Abstract: “The MYC oncogene is a promising drug target for cancer research due to its overexpression in a majority of cancers. However, the MYC protein is considered undruggable. The G-quadruplex structure formed in the promoter region of the MYC oncogene is a transcription inhibitor. Stabilizing the MYC promoter G-quadruplex with small molecules decreases MYC levels in cancer cells causing cell death. Indenoisoquinoline compounds have been shown to induce and stabilize the MYC promoter G-quadruplex and inhibit MYC expression. Currently, there is no commercially available DNA-targeted small molecule compound library. In this study we first establish a DNA-targeted molecule compound library with over 2000 indenoisoquinoline compounds and analogs. We created a physical library with electronic data to easily access the information about each compound. We screened the library in vitro for binding of each compound to the MYC promoter G-quadruplex. Then we used in vivo cellular assays with two different cancer cell lines to determine the compounds’ toxicity to cancer cells and their ability to stabilize the MYC promoter G-quadruplex and downregulate MYC. With these results we discovered 270 hit compounds. Currently we are conducting more detailed studies of these hit compounds to better determine their MYC promoter G-quadruplex binding and MYC inhibition in cancer cells to ultimately develop an in-depth structure-activity relationship for indenoisoquinolines at this promising cancer target.”
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Very interesting and clear poster with a good deal of work. Two questions would be what is the insertion in the Ca46 that prevents quadraplex formation, and also (and not asking for super specific information here) but how closely related are your compounds in the groups of 10? So for instance how close is the structure of C1915 to C1905, or are the compounds grouped into 10s at random?
I am not sure as to what exactly is the make-up of the insertion. It prevents the formation of the MYC promoter G-quadruplex.
The compounds are grouped randomly. Though there is the occasional run in the 2000 compounds where only single changes are made such as a side chain Nitrogen becomes a methyl. But there is no intentional order.
Mercedes,
What an intriguing approach to targeting this gene. You show the G-quadriplex with three loops all containing -NNNN-.
1. What is the sequence variability in humans for these loops?
2. Are any sequences in these regions more prevalent in cancerous cells versus non-cancerous cells?
3. Was sequence variability at these positions included in your experimental design? If so, how?
1. The loops usually contain between 1 and 4 nucleotides which can be any of the four bases. Sometimes when the end base is a guanine, it can even be incorporated into the G-quadruplex and the G on the other end is incorporated into the next loop.
2. These sequences exist in both cancerous cells and non-cancerous. The selectivity exists in the fact that the cancerous cells depend on the MYC protein for their survival whereas normal cells are able to survive with the temporary absence of this protein.
3. The sequence variability was not included in our design because the variability is between different G-quadruplexes such as the ones in the telomeres vs the MYC promoter. Barring random mutation, the MYC promoter G-quadruplex is the same every time.
Hi Mercedes, this is a very interesting poster! A few questions for you—
1) This seems like a pretty big project— what part of this poster did you conduct yourself? What did you enjoy about it?
2) FRET melting—- is this thermal stability assay? how is FRET incorporated in this assay?
3) To clarify, when you say in vivo you mean a whole-cell assay, rather than in animal?
4) What were the “themes” of your derivatization campaigns for library construction? ie what kind of changes were you intalling?
5) How specific/sensitive do you think your assay is? Are you surprised/taken back by the high hit rate of 13.5%?
Thanks!
Aya
Hi Aya, Thank you for your questions.
1. I personally assisted in the library construction and in performing the cell assays. I really enjoy working with the cells and learning about how to take care of them and how sensitive they can be to things like pH change.
2. FRET melting is the thermal stability assay. Essentially we tested the melting temperature before and after treatment with the drug. We considered drugs with a change greater than 5 degrees Celsius as our hit criteria and combined that with our cell assay data.
3. Yes the in vivo assay is a whole-cell assay.
4. As you can see from the diagram of the indenoisoquinoline molecule, there is a variable atom, we also varied the length and make-up of the longer side chain. Some molecules have the final ring open, some have more nitrogen in the rings. Some of the molecules require counter-ions such as HCl or H2S.
5. I was not surprised by our high hit rate as these idenoisoquinoline compounds have already been shown to reduce MYC expression. We also designed the hit library qualifications with getting a number like that in mind. We wanted to be able to do more intensive tests on a smaller and more manageable number but we did not want to leave any potential hits out.