> I did not think you are after inhibitor of translocation. Rather, I
suggested
> to use translocation as a measure in screen. If your staff X specifically
> binds to inactive form of SR, it is almost certain it will inhibit
activation
> by steroids.
And here is a problem that while SR is bound to HSP90, the staff X (peptide)
cannot bind SR. I failed so far to show any in vivo binding
(accumulation/retention), and suspect this is for that particular reason,
whereas in vitro everything works fine. There is certainly a possibility
that I am not using the right methodology (flow cytometry, fluorescent
microscopy) to show retention of X in SR-positive cells versus SR-negative.
Both mentioned techniques might be not accurate and sensitive enough for my
purposes. Moreover, I did not find any references so far on specific
intracellular protein targeting and how the retention of the agent was shown
in vivo. Any pointers here would be also much appreciated. There is a lot of
peptides developed that target surface receptors though, but that seems to
not quite relevant (except for the case of in situ delivery of radioactive
agents to the malignant tissue).
>> In any case, that geldanamycin substance pointed out elsewhere in
> this thread sounds like it might be just what you are looking for.
Geldanamycin is certainly a good stuff to try. However and unfortunately, it
also inhibits caspases, tyrosine kinases and acts as a potential cell death
and apoptosis inducer. In my setup I am treating live cells (Hek293, some
cancer lines) with the FITC-labelled X-peptide (membrane penetrable due to
the presence of specific transduction domain !) and follow retention of
fluorescence after washing the cells the number of times so that X
concentration goes below concentration of SR, and then do flow.
>> >Another
> >problem is that the weaker binding steroids, as well as many other
steroids,
> >are controlled substances and are not available on the market (at least
as
> >far as I know).
>> Not a problem. You can buy any kind of scary drug for research
> purposes if you can prove you really need it.
>> Apart from all that, if the whole idea is tissue-specific targeting of
drugs,
> IMHO, SR binder is not a promising approach. To bind SR, the stuff would
> have to get into the cell to begin with. If so, I can't see how it can be
> called targeting, for the stuff would have to be membrane permeable
> (and specific binding to specific SR would not cause real accumulation
> of the drug).
Se above. There is a lot of peptide sequences available now to delover even
full-size proteins through cellular membranes. Even poly-K and poly-H do the
job in some cases.
>> Standard extracellular receptors and conjugates with their ligands
> seem to fit the bill better (but I guess it's been tried extensively and
> never worked adequately).
Exactly. No room for innovation here :)
Emir
