Anticancer drugs form colloidal aggregates and lose activity

Over the last few years, one of the most interesting findings in drug screening and testing at a preclinical level has been the observation that many drugs form colloidal aggregates under standard testing conditions and nonspecifically inhibit target proteins which they otherwise would not affect. This are large aggregates, a hundred nanometers or more in diameter, and they cause proteins to stick and partially unfold, creating the illusion of inhibition. This leads to false positives, especially in high-throughput screening protocols. And these false positives can be absolutely rampant.

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

The same and not the same: more aggregates in HTS

High-throughput screening (HTS) is now a mainstay of drug discovery and usually the starting point for most drug discovery projects. Industry usually has a lot of resources invested in HTS and therefore needs to be aware of false positives and false negatives that could hamper useful results and lead one down an erroneous path.

Among the many factors responsible for false positives in HTS, one of the most startling and important factors recently unearthed is the non-specific and potent inhibition of enzymes by aggregates of molecules occurring under typical assay conditions. These aggregates are large enough to be observed under a microscope and to be detected by dynamic light scattering. The aggregates adsorb enzyme molecules on their surface, and one of the best tests for detecting their presence is to re-run the enzyme assay under high detergent concentration. High detergent concentrations usually break up the aggregates and lead to a loss of potent inhibition. The phenomenon of aggregation-based inhibition was accidentally discovered by Brian Shoichet's group at UCSF and has been comprehensively explored by him and his students in a series of papers throughout the last decade, although much is still to be known about the exact physical nature of these aggregates. The reason why this has become a big deal is because it has been observed in an unusual number of cases, which leads to the suspicion that much effort might have been already expended in drug discovery campaigns in pursuing such false leads.

In a recent paper, Shoichet and Craik's groups at UCSF accidentally discovered aggregate-based inhibition in discovering inhibitors for the enzyme cruzain which is a part of the metabolic machinery of the parasite responsible for Chagas disease. The authors had started with an initial hit from a virtual screening campaign and were engaged in the usual process of modifying the hit based on SAR. The initial tinkering led to a series of oxadiazole inhibitors which exhibited potent inhibition of cruzain.

However, many of these molecules failed to show activity in cell-based assays. Such a discrepancy between enzyme and cell-based assays can be traced back to many reasons including poor permeability. But in this particular case, kinetic measurements hinted at aggregates of the oxadiazoles that were inhibiting the enzyme. At this stage it was also discovered that unlike the initial hits series, the oxadiazole series had been accidentally assayed under low detergent conditions. The molecules also inhibited another intensely studied enzyme in the Shoichet group- AmpC beta-lactamase. The quintessential test for aggregate-based inhibition, namely increasing the concentration of detergent (Triton in this case), also proved positive confirming the phenomenon. Interestingly the initial set of hit molecules also seemed to exhibit this phenomenon but only in case of AmpC lactamase and not in case of cruzain. In case of cruzain, experiments with differing detergent concentrations proved that the initial set of molecules were equally potent under both conditions, while the oxadiazoles lost activity under high detergent conditions, indicating divergent modes of inhibition between the two sets of molecules.

Finally, note that the aggregation-based inhibition would likely have not been discovered if the oxadiazole series had been assayed under the same low detergent condition as the initial hit series. What seemed like similar molecules turned out to behave very differently under different assay conditions. Sometimes mistakes can reward you with unexpected treasures, and similarity needs to be pried out from the eye of the experimenter. Never underestimate the importance of going wrong (of course revealed only in retrospect).

As the authors narrate, the moral of such studies should not be lost on medicinal chemists, who usually interpret high and low potency of related molecules based on local structural features like hydrogen bonding, electrostatics and hydrophobicity. Aggregation-based enzyme inhibition proves that chemists have to look beyond single molecule structural features toward supramolecular features of several molecules that are interacting with each other. Chemists regularly engaged in HTS campaigns might well keep this valuable piece of advice in mind. Scientific enumeration, it seems, has to always be done at several different levels.

Note: Apologies to Prof. Roald Hoffmann for appropriating the title

Ferreira, R., Bryant, C., Ang, K., McKerrow, J., Shoichet, B., & Renslo, A. (2009). Divergent Modes of Enzyme Inhibition in a Homologous Structure−Activity Series Journal of Medicinal Chemistry DOI: 10.1021/jm9009229

A rash of molecular personalities

Just like human beings, molecules have personalities. And just like human beings, they display those personalities best when they react to a stimulus. For a medicinal chemist, one such stimulus is HTS where one can identify different flavors of molecules through their interaction with protein targets. But this is not always done, and quantitative analysis of molecules in HT screens is lacking. Clearly such analyses will help to identify compositions of such screens and give insight into future screens.

In his latest offering, Brian Shoichet does just that. He and his group set out to identify essentially every molecular character from a colorful screen of about 70000 molecular personalities applied to ampicillin resistant beta-lactamase. Their results are surprising.

Out of 70000, about 1274 showed activity. Shoichet has already extensively documented the alarming frequency of aggregate-forming molecules in common HTS screens. It's a very substantial contribution from his laboratory. In this case, 1204 (95%) of the 1274 turned out to be inhibiting the enzyme through non-specific aggregation. This can be found out by adding detergent, which breaks up the aggregates and gets rid of the spurious activity.

