ABCs of Aggregation

If your molecule looks as if it may be an aggregator:

Watch your slope! Aggregators often cause steep dose-response curves.[1]

Use detergent! Run your enzyme assay with and without detergent. Aggregationbased 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.[2]

Beware of promiscuity! Aggregators are non-specific inhibitors. Run counterscreens 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 IC 50

values with and without detergent and the data from any counter-screens against unrelated enzymes. This information works well in the Supplementary Materials and helps
to convince others that your molecule is not an aggregator.

Frequent Questions
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 IC 50 curve (even up to 0.01% Triton X100). 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 IC 50
values.[2]
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]
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]
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]
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.