnight. A light yellow solid was filtered (604 mg, yield 75%). 1H NMR (DMSO): d 2.39 (s, 3H); 7.36 (m, 1H); 7.46 (t, 2H, 7.8 Hz); 7.61 (d, 2H, 7.8 Hz).
(5) 2-Amino-5-(4-chlorophenyl)-6-propyl-1,3,4-thiadiazine hydrochloride (7C)
Exactly 500 mg of thiosemicarbazide and 1.086 g of 2-bromo-4′-chlorovalerophenone were stirred in 20 ml of dry ethanol under a nitrogen stream. A solid was filtered and the filtrate was reduced in vacuo. The residue was taken up in chloroform, filtered and the filtrate was evaporated in vacuo to a yellow solid (1.22 g; 100% yield) was analyzed by NMR. 1H NMR (DMSO): d 0.90 (t, 3H, 7.5 Hz); 1.59 (m, 2H, 7.5 Hz); 2.76 (t, 2H, 7.5 Hz); 7.54 (d, 2H, 8.7 Hz); 7.61 (d, 2H, 8.7 Hz).
Creation and characterization of isogenic MEU1 and meu1Δ strains
To facilitate small compound screening in S. cerevisiae we first created isogenic MEU1 and meu1Δ strains by single step gene replacement in a strain background deleted for the multi-drug resistance genes PDR1 and PDR524. Characterization of meu1Δ::LEU2 containing cells shows that they lack MTAP activity (Fig. 1A) and are unable to use MTA instead of adenine to complement their ade2 mutation (Fig. 1B). Thus MTAP activity is effectively lost in meu1Δ cells. Growth rates between the two strains were only slightly different, with meu1Δ::LEU2 growing slightly faster that MEU1 cells (Fig. 2).
Figure 1
Figure 1
Characterization of MTAP status in isogenic MEU1 and meu1Δ cells. A. MTAP enzyme activity present in lysates derived from indicated cells. Assays were done as described in methods in quadruplicate and error bars show standard deviation. B. Growth (more ...)
Figure 2
Figure 2
Growth rate comparison of MEU1 and meu1Δ cells. Saturated cultures of isogenic MEU1 and meu1Δ strains were diluted to the indicated OD570 in 3 ml of SC media in test tubes and grown with aeration at 30°C. OD570 and growth was monitored (more ...)
Screening
The basis of our screen was to identify compounds that preferentially inhibited the growth of MEU1cells vs. meu1Δ cells. Our screening strategy involved growing, isogenic MEU1 and meu1Δ cells in 384 well microtiter plates and testing all compounds in duplicate plates (Fig. 3). Each plate had a total of 320 wells that were exposed to drug, 48 wells that were not exposed to drug, and 16 wells that contained media, but no cells. Examination of the growth in untreated wells containing cells revealed that the intra-plate co-efficient of variation (CV) was 11.8% in MEU1 cells and 12.5% in meu1Δ cells. The inter-plate CV for untreated cells was slightly higher, 17.1% for MEU1 cells and 14.5% for meu1Δ cells. To control for this increased plate to plate variability, the data on each plate was normalized by dividing each of the compound treated wells OD570 value by the average of the untreated wells on the same plate.
Figure 3
Figure 3
Outline of methodology used for screening. On the left is a flow chart describing the screening methodology. On the right is a diagram showing the set up of each 384-well plate.
In the primary screen, we examined the growth effects of 30,080 compounds added to each well at a final concentration of 2.5 μM. In general, the vast majority of compounds tested had no effect on yeast cell growth for either strain. Using a 50% reduction in mean growth as a cutoff, we found that 1.8% of the compounds exhibited growth inhibition in MEU1 cells and 2.1% of the compounds exhibited growth inhibition in meu1Δ cells. Searching for compounds that exhibited at least a two-fold increased growth inhibition in MEU1 vs. meu1Δ cells identified 218 compounds, giving the hit rate of ~0.72%. Out of 218 compounds, 80 compounds met the criteria that they had less than 50% inhibition in MEU1 cells and had a CV between the two replicates of <30%. These 80 compounds were then retested using that five times the original concentration of drug and 11 showed differential growth upon retest (Fig. 4A). We then used these 11 compounds for dose-response experiments to determine the relative IC50 of each compound for each strain. Only one compound (1487–1117) containing a 1,3,4-thiadiazine ring exhibited a dose-response curve in which a statistically significant difference between MEU1 and meu1Δ cells could be observed (Fig. 4B, Table 1). Unfortunately, the supplier of the compound library (Chemical Diversity, Inc.) was unable to re-synthesize this compound despite several attempts. Because we suspected the 1,3,4-thiadiazine ring might be important for function, and this part of the molecule was relatively simple to produce, we examined the effect of a series of 1,3,4-thiadiazine compounds that were produced in house on cell growth. These compounds all contained a 2-amino-1,3,4-thiadiazine ring and a phenol ring with modifications at either 3 or 4 position (Fig. 5). We found that loss of MTAP resulted in increased growth inhibition for all but one of the compounds examined. The calculated IC50 for each compound are shown in Table 1. Interestingly, the one compound that failed to show differential effect was compound 93A, which was the only compound to lack a halogen modification of the phenol ring.
