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Anticancer Screens for hTopo I, IIβ(Inhibitors, Poisons).

In Vitro Screening Services
Vivo Screening Services

Anticancer Screening: TopoGEN offers powerful and tractable services to identify Topoisomerase-targeting drugs. Topo targeting drugs can be subdivided into two large groups:
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  • Catalytic Inhibitory Compounds (or CICs) These drugs block or restrict catalytic activity of the enzyme. Typically, CICs will act at steps 1 or 2 in the overall reaction sequence (see Fig. 1). By preventing the non-covalent (electrostatic or ionic bridge) complexes which are necessary to proceed to the cleavage complex (step 3), the reaction is effectively blocked. There are a number of topo II CICs (ICRF-187) and many fewer topo I CICs. Note that CICs can be highly non-specific since even elevated NaCl can be a CIC. In other cases, strong DNA intercalators may interfere with the catalytic reaction sequence. Many in the field simply view a CIC as any agent that can interfere with the ability of a poison (like CPT and topo 1) to form cleavage complexes (ie, can ‘reverse or block’ the action of an IFP). This sort of dual drug testing approach can be complicated and we at TopoGEN have the view that it is better to directly test CICs in the absence of other drugs. We offer this as a service
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  • Interfacial Poisons (or IFPs) Interfacial Poisons (or IFPs): These agents are technically poisons because they inhibit the re-ligation step in the sequence (‘poison’ the reaction sequence). Consequently, the enzyme becomes covalently trapped on the DNA target sequence and concurrently DNA cleavage is stabilized (single strand nice with topo I and double strand DNA break for topo II). Mechanistically, it appears that IFPs intervene stereochemically in the cleavage complex, forming what is called a ‘ternary complex’ Topogen Reaction((topo+drug+DNA) wherein the IFP drug induces a mis-alignment of the cleavage intermediate making it unable to re-ligate. When testing IFPs (see Step 3, Fig. 1) it is vital that you perform a proteinase K digestion step, otherwise the cleavage products will shift in the agarose gel, making interpretation impossible.

    Note that some drugs may display mixed modes of action, behaving as both CIC and IFP. In most cases, the inhibitory and cleavage maximal effects may occur at different concentrations of drug. Thus, they may titrate very differently.
Our Screening Services: Independent Validation, Expediency, Value.

At TopoGEN, we developed, trademarked and patented many of the technologies associated with drug effects on DNA. We are leading experts at drug testing and assessment of drug action from a mechanistic standpoint. When you engage our services, we will describe and lay out a logical/rational approach that is rich in content and strongly mechanistic. We offer independent consultants who can validate our findings. Our services offer expediency and value as well. We can significantly accelerate the pace of your research, produce publication quality data and you will own the intellectual property that comes with our studies. We routinely enter into confidentiality agreements on projects, so your results are protected. Our in-house testing program is flexible and efficient. The following are important points associated with our services:

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  • All positive and negative controls We demonstrate internally that all testing platforms are perfectly functional. As a result, there is no guessing. Your data will be clear and unambiguous. We stand behind our findings and certify the data.
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  • All markers will be included To document and validate the results. Again, we certify tractable results that will be internally controlled.
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  • Testing Flexibility We tailor each contract to offer maximum flexibility and coordinate with you over the course of the project to ensure intellectual flow of ideas and results. We will advise you on the best course of action with all hits. You can select the level of detail for the project that best suits your needs
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  • Over-run Flexibility In most if not all projects, there will be a need to re-examine, re-test or alter the course. How do we correct for work-load adjustments in the mid-stream? Our solution: we include on all projects, a small indirect “over-run fee” that will allow us to perform additional key experiments without asking for additional funds. This is an advantage for clients and ensures that our high standards of data quality will be maintained. For example, if additional testing regimens are required, we carry out the service automatically. Importantly, there is no guessing on the budget and you will not pay additional fees.
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  • Consulting We are a team of topo experts. All projects include email and direct SKYPE support over the course of the project. Our scientific staff will discuss and suggest future prospects or experiments.
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  • Reporting For complete flexibility and to ensure cost-effective contracts, we offer two levels of reporting. At a basic level, included in all contracts, we provide well-documented data (powerpoint, PDF or password protected cloud account). We draw conclusions, presented as bullets, on each slide and make a recommendation for further development and maturation of the project. For clients who wish to have a & ´manuscript´ style report, we offer fully referenced publication style reports. These reports are professionally prepared and are suitable for presentation to regulatory agencies. These publication style reports can be reviewed independently by third party opinion leaders in the field and the reviews appended to the final report.
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  • Hands-on Experience The company has been actively engaged in contract drug testing since the 1990s and we have the experience to get results that will enable ‘go/no go’ decision making on drug development.
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  • Expedience In most cases, the project will be completed over a period of several business days. We will clearly stipulate how long the project will take and we deliver on time.

