What's striking is the sheer ubiquity of this phenomenon which has been observed with all kinds of drugs under all kinds of conditions; while the initial observation was limited to isolated protein-based assays, the phenomenon has also been seen in simulated gastric fluids and in the presence of many different kinds of proteins like serum albumin which are found inside the body. The colloid spirit seems to emphatically favor a shotgun approach.
Now a team led by the brother-sister duo Brian and Molly Shoichet (UCSF and Toronto) has found something that should give drug testers further pause for thought; they see some bestselling anticancer drugs forming colloids (shown above) in cell-based assays to an extent that actually diminishes their activity, leading not to false positives but to false negatives. They test seven known anticancer drugs in cell assays both under known colloid forming conditions along with conditions that break the colloids up. This is not as easy as it sounds since it involves adding a detergent which would usually be too toxic to cells; fortunately in this case they find the right one. Another interesting finding is the re-evaluation of a popular dye used to study "leaky" cancer blood vessels; unlike the previously proposed mechanism, the current study seems to suggest that the dye too forms large aggregates and nonspecifically inhibits the protein serum albumin.
The testing essentially reveals that the drugs when they form colloids basically show activity that's so low as to be negligible and equivalent to the controls. That's a self-(un)proclaimed false negative. Now anybody who deals with error analysis knows that false negatives are fundamentally worse than false positives since by definition they cannot even be detected. The present study raises the pertinent question; how many promising drugs might we be missing because they form aggregates and lower the observed response in cells? And since the colloid forming phenomenon has been shown to be so ubiquitous, could it possibly be influencing the mechanism of action of all kinds of drugs inside the body? And in what ways? It's a fascinating question, and one of those that continues to make basic research in drug discovery still so interesting.
Image source and credit: ACS
I am not very familiar with the drug discovery process. Do all potential drugs have to pass a cell-based assay test, or is it enough with some other technique like thermophoresis?
ReplyDeleteYes, all potential drugs have to pass a cell-based assay test if you want to proceed with clinical trials.
DeleteI'm going to be very lazy an post exactly what I posted over at In the Pipeline. The discussions here take place on a more relaxed timescale and the participants tend to be more interested in the fundamental science.
ReplyDeletePlasma protein binding is measured routinely in Drug Discovery and this study has potential implications for how we interpret these measurements. I am not too familiar with the fine details of PPB assays but I seem to recall a sensitivity to lipid concentration in the assay media (perhaps somebody who knows about this could comment). Also people have looked at binding to Albumin using SPR which would provide insight into binding stoichiometry. In general, I would like to see aqueous solubility measured in protein free media for studies of aggregation.
On an unrelated note, I'm not sure if a cysteine protease such as Cruzain is an ideal enzyme with which to study aggregation phenomena since inhibition can result from oxdidation of the catalytic cysteine. I need to look art some of the earlier studies but, in the meantime, does anybody know whether researchers have looked much at the effect of these detergents on enzyme activity in the absence of inhibitors?
Rephrasing Freeman Dyson, "I would rather be lazy than uninteresting"! Your point about PPB is quite interesting, I don't think they routinely run PPB binding tests in the presence and absence of detergent and it would be especially useful to quantify how much of a difference the detergent makes (since congeneric series of drugs sometimes result in PPB binding differences of less than 1%). The question about cruzain is intriguing too; I know the Shoichet group has found cruzain to be a general determinant of aggregation-based inhibition for a variety of drugs, but the possibility of detergent action in the absence of inhibitors certainly seems to be worth investigating. At the very least it would be a simple control.
DeleteI think that I've shared this link here before. There is more to interference than aggregation and cysteines can be especially spiteful in this regard.
Deletehttp://dx.doi.org/10.1016/j.bmcl.2003.12.014
Intriguing. It seems like the ideal chemical experiment would involve testing every reagent, substrate and product against every other to see if anything cross-reacts.
DeleteCompletely irrelevant observation - I'm now going to have to wonder the next time I see "EPR" somewhere if it's referring to Einstein-Podulsky-Rosen, electron paramagnetic resonance, or enhanced permeability & retention.
ReplyDeleteFrom my non-drug-discovery background in working with enzymes, enzyme substrates, and detergents, I don't fathom envying anyone who tries to follow up on this sort of thing to formulate a product. Especially if they also have to deal with P-glycoprotein activity as well!
As the tagline says, "Aggregation: When drug discovery doesn't provide enough challenges and your heart yearns for more."
ReplyDelete