Figure 4
Figure 4
Effect of compounds in MEU1 and meu1Δ cells in secondary screening. A. Saturated cultures of MEU1 or meu1Δ cells were used to inoculate 100 μL of media in 96-well plate at a final OD570 of 0.030 for MEU1 and 0.015 for meu1Δ (more ...)
Table 1
Table 1
Yeast IC50 values
Figure 5
Figure 5
Dose response curves in MEU1 and meu1Δ cells for the indicated 2-amino-1,3,4-thiadiazine ring containing compounds. Dose response curves were constructed as described in methods. Structures are shown in each panel on the left.
Studies in isogenic MTAP+ and MTAP human cells
We next tested the compounds identified as having differential growth inhibition in yeast in isogenic MTAP+ and MTAP human cell lines. These cells were created by expressing MTAP in a MTAP deleted HT1080 fibrosarcoma cell line (see Methods). For each compound we performed dose response experiments and calculated IC50. In addition, we tested three other cytotoxic agents used to target de novo nucleotide biosynthesis, including 5-flurouracil, 6-methylmercaptopurine ribonucleotide (6-MMPR), and alanosine (Table 2). All of the experimental compounds tested had reduced IC50 values in MTAP vs. MTAP+ cells, with four of the five compounds having no overlap within the 95% confidence interval. None of the control compounds showed statistically significant differences in IC50.
Table 2
Table 2
HT1080 IC50 values
In the work described here, we have performed a chemical genetic screen designed to find compounds that can preferentially inhibit the growth of yeast cells deleted for MTAP. Using this screen we have identified a series of compounds containing a 1,3,4-thiadiazine ring that have enhanced growth inhibition in both yeast and human cells lacking MTAP activity. The technology used to perform the screening was simple and did not require large amounts of expensive robotic equipment. To differentially screen 30,080 compounds in duplicate required approximately 200 plates and could be performed by a single person in a few weeks. However, there are some limitations to the approach. First, there was a high rate of false positives in the primary screen, and it was essential to include secondary and tertiary screens to confirm potential hits. We suspect that the high rate of false positives may be related to the variability of drug transferred to each well by the disposable pin transfer units. A second limitation of the approach is that only one concentration of the starting compound was tested. Since most of the compounds tested have relatively steep dose response curves, compounds that may be differentially effective may be missed either due to too high or too low drug concentrations. A third limitation concerns the ease of re-synthesis of “hit” compounds. It is sometimes difficult to re-synthesize the compound in larger quantities, as was the case in our initial hit compound, 1487–1117. A final limitation of this approach is that the actual molecular targets of the drug molecules identified are unknown and determination of the target(s) would not be trivial.
The compounds identified as having increased growth inhibition in meu1Δ yeast contained a 1,3,4-thiadiazine ring and a phenol ring. Compounds containing these moieties have been shown to have a variety of biological effects including anti-microbial activity, anti-inflammatory activity, act as phosphodiesterase inhibitors, and metalloprotease inhibitors 23,2527 In yeast, the halogenation of the phenol ring appeared to be important, as the one compound lacking halogenation, 93A did not show differential growth inhibition. Interestingly, halogenation of the same position of the phenol was shown to increase the effectiveness of related compounds in both an anti-inflammatory assay and as an inhibitor of matrix metalloproteases.23,27 It should also be noted that the parent compound 1487-117 was the most potent with regards to growth inhibition in yeast in both MEU1 and meu1Δ cells. This compound is distinguishable from the other compounds in the series by two modifications: 1) the addition of a brominated phenol group attached to the amino group hanging off C2 of the thiadizaine ring and 2) alkylation of the N4 position of the same ring. Further studies will be required to understand the importance of these modifications with regards to the increased potency of this compound.
