Fwd: Intein control in a targeted cancer therapy
Mac Cowell
From: Scott Tarone <sta...@synapticsynthase.com>
Date: Sun, Jun 30, 2013 at 7:16 PM
Subject: Intein control in a targeted cancer therapy
To: con...@diybio.org
Hello,
I’m not sure if your group is the right place for me to look for help, but I thought it worth a shot.
I am self-taught in the biosciences… Neuroscience, Genetics, Immunology, and Targeted Cancer Therapies. After watching a presentation on Chimeric Antigen Receptors on the NIH site, I had an idea on another approach that would remove the limitations inherent in the technique presented (though it was a fantastic step forward, and very successful). My approach is based on a constitutively active Caspase 3 mutant controlled by a molecular switch.
Long story short… I just wrote up a few paragraphs on my idea and sent it off to a few people in the field. One of them (a senior scientist from Pfizer) got back to me a few months later and said I may be on to something and that I needed to write a paper. I did that and then wrote an article on that paper with him and that was published in a small time online journal last year. Since then my paper and the article have been download several hundred times.
I didn’t provide the links or an attachment as my first attempt to send this email was blocked by spam filters.. I will provide that information if you have interest. In the interim, you can search for “intracellular Caspase modulating Chimeric Antigen Receptor”.
Thank you very much for your time…
Take care,
Scott
InteinHereAndThere
Nathan McCorkle
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Josiah Zayner
On first writing a scientific paper I would suggest doing a thorough literature search to uncover what people have done before. I also would suggest reading current literature. 32 Citations is scant for a theory paper and only ~5 of them are have been published in the last 3 years(all in 2010). This has no general bearing on the quality of a paper but is definitely a red flag because alot happens in Science in 3 years.
What you are proposing is not worded clearly but let me try. You want to create a T-cell that has an extracellular protein that will bind a cancer cell and then cause the uptake of a active caspase to kill the cell? All of that is daunting. One cannot just do this so easy.
Similar attempts have been tried by Adam Arkins group:
I just saw a talk by him recently. The bacteria kill the host even when having layers of specificity to target cancer cells. So this is no easy task.
Induction of apoptosis of cancer cells my using a monoclonal antibody to EGF to activate caspase, been done:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2363248/
Synthetic antibody work by Koide, Sidhu, Kossiakoff
Specifically targeting cancer cells, so many papers on that:
http://scholar.google.com/scholar?hl=en&as_sdt=0,14&q=specific+targeting+cancer+cells
Including some new cool work by Kobi Benenson
http://www.sciencemag.org/content/333/6047/1307.short
Protein design by David Baker and many others
Sometimes the problem why we don't see an accomplishment in Science is not that people don't have good ideas. The problem is that everything people have tried has not worked. Sure, people can have a treatment target a cancer cell in culture and have a protein taken up and activated. That's not real life. It is far from it.
Some statements you make in the work are completely wrong "However, with regard to oncogenes, the research suggests it isn't the function of the protein product that is necessarily altered in an oncogenic mutation, but rather a change in transcription regulation". Even the papers you cite disagree with this! Go here for a nice summary and tons of citations: http://en.wikipedia.org/wiki/Oncogene
"The interaction of forces at the atomic and quantum level that are the fabric in which proteins fold are not well understood. It is for this reason that, just as we confirm the very existence of proteins, an indirect path must be followed to engineer them."
Protein folding is very well understood. Read works by Firsht, Sosnick, Baker, DE Shaw.
If we don't understand the forces how can Scientists run simulations that recapitulate folding experiments to a high degree:
http://www.sciencemag.org/content/334/6055/517.short
Sure we don't understand everything about protein folding and have lots of trouble folding multi-domain proteins but "not well understood" is completely false. More like "not an easy way to calculate". Calculating changes in nuclei polarization or QM/MM requires so much computational power. Our understanding is great, it is the computation that is having a hard time keeping up.
"Marc Ostermeier wrote in 2009 given that allosteric effects depend on the “protein’s sequence,structure, and energetics” one might reasonably conclude that computational modeling would be required to engineer the same. However, it has been rational design strategies that have proven most often successful. This is due, he continues, to the “modularity of protein function”"
Rational design strategies have not proven the most successful. I work on designing new protein function. Computational modelling is so far beyond rational design. That is like saying a human is better at maths than a computer. Most every major modern protein design strategy that has worked has been done with a computer.
http://www.sciencemag.org/content/329/5989/309.short
http://www.sciencemag.org/content/302/5649/1364.short
http://www.sciencemag.org/content/319/5868/1387.short
"Upon encountering its tumor-specific ligand the resultant binding will be a conformational change that induces the intein self-excision and extein ligation. "
I feel like I need a meme here so:
http://cdn0.meme.li/instances/300x300/39360153.jpg
Inducing conformational change is one of the most complicated things in protein engineering. It is a huge goal of most protein engineers. The interaction networks that induce conformational change are so complicated and multi-noded. This in itself would be a huge feat much less all the other aims in the writing.
