NCI-60

NCI-60 One-Dose Screen

General Description

As of early 2007, all compounds submitted to the NCI-60 Cell screen are tested initially at a single high dose (10-5 M) in the full NCI-60 cell panel. Only compounds which satisfy a pre-determined threshold of anti-proliferative activity (e.g. growth inhibition) in a minimum number of cell lines are advanced to the five-dose screen. The criteria for progression to the five-dose screen is selected to efficiently capture compounds with anti-proliferative activity based on careful analysis of historical DTP screening data. The threshold for advancing to the five-dose screen may be updated as additional data becomes available.

Interpretation of One-Dose Data

The one-dose data is reported as a mean graph of the percent growth inhibition of treated cells and is similar in appearance to mean graphs from the five-dose assay. The number reported for the one-dose assay is growth inhibition relative to the no-drug control, and is based on the number of live cells at time zero. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). These measures also apply to the five-dose screen. For example, a value of 100 means no growth inhibition. A value of 40 means 60% growth inhibition. A value of 0 means no growth inhibition. A value of -40 means 40% lethality. A value of -100 means all cells are dead. Information from the one-dose mean graph is available for COMPARE analysis.

NCI-60 Five-Dose Screen

Compounds that exhibit significant growth inhibition in the one-dose screen are evaluated against the NCI-60 cell panel at five concentration levels.

The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μL at plating densities ranging from 5,000 to 40,000 cells/well, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37°C, 5% CO2, 95% air and 100% relative humidity for 24 hours prior to addition of experimental drugs.

After 24 hours, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Additional 4-fold, 10-fold, or ½ log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium to yield the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48 hours at 37°C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same, except the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations. Percentage growth is calculated as:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz

[(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz.

Three dose response parameters are calculated for each experimental agent. Growth inhibition of 50% (GI50) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment is calculated from [(Ti-Tz)/Tz] x 100 = -50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.

Compound Submission for NCI-60 Evaluation

** The National Cancer Institute is not currently accepting compound submissions for the NCI-60 screen that originate from outside the United States and U.S. Territories. Updates to this policy will appear on this website. **

NCI-60 Submission Request Procedure (Account Setup and Compound Submission)

All compound submissions for NCI-60 screening are processed through the DTP Compound Submission Application. Submitters may use this tool to:

• submit requests
• process confidentiality disclosures and material transfer agreements
• view packing/shipping details
• check status of their submissions
• access results

Requests for assistance or questions regarding the request procedure should be sent to Keania Colbert (keania.colbert@nih.gov).

User Account Setup

Investigators interested in submitting compounds for NCI-60 evaluation are required to complete a one-time account setup through the DTP Compound Submission Application prior to screening request. During account setup, investigators will create a User ID and Password then receive a Supplier Code. Information required for account setup is outlined below:

• name
• institution
• address
• e-mail address

Requests for assistance or questions regarding account setup should be sent to Keania Colbert (keania.colbert@nih.gov).

Compound Submission

** The National Cancer Institute is not currently accepting compound submissions for the NCI-60 screen that originate from outside the United States and U.S. Territories. Updates to this policy will appear on this website. **

Once submitters have created their account, they may initiate a request by selecting "Submit Compounds" within the DTP Submission Application. Information required is listed below:

• chemical structure
• stereochemistry
• molecular weight
• physical state
• institutional signatory authority name
• institutional signatory authority e-mail address

Chemical structures of individual entries must be submitted for each compound. Structural information can be entered using either the embedded chemical drawing program or added as a mol file.

Once submitted, the request is routed to the requester's designated signatory authority. The submitter's institutional signatory authority is required to agree to an online standard, non-negotiable, Confidentiality Disclosure/Material Transfer Agreement (CDA/MTA). The submission request is then forwarded to DTP staff for review upon agreement to the CDA/MTA. If the CDA/MTA is not executed by the submitter's institutional authority within 30 days, the request will expire and must be resubmitted. The following minimum information is required at time of submission request:

If compounds are of particular interest based on a biological hypothesis or data, or similar chemical analogs, it is recommended that a brief explanation be entered in the appropriate field to strengthen the case for selection. After the DTP selection process is complete, the supplier receives e-mail notification of a decision and if selected, instructions for mailing the physical sample(s) to the NCI Repository. Investigators should not send in any samples until instructed. The sample size requirement is 10-15 mg, but for relatively inaccessible natural products, 5 mg is acceptable.

After compounds are received at the NCI Repository, they are assigned an identifying NSC number, inventoried and placed into the NCI-60 screening queue. All subsequent communications regarding a compound should include its NSC number.

Any unused sample will be subject to the terms of the CDA/MTA.

There is a limit of 10 structure submission requests per 30 day period.

Compound Selection Guidelines

Structures are generally selected for screening based on their ability to add diversity to the NCI small molecule compound collection. The submission of novel heterocyclic ring systems is particularly encouraged. In addition, NCI-60 encourages the submission of compounds with drug-like properties utilizing the concept of privileged scaffolds (1,2,3) or structures based on computer-aided design. The program is not intended to handle extensive supplier SAR studies that include large numbers of similar analogs. If analogs within a series are to be submitted, it is recommended that suppliers initially pre-select only the compounds within an analog series which will most efficiently provide the greatest information. This will ensure that the structures of most interest to the supplier are selected. If initial submissions show activity, they can provide a basis for the consideration of future analogs.