The reason for the increased sensitivity of MTAP cells to these compounds is unknown. MTAP is highly conserved in evolution, but at the cellular level seems to be entirely dispensable for growth. MTAP's known enzymatic activity is to provide the cell with adenine and methionine from MTA. However, in the yeast assay described here, adenine and methionine are supplemented in the media, so interference with the de novo production of these metabolites is not likely to result in a growth phenotype. However, it may be that MTAP has other as yet undescribed functions. Interestingly, the yeast MTAP orthologue, MEU1 (Multi-copy enhancer of UAS1), was initially identified in a genetic screen designed to identify transcription factors regulating the Adh2 gene.16 Also, microarray experiments show there are over 300 transcripts that are differentially regulated between MTAP+ and MTAP HT1080 cells (WDK, in preparation). Taken together, these observations suggest that loss of MTAP may have more wide ranging effects on cellular physiology than previously thought. It is possible that the compounds described here have increased sensitivity in MTAP cells because it affects one of these other MTAP regulated pathways.
Even though the screening system described here was successful in identifying compounds that had some increased potency in inhibiting the growth of MTAP yeast and mammalian cells, the differential IC50 of these compounds was relatively modest. Furthermore, in HT1080 cells none of the compounds had IC50 values less than 10 μM. However it is possible that further optimization of the compounds may lead to increased potency and potential usefulness. Alternatively, screening a much larger compound library using isogenic MTAP+ and MTAP human cell lines may reveal new compounds with greater selectivity and potency. The feasibility of this approach is strengthened by the findings presented here.
ACKNOWLEDGMENTS
This work was supported by grants for the NIH (CA131024), The American Cancer Society (RSG-03-157-01-GMC), and an appropriation form the Commonwealth of Pennsylvania. We also acknowledge the contribution of the Fox Chase Cancer Center DNA sequencing facility.
1. Toohey JI. Methylthioadenosine nucleoside phosphorylase deficiency in methylthio-dependent cancer cells. Biochem Biophys Res Commun. 1978;83:27–35. [PubMed]
2. Subhi AL, Tang B, Balsara BR, Altomare DA, Testa JR, Cooper HS, et al. Loss of methylthioadenosine phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer. Clin Cancer Res. 2004;10:7290–7296. [PubMed]
3. Hustinx SR, Hruban RH, Leoni LM, Iacobuzio-Donahue C, Cameron JL, Yeo CJ, et al. Homozygous deletion of the MTAP gene in invasive adenocarcinoma of the pancreas and in periampullary cancer: a potential new target for therapy. Cancer Biol Ther. 2005;4:83–86. [PubMed]
4. Schmid M, Malicki D, Nobori T, Rosenbach MD, Campbell K, Carson DA, et al. Homozygous deletions of methylthioadenosine phosphorylase (MTAP) are more frequent than p16INK4A (CDKN2) homozygous deletions in primary non- small cell lung cancers (NSCLC) Oncogene. 1998;17:2669–2675. [PubMed]
5. Brat DJ, James CD, Jedlicka AE, Connolly DC, Chang E, Castellani RJ, et al. Molecular genetic alterations in radiation-induced astrocytomas. Am J Pathol. 1999;154:1431–1438. [PubMed]
6. Stadler WM, Olopade OI. The 9p21 region in bladder cancer cell lines: large homozygous deletion inactivate the CDKN2, CDKN2B and MTAP genes. Urol Res. 1996;24:239–244. [PubMed]
7. Garcia-Castellano JM, Villanueva A, Healey JH, Sowers R, Cordon-Cardo C, Huvos A, et al. Methylthioadenosine phosphorylase gene deletions are common in osteosarcoma. Clin Cancer Res. 2002;8:782–787. [PubMed]
