One of the odd things about science is that you spend a good amount of time trying to prove that you don't know what you're talking about. At least, if you're doing it right, you should.
Take the first part of a drug discovery project, for instance. Most of them have a "primary assay", which is usually done against a purified protein in vitro, under fairly artificial conditions. Compounds that meet some standard of activity against that target then move on to the secondary assay, which is supposed to be aimed at the same process, but now it's done in living cells. That's a much tougher test. (It's a big leap from pure proteins to cells, about the same size as the leap from cells to whole animals.)
The hope is that the two assays will correlate with each other, but it's never a perfect fit. Generally, what you see is some of the active compounds dropping out for no apparent reason in the cell assay. If your target is in the cytoplasm, then there's always the possibility that these compounds don't penetrate into the cell as well as the others. Or they make it in, but are pumped right back out before they can get anything accomplished. Or perhaps they find some other (even tighter) binding site once they're inside, on some protein unrelated to the readout of your assay. There are always plenty of ways to explain these misfires.
And that's fine, as far as it goes. But if you don't double back and check these things out occasionally, you run the risk of fooling yourself. If your two assays don't correlate very well, it might be that cell penetration is lousing things up, sure - and it might also be that your assays aren't measuring the same thing. Or it could be that your target from the first assay isn't as important as you thought it was. These are the sorts of thing you really ought to be sure about.
So you need to keep yourself honest. Take some of your not-so-good compounds, the ones you'd normally discard after the first cut, and take them on to the cell assay regardless. They'd better not work! Test some of the compounds on a closely related cell line that doesn't have your target in it, if you've got some - is your target really the reason for the activity you're seeing?
Most of the time, you'll find that things are just fine. The inactive compounds really are inactive all the way through. But I've seen the exceptions occur, and more than once. You don't want to wait any longer than necessary to find out that your project is a dud. And worse yet, you really don't want someone else to find out for you. It leads to some of those awkward scenes we'd all rather avoid.
1. Jasper on August 19, 2005 10:47 AM writes...
I’ve often wondered why no one questions the overall strategy of the molecular target approach to drug discovery? Here’s the ‘typical’ scenario for drug discovery:
Select ‘hot’ new target from literature reference, genomics disease database, or knock-out mice -> clone, isolate and establish activity in ‘purified’ target -> screen, select series compounds -> SAR against ‘purified’ target -> secondary biology models -> animal models -> lots of hand-wringing and triage at this point when no efficacy produced.
In my mind it’s too easy to say this due an invalid target. One can’t even generally claim the target is non-drugable. Too many times the chemist produce compounds with nM efficacy by in vitro assays. So is it all just poor bioavailability? Sometimes, but it doesn’t explain all of the failures.
If one accepts a crisis is going on inside the pharmaceutical industry, then the current strategy is somehow faulty. But at what point? Have pharma scientists embraced too readily the genes to drugs concept? Perhaps over or under regulation of genes doesn’t predict functional responses quite as well as the molecular biologist would want. Or is it what I believe – that the single target approach is untenable with whole systems biology? It is the rare protein indeed that cells can’t find a work-around or alternate pathway to partially or wholly compensate.
I’m of the opinion that drug discovery must go back to being a biological science rather than being looked at as an engineering process.
Permalink to Comment2. David on August 19, 2005 12:19 PM writes...
It should also be noted that even primary assays can show poor correlation depending on which technology is chosen. The much touted Z' factor is only a measure of internal consistency. Matthew Sills from Novartis demonstrated this in his article "Comparison of Assay Technologies for a Tyrosine Kinase Assay Generates Different Results in High Throughput Screening" JOURNAL OF BIOMOLECULAR SCREENING Volume 7, Number 3, 2002
Permalink to Comment3. Julian Shillcock on August 21, 2005 8:02 AM writes...
I have the following (slightly relevant) question about the drug discovery process; even an approximate answer would be interesting to me. What fraction of potential drug molecules fail as a result of the molecule/target interactions, and as a result of not getting the molecule to the target in sufficient quantities; and what fraction of the resources expended by a company to develop the drug molecule are devoted to developing the molecules and to optimising the delivery mechanism? If molecules fail in the secondary (living cell) assay, is the time even taken to find out if the failure resulted from the molecule's failure to bind to the target or to its inability to be delivered? (In the interests of honesty, I should point out that I have a pet theory about this, and the answer is of more than academic interest to me.)
Permalink to Comment4. Chris on August 21, 2005 10:37 PM writes...
Derek,
My impression of your blog is overwhelmingly positive so take the following point as constructive. I think the quality of writing would benefit greatly from the use of specific examples. For instance, you say that many compounds can hit in a screen of in vitro activity but fail in a cell-based assay for a variety of reasons. I'd be curious to know some examples of drug discovery programs which ran into these issues. What was the target, the cell line, the assay, the molecule. Obviously one can go into excruciating detail but on the other hand the general nature of these posts leaves one wanting details. It is understandable that your ability to give details is limited by the proprietary nature of your work.
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