Field of Science

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


  1. Covalency should not be equated with irreversibility. I'm not particularly famiiar with the MS experiments (and can't currently see the article) but I'm assuming that they will be unable to detect a protein adduct if the off-rate is too high. One question you need to be asking is whether covalent inhibition is more common for hydrolytic enzymes.

  2. Good point. Although in this study they turn out to be irreversible too. I would think that covalent inhib is more common for hydrolytics, but I would like to see a survey.


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