Many many other things I will not touch on for the sake of brevity as I am sure you are understanding my point.
Lots and lots of writing is uncited and aims are proposed as if they can just be done easily. Your goal in a writing such as this is that unless a statement made is an undeniable truth have a citation. READ the citations also. It is very apparent that you perhaps only read the title or abstract of many of these papers or chose to ignore much of what is written in the papers.
Good, to try your hand at some scientific theory work but I would recommend staying away from experimental theory unless you can test the experiments. One is so far removed from the experimental process that they have a hard time coming to terms with what it really takes to perform an experiment.
With your motivation I would suggest investing more in computational theory, thermodynamics theory or physical chemistry. You seem interested in it from the writing and it is an area that in many cases one can directly compare their work to experiment to validate it.
Hopefully this doesn't discourage you to stop learning and instead encourages you to to learn more and think deeply and Scientifically minded about things you propose.
Remember 90% of experimental planning is a good literature search and reading.
matt harbowy
The forces involved in protein folding are not "well understood"- the most successful structure predictors are ones that use established crystal structures and motifs. While leaders in the field believe quite rightfully and honestly they have cracked this; what they believe, and finished solutions, are two very different things.
-matt
"Be a molecular model... Or just look like one!"
Josiah Zayner
The paper you found the link on is a paper simulating and recapitulating experimental protein folding for fast folding single domain proteins.
Read about force fields used in computational chemistry and molecular dynamics simulations.
Google "force fields and protein folding"
http://www.ncbi.nlm.nih.gov/pubmed/21539772
"To that end, we performed equilibrium simulations of a fast-folding variant of the villin headpiece using four different force fields. In each simulation, we observed a large number of transitions between the unfolded and folded states, and in all four cases, both the rate of folding and the structure of the native state were in good agreement with experiments."
The lab I work in uses an ab initio C-beta side chain model that works off of statistical potentials and forces and they can fold proteins and predict folding pathways.
http://www.pnas.org/content/106/10/3734.short
Yes, Protein Biophysicists and Biochemists do know a fair bit about protein folding.
If you want to argue about how much we know about protein folding I don't think this is the thread. If you want to add to the critique of the manuscript the OP posted that would probably be more useful.
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InteinHereAndThere
Hello,
First, my thanks for taking the time to review and comment. I actually wasn't looking for a critique, but rather someone to work with me to develop and execute assays. Sigma Aldrich may be willing to produce my zinc-finger configuration pro bono… I've already been invited up to the Institute for Advanced Biotechnology and Medicine to discuss my paper, and I've conversed with Dr. June and Dr. Wender (Bergstrom Professor of Chemistry, Stanford) among others about my proposed therapeutic and none expressed any concerns regarding neither the fundamentals nor feasibility of the approach I put forth.
I am surprised that you came to the conclusion that my proposed therapeutic involves a T Cell... it does not, it is a single chimeric protein having nothing to do with any lymphocyte… my apologies if my writing wasn't clear.
Also, again, although I appreciate your time, I take offense at your inferring that I simply read abstracts; I assure you I read several hundred pages (and a textbook or two) of research over several months and incurred a large expense acquiring those papers despite the fact that my tax dollars paid for much of the research.
I also disagree with (though appreciate :) ) just about every one of your assertions. I'll address the rest tomorrow, but just for starters… Take the Oncogenes... I wrote "but rather a change in transcription regulation, a substantial decrease in the ubiquitination-proteasome turnover rate, or its active state changing from induced to constitutive that is the actual driver of oncogenesis (8) (9). "
Each of those three assertions are accurate reflections of those made in the research I cited. In fact you wouldn't even have to read passed the title of the papers… referring to c-Myc; "hot spot mutations in lymphomas result in inefficient ubiquitination and decreased proteasome-mediated turnover"
even the Wiki article you linked states:
"an increase in protein (enzyme) activity… and a loss of regulation" and all but 3.2 specifically refer to regulation, not a change in primary function and even 3.2 refers to a change in state to constitutively active…
Thanks again,
Scott
On Monday, July 1, 2013 4:47:47 PM UTC-4, Mackenzie Cowell wrote:
Josiah Zayner
You say in this sentence that it isn't the function of the protein but transcription regulation. Do changes in transcription cause cancer? Yes. Do changes in protein function cause cancer? Yes. Your statement is wrong then or very missleading. Gleevec one of the most successful cancer drugs targets protein function!