Highly flexible acyclic analogs with accompanying entropic liabilities are generally not accepted. Submission of structures containing problematic linkages or functional groups for successful drug development (e.g., nitro, nitroso, -N-N-, -N=N-, imine, semicarbazone, thioamides, thioureas) are discouraged. Analogs related to well-studied agents (e.g., brefeldins, anthracyclins, taxanes, camptothecins, combretastatin, aminoacridines, platinum-based agents) which have been the subject of thorough SAR investigations are also generally not selected without providing a rationale (or preferably data) for improved or novel biological or chemical properties which would support continuing development. As a service, the program also supports NCI-60 cell screen characterization (e.g., tumor selectivity, COMPARE data) of important individual late pre-clinical or investigational clinical agents.

Pan-assay interference compounds (PAINS) with activity that does not generally depend on a specific, drug-like interaction between molecule and protein are also discouraged. A list of the most notorious PAINS include, but are not limited to, these chemotypes (4):

• toxoflavins
• iosthiazolones
• curcumin analogs
• hydroxyphenyl hydrazones
• ene-rhodanines
• phenol-sulfonamides
• enones
• quinones
• catechols

The following classes of materials are generally not accepted:

• radicals
• high MW pegylated or similarly derivatized agents
• multiple pharmacophores linked by a tether(s)
• physical mixtures of active components
• reactive molecules, thermal or photo labile molecules (e.g., mustards, acyl halides, α-halo carbonyl compounds, reactive Michael acceptors)
• nanoparticle formulations

Data Retrieval and Testing Decisions

Submitters are sent an e-mail alert once a testing decision or testing data is added to their account data. Testing decisions are accessible by logging into the DTP Compound Submission Application. The submitter should find a link to results posted to the "Action Items" section and follow the link to access decisions and results.

Testing Decisions include:

• no further testing
• selected for five-dose testing
• re-test in NCI-60

Requests for assistance or questions regarding data retrieval should be sent to Keania Colbert (keania.colbert@nih.gov).

Frequently Asked Questions: NCI-60 Submission

1. Why is there a limit on the number of submissions?
   A limit is instituted due to the demand for the NCI-60 testing resource. It allows for a steady flow of submissions and a reasonable turnaround time for return of testing data.
2. How long will it take to obtain results?
   In general, initial 1-dose results are available within 8 weeks of receipt of sample. Results of 5-dose testing are dependent on Program priorities.
3. How does one acknowledge the NCI's contribution in a manuscript or other publication?
   Investigators shall add the following statement to the acknowledgement section of their publication: "The PI wishes to thank the National Cancer Institute Developmental Therapeutics Program (NCI/DTP) for providing screening data of compounds present in this (poster, manuscript, presentation, etc.). Specifically, NSC #: X, Y, Z , etc."
4. Are mixtures of compounds allowed?
   No, only single pure compounds are acceptable.
5. May solutions be submitted?
   No, only neat compounds (solid or oil) may be submitted.
6. What if my compound is not stable to storage or solution?
   A request to store cold and solubilize immediately prior to testing can be included in the submission storage and/or comments section.
7. Can pre-plated samples be submitted?
   No, we only accept vialed samples.
8. Can I submit compounds without the revealing the structure?
   No, the chemical structure, including stereochemistry, is mandatory.
9. What is an NSC number?
   This is a compound identifier assigned by DTP to identify an agent or product (e.g., small molecule or biological agent). It is an abbreviation for Cancer Chemotherapy National Service Center number. Most structures have a unique NSC number but over the years a small percentage of structures or agents may have been assigned more than one NSC number. In the case of salts, different salt forms (e.g., HCl, HOAc) are designated with separate NSC numbers. An NSC number will be assigned to your compound once the request is selected AND the material is received. You will need the NSC number to retrieve your testing data and it should be used in all communications with NCI regarding your compound.
10. I have submitted a request; it has been 10 business days and I have not received a selection decision. What should I do?
    Initially, check with your designated signatory authority to confirm that they have received our email and agreed to our required CDA/MTA. If confirmed and more than 10 days have elapsed since, please contact Keania Colbert (keania.colbert@nih.gov) for follow-up.
11. We would like to negotiate modifications to the standard CDA/MTA. Whom may I contact?
    No changes are allowed to the CDA/MTA.
12. Can I submit requests independently as a postdoc or graduate student?
    No, in order to be eligible for an account you will need to be a faculty member. Your use of the account will be governed by the account holder.
13. If my compound is selected for the 5-dose test, should I prepare an additional sample to send?
    No, the initial 10-15 mg sample you provided is sufficient to create test solutions for both the 1-dose and 5-dose assays.
14. What should I do, if I forget my DTP account username and password?
    Contact Keania Colbert (keania.colbert@nih.gov) who will reset your account.