Doug Auld, Ph.D. Novartis Institutes for BioMedical Research

Aggregation of Compounds to Form Colloidal Bodies That Interfere With Assays Is One of the Prominent Artifacts Found in Biochemical Assay

Aggregation of compounds to form colloidal bodies that interfere with assays is one of the prominent artifacts found in biochemical assays. Models to predict aggregating compounds have been reported, but many of these models often under-predict true aggregators and over-predict non-aggregators. This paper from the Shoichet lab describes a simple algorithm based on similarity to 12,600 known aggregators. The ∼12,000 known aggregators show a wide range of physicochemical properties and chemical diversity, but 80% of these have cLogP values >3. The prediction of aggregating compounds is based on chemical similarity (Tanimoto coefficients, Tcs) to these known aggregators along with a criteria of cLogP >3. The lower limit of the chemical similarity based on the Tcs was set at 0.85. Compounds with reported potencies in the 0.1–10 μM range were considered for testing. Prospective testing of predicted aggregators involved two experiments: detergent sensitive inhibition of either AmpC β-lactamase or malate dehydrogenase, and detecting colloidal particles by dynamic light scattering (DLS; see figure). Comparing the percentage of aggregators in the published literature to the total number of purchasable compounds shows a 10-fold enrichment in the published literature. Furthermore, using the prediction algorithm, one finds that since 1995, the number of likely aggregators has increased in the medicinal chemistry literature. These findings support that aggregators have been selected for lead discovery efforts over the past 10 years. The paper also demonstrates that the type of compounds that aggregate are chemically diverse and that many useful compounds (including drugs) may aggregate at high concentration in a certain buffer condition but still show genuine inhibition of the target protein at lower compound concentrations. Therefore, the authors emphasize that the “aggregation advisor” tool provided is not meant to remove aggregators from screening collections but to inform and help researchers to consider this mechanism of inhibition when interpreting their assay results.


Example experimental data from Figure 1 of the paper. Dynamic light scattering (DLS) curves (first and third columns) and β-lactamase inhibition concentration–response curves with (red) and without (black) Triton X-100 (second and fourth columns), for characteristic new aggregators identified here. For some compounds where inhibition was fully reversed by detergent addition, only points at the highest inhibitor concentration were determined and are shown (arrows with red points). For a larger graphic click here.

* Abstract from J Med Chem 2015;58:7076–7087

Colloidal aggregation of organic molecules is the dominant mechanism for artifactual inhibition of proteins, and controls against it are widely deployed. Notwithstanding an increasingly detailed understanding of this phenomenon, a method to reliably predict aggregation has remained elusive. Correspondingly, active molecules that act via aggregation continue to be found in early discovery campaigns and remain common in the literature. Over the past decade, over 12 thousand aggregating organic molecules have been identified, potentially enabling a precedent-based approach to match known aggregators with new molecules that may be expected to aggregate and lead to artifacts. We investigate an approach that uses lipophilicity, affinity, and similarity to known aggregators to advise on the likelihood that a candidate compound is an aggregator. In prospective experimental testing, five of seven new molecules with Tanimoto coefficients (Tc's) between 0.95 and 0.99 to known aggregators aggregated at relevant concentrations. Ten of 19 with Tc's between 0.94 and 0.90 and three of seven with Tc's between 0.89 and 0.85 also aggregated. Another three of the predicted compounds aggregated at higher concentrations. This method finds that 61 827 or 5.1% of the ligands acting in the 0.1 to 10 μM range in the medicinal chemistry literature are at least 85% similar to a known aggregator with these physical properties and may aggregate at relevant concentrations. Intriguingly, only 0.73% of all drug-like commercially available compounds resemble the known aggregators, suggesting that colloidal aggregators are enriched in the literature. As a percentage of the literature, aggregator-like compounds have increased 9-fold since 1995, partly reflecting the advent of high-throughput and virtual screens against molecular targets. Emerging from this study is an aggregator advisor database and tool (, free to the community, that may help distinguish between fruitful and artifactual screening hits acting by this mechanism.

Doug Auld, Ph.D., is affiliated with the Novartis Institutes for BioMedical Research.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a "Literature Search and Review" column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the "Literature Search and Review" column, a paper published in Journal of Medicinal Chemistry titled "An aggregation advisor for ligand discovery." Authors of the paper are Irwin JJ, Duan D, Torosyan H, Doak AK, Ziebart KT, Sterling T, Tumanian G, Shoichet BK.

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