Time for a personal post

yes i would like to make a personal post. ....

well, i have more or less kept quite active. I feel very distant from my body. As if it has become somewhat untouchable...

um, well i really don't have much to say.

Dinucleoside tetraphosphates...continued

So last time we (rather hastily) established what excision is and what TAMs do.

So in this paper (Meyer et al. 2006) instead of chainterminating the DNA chain with a regular chain-terminator, they used a dinucleoside tetraphosphate. This compound basically consists of two nucleotides (which could be chain-terminators) and 4 phospahtes in between them. Or, if you look at it in a more relevant way, these compounds basically represent the intermediate product that is formed at the instant of excision. So imagine if you had a ddTMP as the chain-terminator sitting at the 3' terminus of the primer, and ATP came in as a PPi donor, then as ATP donates its two phosphates, the intermediate product there would be AppppddC. And of course immediately after that, we have the release of the ddC (in the form of ddCTP) and AMP (the old ATP which has now lost two phosphates).

Ok so now that we have established what the dinucleoside tetraphosphate is, we can ask what would be the advantage of its use? Basically, they show that this compound can pretty much inhibit DNA synthesis by RT as well as a regular chain-terminator. More importantly, they show that this compound binds TAMs RT better than WT RT. Mechanistically, this is relevant because it supports the model in which TAMs promotes the binding of the incoming PPi donor better as compared to WT (WildType) RT.

But what makes this compound clinically relevant is the fact that they can selectively bind a group of mutants better than WT. So hypothetically, let's say you administer AZT to a patient. That selects for TAMs which are AZT resistant. Well you can then administer NppppddNs which inihibits what you just selected for! So the two families of drugs can work synergistically in that regard.

One problem however is delivery. How is it going to get into the cell? The paper addresses this point in their discussion.

But I am getting tired of this stuff now.

The paper also discusses how they synthesize the compounds and the mechanism of ddNMP transfer to the DNA chain.

Man, i just got very impatient with this stuff all of a sudden. Next time i'll try to write more consisely with a clear structure rather than a rant. That way i'll make my point without getting bored.

ok back to the lab now.

Dinucleoside triphosphates may be more effective against TAMs than regular chain-terminators

The latest paper I have read was published by the W. Scott group in 2006 (Meyer et al. AAC) ( yeah i know that is not proper refrencing. i get better eventually...ma~nana, ma~nana). Here is a little background scoop before we get into the paper. So Regular chain-terminators (i.e. NRTIs) bind wildtype RT and get incorporated into the elongating DNA chain. But! what is special about RT is that even though the forward reaction (i.e. DNA elongation) is favored, the back reaction (e.i. excision; the removal of an incorporated nucleotide) can happen as well. Now obviously under normal conditions this reaction is significantly unfavored. But! if the forward reaction happens to be slowed down (in this case let's say because of the incorporation of a certain chain-terminator), then the excision reaction becomes more relevant.
Now a number of NRTI-resistance associated mutations function by promoting this reaction. Very popular example of this is TAMs (Thymidine-analogue associated mutations).
So then the next question is...well...how? How are these mutations favoring excision?
To answer that question, we have to have a quick review of teh excision reaction itself. Now when a nucleotide is being incorporated, it has three phosphates right? and two of those get released (hydrolysis) and the nucleic acid and one phosphate (5') remain in the growing DNA chain. The excision reaction is exactly the opposite of that. It is when two phosphates come in and bind to the remaining phosphate on the nucleic acid and hence a nucleotide is released. So relevant to what we are saying here is, where are those two phosphates coming from? in vitro, this can be a simple pyrophophate (PPi, i.e. a molecule consisiting of two phosphates). But it seems that a much more relevant in vivo PPi donor is ATP (Adenosine triphosphate). So the way I like to think about it is that ATP basically backs up the driveway from where PPi normally leaves.

So! to make the story short, if somehow a mutation caused ATP to bind more favorably as a PPi donor, then that mutation could theoretically promote excision. And we thikn this is what is happening with TAMs.

ok so this is the background info necessary for getting into the paper. why don't i continue with the actual paper at our next session.

p.s. i will try to reference the statements that i make. Because 1) it will be better for you 2) it will help me with remmebering who said what where and why.

time well wasted...no regrets!

I was going to post the follow-up to the L234F business tonight and talk about another article that I am reading. But...instead...i watched 19 episodes of The Office (US version).

L234F: what is your deal?

Ok so my research for L234F has been futile thus far. I have only found one article on it. It is about capravirine, a novel NNRTI which has failed in clinical trials. Sato et al. (antiviral reseach, 2006) report that this NNRTI selects for the usual NNRTI resistant mutations. But in addition it also selects for L234I. this mutation has not been associated with any other NNRTIs. Interestingly, the presence of this mutation, along with a few other NNRTI mutations, has an antagonistinc effect on T215Y (AZT resistance) and vice versa. This point is of interest to me. It is also important to note that this is the only mutation that relates to my research which is not an NRTI associated mutation.

I also know from conversations that L234 is in the linking area between p66 and p51.

So what remains to be addressed:
1. where is L234F in RT exactly?
2. is it an NRTI associated mutation as well?
3. Leucine is converted to phenylalanine. what's the difference between these amino acids?

ok today was ok to be the day that i write here about that L234F article that i have been reading for the past...month. But i didn't get a chance to do it because i did other fun things instead. But tomorrow....tomorrow...i promise you science. I promise you. I promise you so bad.

My appartment has been feeling rediculously good this past week. I don't want to leave it. well i only want to leave it for short periods of time.

but tomorrow...oh you watch out. I'll L234F this place up so bad. it's not even going to be funny.


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