So now there were 70 detergent-insensitive compounds. How many of these were true, reversible binders? 25 of these were beta-lactams, and since they are covalent modifiers of the enzyme and known chemical scaffolds, they were not considered further. So out of the remaining ones, 25 were re-synthesized and were found to be false positive through lack of reproducible activity. There were now 20 non beta-lactams. Out of these 9 were again found to be aggregators- the earlier screen had skipped them because of low detergent concentration.

That left 12 molecules. After some more scrutiny, these were all found to be covalent, irreversible modifiers of the enzyme. A neat and simple trick can be used to identify covalent modification; mass spectra of the modified enzyme are clearly different from the apo enzyme.

So how many non-covalent, reversible inhibitors of beta-lactmase were found? Zero.

To shed some more light on this strange phenomenon, the authors turned to docking with DOCK. To make sure the program can identify reversible binders, some known binders were seeded among the unknown binders. After docking and observing that the first 500 hits contained the known binders, 16 out of these 500 compounds were selected based on structural diversity and then assayed. Interestingly, two among these compounds were found to inhibit the enzyme at IC50 values of >100 µM. No wonder the initial screen had missed these phthalimide culprits- the highest concentration in the screen was 30 µM.

In other studies, they also did some SAR on the hits and verified the docking poses by obtaining crystal structures. There are other interesting details in the paper.

But even if the study did not unearth reversible, potent, novel binders, it is of course still very instructive. It tells us about the variety of beasts existing in HTS. It also again sheds light on docking as a valuable complement to HTS. In this case, 70000 compounds may been too less for assaying, and 30 µM must have been two low a threshold for finding hits. In any case, higher thresholds for testing are limited by practical difficulties, including material availability and solubility. But what is valuable is that given due effort, we can identify compounds that give false positive results in screens through novel mechanisms- in this case by aggregation (detected by detergent addition) and by covalent modification (detected by mass spec)

There are clearly some notorious and dirty candidates in HTS screens- more than everyone would be comfortable with- and this study provides a good model for being on one's guard and seeking to identify them as thoroughly as possible. When we lay down the red carpet, we want only the cream of the crop, not asses disguised as lions.

Babaoglu, K., Simeonov, A., Irwin, J.J., Nelson, M.E., Feng, B., Thomas, C.J., Cancian, L., Costi, M.P., Maltby, D.A., Jadhav, A., Inglese, J., Austin, C.P., Shoichet, B.K. (2008). Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens against β-Lactamase. Journal of Medicinal Chemistry DOI: 10.1021/jm701500e

Interview with Brian Shoichet: aggregation-induced inhibition

Ok, now that we have gotten past the Nobel mania (or maybe not; go Somorjai), we can hopefully come back to real life. I was reading an interview with Brian Shoichet, who is one of the most promising stars in the areas of screening, docking, and structure-based design. He has gotten his fingers in many pies, both computational and experimental.

However, it was somebody's comment about the pharmaceutical industry thinking that "Shoichet deserves a heroes prize" that got me looking at his work, and I quickly learnt the reasons for that quote. As we all know, one of the biggest or perhaps the biggest problem facing HTS in industry is false positives. A lot of times, molecules that are found to be active in an assay fail to be active later. If industry could weed out such nuisances ahead of time, a lot of time, money and energy could be saved.

Shoichet, after a lot of interesting initiation and investigation, came up with one simple reason for why molecules may be showing false colours; because they form colloidal aggregates that somehow inhibit the proteins in the assay. If these are broken up say with detergent, the individual molecules no longer show activity. Thus, a relatively simple physical phenomenon is responsible for these molecules showing false activities. Such molecules were detected in earlier assays by some characteristics, mainly very steep dose-response curves and flat SAR; changes in structure usually causing very small changes in activity. They are also often promiscuous inhibitors. But nobody knew what was exactly happening and all the analysis was post-"mortem".

The first step in Shoichet's lab was the elucidation of this aggregation-induced inhibition. The aggregation can be detected with dynamic light scattering (DLS). The more challenging and useful step is to be able to come up with a list of chemical scaffolds that are likely to show this phenomenon, so that one can watch out for them beforehand. Before that, one would also need to know the exact mechanism of aggregation-based inhibition. In case of some molecules, there is some structural correlation, flat aromatic dye-like molecules being prone to aggregation for example by stacking. But many other scaffolds seem more diverse and at first glance show no common functionalities. Ths phenomenon is linked by common physical forces, not chemical ones. The details are not known but continue to be worked out.

Shoichet's lab continues to make progress, and he has recently come up with a screen for detecting such aggregation-based inhibitors (DOI: 10.1021/jm061317y). There are two major conclusions from the study; first, that breaking up aggregates with detergents can be a good way of identifying them, and secondly that aggregation may be a much more common phenomenon for false positives in screens than was thought before. This fact may be extremely significant for industry and could potentially save a lot of time, money and labour beforehand.

In other quite different work (DOI: 10.1038/nature05981), Shoichet also made the cover of Nature, when he used docking and structure-based design to predict the function for an enzyme whose function was unknown, based on substrate docking and analysis. The strategy used was quite clever; docking thousands of high-energy forms of metabolites rather than the metabolites themselves to know which ones would optimally interact with the active site. In this particular case, the "optimum interaction" pointed to a deamination, and the protein of unknown function indeed experimentally turned out to be a good deaminase.

All in all, a very promising chemist and I believe one to watch out for. Unfortunately, the interview itself is published in the journal Assay and Drug Development Technologies, not one which libraries usually subscribe to (I got it through ILL). But here's the DOI anyway (DOI: 10.1089/adt.2007.9996)

Also, again, check out his Colbert-style interview on youtube.