8. Dreyling MH, Roulston D, Bohlander SK, Vardiman J, Olopade OI. Codeletion of CDKN2 and MTAP genes in a subset of non-Hodgkin's lymphoma may be associated with histologic transformation from low- grade to diffuse large-cell lymphoma. Genes Chromosomes Cancer. 1998;22:72–78. [PubMed]
9. M'Soka TJ, Nishioka J, Taga A, Kato K, Kawasaki H, Yamada Y, et al. Detection of methylthioadenosine phosphorylase (MTAP) and p16 gene deletion in T cell acute lymphoblastic leukemia by real-time quantitative PCR assay. Leukemia. 2000;14:935–940. [PubMed]
10. Hori Y, Hori H, Yamada Y, Carrera CJ, Tomonaga M, Kamihira S, et al. The methylthioadenosine phosphorylase gene is frequently co-deleted with the p16INK4a gene in acute type adult T-cell leukemia. Int J Cancer. 1998;75:51–56. [PubMed]
11. Karikari CA, Mullendore M, Eshleman JR, Argani P, Leoni LM, Chattopadhyay S, et al. Homozygous deletions of methylthioadenosine phosphorylase in human biliary tract cancers. Mol Cancer Ther. 2005;4:1860–1866. [PubMed]
12. Chen YJ, Lin SC, Kao T, Chang CS, Hong PS, Shieh TM, et al. Genome-wide profiling of oral squamous cell carcinoma. J Pathol. 2004;204:326–332. [PubMed]
13. Illei PB, Rusch VW, Zakowski MF, Ladanyi M. Homozygous deletion of CDKN2A and codeletion of the methylthioadenosine phosphorylase gene in the majority of pleural mesotheliomas. Clin Cancer Res. 2003;9:2108–2113. [PubMed]
14. Wong YF, Chung TK, Cheung TH, Nobori T, Chang AM. MTAP gene deletion in endometrial cancer. Gynecol Obstet Invest. 1998;45:272–276. [PubMed]
15. Kamatani N, Nelson-Rees WA, Carson DA. Selective killing of human malignant cell lines deficient in methylthioadenosine phosphorylase, a purine metabolic enzyme. Proc Natl Acad Sci U S A. 1981;78:1219–1223. [PubMed]
16. Donoviel MS, Young ET. Isolation and identification of genes activating UAS2-dependent ADH2 expression in Saccharomyces cerevisiae. Genetics. 1996;143:1137–1148. [PubMed]
17. Christopher SA, Diegelman P, Porter CW, Kruger WD. Methylthioadenosine phosphorylase, a gene frequently codeleted with p16(cdkN2a/ARF), acts as a tumor suppressor in a breast cancer cell line. Cancer Res. 2002;62:6639–6644. [PubMed]
18. Subhi AL, Diegelman P, Porter CW, Tang B, Lu ZJ, Markham GD, et al. Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites. J Biol Chem. 2003;278:49868–49873. [PubMed]
19. Parsons AB, Lopez A, Givoni IE, Williams DE, Gray CA, Porter J, et al. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell. 2006;126:611–625. [PubMed]
20. Sherman F. Getting started with yeast. Methods Enzymol. 2002;350:3–41. [PubMed]
21. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001;46:3–26. [PubMed]
22. Tang B, Li YN, Kruger WD. Defects in methylthioadenosine phosphorylase are associated with but not responsible for methionine-dependent tumor cell growth. Cancer Res. 2000;60:5543–5547. [PubMed]
23. Schroder J, Henke A, Wenzel H, Brandstetter H, Stammler HG, Stammler A, et al. Structure-based design and synthesis of potent matrix metalloproteinase inhibitors derived from a 6H-1,3,4-thiadiazine scaffold. J Med Chem. 2001;44:3231–3243. [PubMed]
24. Balzi E, Wang M, Leterme S, Van Dyck L, Goffeau A. PDR5, a novel yeast multidrug resistance conferring transporter controlled by the transcription regulator PDR1. J Biol Chem. 1994;269:2206–2214. [PubMed]
25. Abdel-Rahman RM. Role of uncondensed 1,2,4-triazine derivatives as biocidal plant protection agents--a review. Pharmazie. 2001;56:195–204. [PubMed]
26. Skoumbourdis AP, Leclair CA, Stefan E, Turjanski AG, Maguire W, Titus SA, et al. Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors. Bioorg Med Chem Lett. 2009;19:3686–3692. [PMC free article] [PubMed]
27. Tozkoparan B, Aytac SP, Aktay G. Novel 3,6-disubstituted 7H-1,2,4-triazolo[3,4-b][1,3,4]thiadiazines: synthesis, characterization, and evaluation of analgesic/anti-inflammatory, antioxidant activities. Arch Pharm (Weinheim) 2009;342:291–298. [PubMed]