ABCs of Aggregation
If your molecule looks as if it may be an aggregator:
- Watch your slope! Aggregators often cause steep dose-response curves.
- Use detergent! Run your enzyme assay with and without detergent. Aggregation-based inhibition may be attenuated by adding non-ionic detergents such as Triton X-100 to the reaction buffer. Note that there is no certain amount of detergent that will completely reverse inhibition in all cases; it is wise to always run at 2 different detergent concentrations.
- Beware of promiscuity! Aggregators are non-specific inhibitors. Run counter-screens against unrelated enzymes to ensure your inhibitor is specific to your target. This tactic may be very useful for anyone running in vivo or cell-based assays that cannot tolerate detergent.
When publishing a novel inhibitor, it is helpful to include such information as the IC50 values with and without detergent and the data from any counter-screens against un-related enzymes. This information works well in the Supplementary Materials and helps to convince others that your molecule is not an aggregator.
0. Does this website predict aggregators?
No. Aggregation is a complex phenomenon that depends not only on molecular properties but also concentration, buffer, temperature, pH and perhaps other variables. Prediction of aggregation is difficult. The same compound may aggregate under one set of conditions, or at one concentration, and not at another. Thus we use the phrase "Aggregator advisor" to indicate that we are giving you advice, not a prediction. The only way to know for sure is to perform experimental controls (see elsewhere on this page).
1. What does a typical small molecule aggregator look like?
Aggregators are often hydrophobic, highly conjugated molecules. However, a very diverse array of compounds have been reported to aggregate, and it is difficult to tell simply by looking at it whether a compound will aggregate. To browse molecules that have been reported to aggregate, please see our Gallery.
2. At what concentrations do small molecules aggregate?
Small molecules aggregate in the micromolar range. The aggregates themselves end up at femtomolar concentrations.
3. Can I see aggregates? How big are they?
Aggregates are not visible to the naked eye but can be detected using light scattering methods. They are typically tens of nanometers in diameter.
4. Can a molecule aggregate under some conditions and not others?
Yes. Aggregation is dependent on many factors, such as concentration, solvent pH and ionic strength, concentration of DMSO, and presence of detergent. [3-6]
5. I run my assay with 0.001% Triton X-100. Am I controlling properly for aggregation?
No. Aggregate formation and aggregation-based inhibition may still occur in low levels of detergent, and it is still possible to obtain a full IC50 curve (even up to 0.01% Triton X-100). The fundamental concept is that increasing the amount of detergent will increase the IC50 value of an aggregator (and will have no effect on a reversible, competitive inhibitor). Therefore, it is critical to perform assays at varying detergent concentrations (i.e., no detergent vs. 0.001% or 0.001% vs. 0.01%) and compare the resulting IC50 values.
6. Is it possible for a molecule to aggregate under certain conditions but still be a (true) competitive inhibitor?
Yes. Aggregators have a Critical Aggregation Concentration (similar to a CMC for micelles). At concentrations below the CAC, all compound remains in monomeric form and can act as a true competitive inhibitor.
7. How do aggregators inhibit enzymes?
A small molecule aggregate, or large colloid, adsorbs protein to its surface and inhibits enzyme activity by causing partial denaturation.
8. Can molecules aggregate in vivo?
We are currently investigating the potential of small molecules to aggregate in vivo. Preliminary research showed that oral drugs are able to form aggregates in simulated intestinal fluid, supporting the notion that in vivo aggregation may occur.
9. I know of compounds that aggregate that you have not included. What should I do?
Please email jji at docking.org with your evidence. We will credit you on this website if we use your tip. Thank you.
Literature Cited1. Shoichet, B.K., Interpreting steep dose-response curves in early inhibitor discovery. J Med Chem, 2006. 49(25): p. 7274-7. 2. Feng, B.Y. and B.K. Shoichet, A detergent-based assay for the detection of promiscuous inhibitors. Nat Protoc, 2006. 1(2):p. 550-3. 3. Frenkel, Y.V., et al., Concentration and pH dependent aggregation of hydrophobic drug molecules and relevance to oral bioavailability. J Med Chem, 2005. 48(6): p. 1974-83. 4. Coan, K.E. and B.K. Shoichet, Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J Am Chem Soc, 2008. 130(29): p. 9606-12. 5. McGovern, S.L., et al., A common mechanism underlying promiscuous inhibitors from virtual and high-throughput screening. J Med Chem, 2002. 45(8): p. 1712-22. 6. Ryan, A.J., et al., Effect of detergent on "promiscuous" inhibitors. J Med Chem, 2003. 46(16): p. 3448-51. 7. McGovern, S.L. and B.K. Shoichet, Kinase inhibitors: not just for kinases anymore. J Med Chem, 2003. 46(8): p. 1478-83. 8. Coan, K.E., et al., Promiscuous aggregate-based inhibitors promote enzyme unfolding. J Med Chem, 2009. 52(7): p. 2067-75. 9. Doak, A.K., et al., Colloid formation by drugs in simulated intestinal fluid. J Med Chem. 53(10): p. 4259-65. 10. Ferreira, R.S. et al., Complementarity between a docking and a high-throughput screen in discovering new cruzain inhibitors. J Med Chem. 53(13): p. 4891-905.
For data sources currently indexed by this site, please click here (format is index number, DOI (if available), citation) For a list of known aggregators in raw text form, please click here (format is SMILES, identifier, index into aggref.txt, above. tab delimited) . Right click to save to file.
CreditsContributors of data to this website - in addition to the data sources and literature cited above - include:
- Brian Shoichet
- Allison Doak
- Kristin Ziebart
- Your name could be here, if you send us your evidence substantiating aggregation, or you send us a publication that establishes aggregation.
Command Line ProgramHere is a python program, some sample data to test it with, and a README file. Right click to save to your local disk.
Notify Us of New AggregatorsIf you discover an aggregator that is not in our database, we would be grateful if you would send it to us. To be included in Aggregator Advisor, we would appreciate two pieces of evidence. This could be
- Evidence that the compound inhibits two very different proteins
- Dynamic light scattering evidence of aggregates
- Evidence that inhibition is sensitive to non-ionic detergent