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  • Expedience In most cases, the project will be completed over a period of several business days. We will clearly stipulate how long the project will take and we deliver on time.
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  • A Selection of Enzyme Targets We can test prokaryotic and eukaryotic systems with highly purified enzymes that are well-controlled reagents. We can provide additional documentation of purity and QC on all preparations. The following enzymes or tractable antibodies are currently in our inventory:
    • Human DNA Topoisomerase I
    • Human DNA Topoisomerase IIα
    • Human DNA Topoisomerase IIβ (in vivo or ICE screens only, see next item below)
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  • Advanced testing in living cells TopoGEN is a pioneer in testing agents that operate in the context of the cell. For eukaryotic systems, we can perform rigorous testing in tissue culture cells. We offer a variety of cell lines suitable for topo I, IIα and IIβ testing. For example, we can test your drug (either as CIC or IFP) in virtually any cell line of interest.
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  • The “Dual Gel Analytical System“ TopoGEN is unique in offering this service as part of our routine testing regimen. In all projects, we design our experiments to garner new information about your compound. For example, all gels are run in duplicate. One is a non-EB gel system, used to demonstrate unambiguous catalytic activity and the other gel is an EB containing gel to parse clearly the cleavage products. Since the reactions come from the same tube, we can be sure that the results are reproducible and accurate. Importantly, the non-EB gel system also reports whether the drug is a DNA intercalator, which may suggest genotoxicity in cells.
Some Sample Data: Identifying CIC and IFC , Active Agents.

Detecting a Topo I CIC active agent.
In this analysis, the client asked if we could test whether his compound was acting specifically as a CIC (catalytic inhibitor). We designed the experiment to test this idea (see Fig. 2). The customer also requested that we NOT use camptothecin (CPT) in this analysis (some researchers test for CIC drugs by testing whether the drug can reverse CPT induced cleavages). To avoid the use of CPT in this analysis, we used very high input levels of topo I which enhances topo I cleavage activity in the absence of CPT, due to the excessive amount topo I relative to DNA. As shown in Fig. 2, reactions containing more than 25 units of topo I accumulate the nicked open circular cleavage products (“OC” in Fig. 2, lanes 1-5). Under these conditions, there is such a large stoichiometric excess of enzyme, when we titrate 100 units of topo I against CPT concentrations (lanes 6-10) we can see double strand DNA breaks because one enzyme molecule is acting contiguous to another which converts single strand nicks into DS breaks (see “Lin” in lane 10). These data define an excellent model to search for topo I drugs that act as CICs and we can readily avoid mixing the test drug with CPT in the same reaction. As shown in lanes 11-16 (Fig. 2) the actual yield of the cleavage product (OC DNA marker lane 1) is strongly reduced with increasing amount so the test drug. In this case, we calculate an IC 50 value of roughly 2.4 uM.

Figure 2
Analysis of a novel Topo I CIC active agent that inhibits the ability of the enzyme to induce DNA cleavages. Reactions were carried out as described in the Topo I Drug screening kit (TG1018). All reactions were incubated with indicated reactants for 30 min at 37oC, terminated and digested with Proteinase K included in the kit. Gels containing EB (0.5 ug/ml) were run at 2 v/cm until the tracking was 80% migrated down the gel. In this experiment, we titrated excess topo I input levels in the absence of any CPT to establish conditions were nicked DNA (OC) were trapped in the absence of an IFP such as CPT. The data show that excessive input levels of topo I (lanes 2-5) facilitate formation of nicked OC DNA products in the absence of CPT. Addition of CPT greatly enhances this result (lanes 6-10) and induces multiple DNA nicking of the plasmid (pHOT1) substrate, forming linear DNA products (“Lin”, lane 10). Increasing concentrations of test drug (lanes 11-16) clearly reduce the yield of OC DNA showing that this test compound is a CIC class of drug. The fact that it cannot induce linears or fully convert the small amount of relaxed (Rel) DNA to OC forms, further documents that this particular compound is not an IFP class of drug.

Detecting a Topo I IFP active agent: An example of our Dual Gel Analysis System
In this analysis, we tested whether a drug might be an IFP, CIC or intercalator using our dual gel system. The controls, VP16 (a topo II IFP) and CPT (topo I IFP) clearly show that the reaction is reporting accurately since the topo II IFP drug does not induce cleavages while the topo I IFP drug (CPT control) clearly induces accumulation of nicked OC DNA (red ring in Panel B). These data clearly show that test drug X is neither a CIC nor an IFP. Additionally, the test drug does not alter the distribution of topoisomers and is not a DNA intercalator. Gel A is important since it reveals that the enzyme is highly active under the conditions of the experiment (as attested by the loss of SC DNA and conversion to topoisomers). Gel B is important because it clearly resolves nicked OC DNA from the other reactants (red ring, blue arrow). Note that in Gel B (EB gel) the resolution of SC and relaxed DNA species is very poor, which is normal; however, Gel A clearly shows that the enzyme was highly active.

Figure 3
Dual Gel Analysis for Topo I CIC, IFP and Intercalation. Reactions were assembled with control drugs (VP16, CPT) and test drug X (titrated over a wide range indicated). Markers (left two lanes) were included for reference: OC: is nicked OPEN CIRCULAR pHOT1; Lin is linearized pHOT1 DNA marker (restricted with EcoR1); SC is the supercoiled DNA substrate. These forms are sometimes referred to as Form II, III, and I, respectively. Reactions were carried out (30 ul Final volume) and terminated by digestion with proteinase K. Sample loading dye was added (6 ul) and each reaction divided in half (15 ul each). Two gels were prepared, with and without 0.5ug/ml EB. Each lane was loaded with 15 ul and gels were electrophoresed at 2 volts/cm for 90min. Gels were removed and stained with EB for 20 min, (gel A only) followed by de-staining for 15 min in water. Gel B was destained for 20 min in water. Both gels were imaged on an automated system.

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