All protein function can indirectly or convolutedly be related to gene regulation but I don't think that is what you were tryign to say.
Scott, what you quote from Wikipedia:
"The proto-oncogene can become an oncogene by a relatively small modification of its original function. There are three basic methods of activation: A mutation within a proto-oncogene, or within a regulatory region (for example the promoter region), can cause a change in the protein structure, causing
- an increase in protein (enzyme) activity"
The paper title you cite, Inefficient ubiquitination is protein function...
None of that has anything to do with transcription.
"Receptor Tyrosine Kinases add phosphate groups to other proteins to turn them on or off. Receptor kinases add phosphate groups to receptor proteins at the surface of the cell (which receive protein signals from outside the cell and transmit them to the inside of the cell). Tyrosine kinases add phosphate groups to the amino acid tyrosine in the target protein. They can cause cancer by turning the receptor permanently on (constitutively), even without signals from outside the cell."
Constitutively turning a protein on is a change in protein function not transcription regulation.
Glad to hear Sigma wants to produce your Zinc-finger configuration pro-bono. You should go with that.
You should send your paper off for peer-reviewed publication. I see you have a similar "paper" "published" in the journal of "Technological Innovations in Life Sciences", which has published 6 articles 3 of which are written by the Editor and maybe 3 of which are actual papers in its 5 year history. None of its articles have ever been cited and none of its articles have ever been written by Academic Faculty. Sounds a bit sketchy...
Send your article to PLoS One. They have an option that allows you to wave the publication costs.
Sorry you had to spend so much money for Scientific articles. If you search this list there are many ways to obtain them for free, including going to the library at your local university.
I look forward to hearing your response to my critiques.
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InteinHereAndThere
Hello...
I wrote "it isn't the function of the protein product that is necessarily altered ". I don't believe that implies that there are no exceptions. As to the quote from Wiki... I agree, the sentence needs to be corrected... and yes, of course, I know that ubiquitination is on the Omega end and transcription is on the Alpha, but the general sense of the text conveys that cancer is a disease of de-regulation, whether that be of expression or removal of activity control.
This is just one example, but it's far reaching…
--"MYC was among the first oncogenes to be identified, and its deregulated expression is widespread in human cancer (Vita and Henriksson 2006)."
Oncogene addiction: setting the stage for molecularly targeted cancer therapy
Sreenath V. Sharma and Jeffrey Settleman
On activity, regarding BCR-ABL, Sharma and Settleman write "The ABL and platelet-derived growth factor receptor (PDGFR) tyrosine kinases, which are targets of imatinib, are often activated by chromosomal translocations (BCR-ABL, TEL-ABL, TEL-PDGFR).
I disagree that a change resulting in constitutive activation of a protein that previously wasn't constitutes a change in function. If a protein phosphorylates, that is its function, whether it needs requires a reaction or not. I do agree, of course, that this change has nothing to do with transcription of its gene. However, that change is a change in the overall regulation of the cell.
------------------
I would be very happy to get my paper before a peer review, as that was a suggestion made by others. However, I've contacted several, including PlosOne and all but one said no outright because I wasn't a member of a lab or employed by a pharma. The one exception was the new journal from Nature Publishing... they agreed to fund the peer review for me, however, I have yet to hear a reason for rejection even though it was due to complete almost 2 months ago. My attempts to contact one of the reviewers have been rejected.
As to my article that was published... I co-wrote that with a former Pfizer senior scientist and adjunct professor at U Conn (who has presented at the New York Academy of Science). I didn't know much about how journals and peer review worked at the time and I believe they were doing what they could to start a new journal. I was asked to co-author the article so I did.
----------------------
As to protein structure…
"Directed evolution circumvents our profound ignorance of how a protein's sequence encodes its function by using iterative rounds of random mutation and artificial selection to discover new and useful proteins.
…
Notwithstanding significant advances, a molecular-level understanding of why one protein performs a certain task better than another remains elusive.
…
Thus predicting the amino acid sequence, or changes to an amino acid sequence, that would generate a specific behavior remains a challenge, particularly for applications requiring high performance (such as an industrial enzyme or a therapeutic protein). Unfortunately, where function is concerned, details matter, and we just don't understand the details."
- Exploring protein fitness landscapes by directed evolution
Philip A. Romero
Frances H. Arnold (CAL Tech)
On Monday, July 1, 2013 4:47:47 PM UTC-4, Mackenzie Cowell wrote: