T HE S CREENERS WERE CREATED BY MAN

T HEY EVOLVED

A ND THEY HAVE A PLAN

Drug Screening Test lots of compounds for inhibition against a target

High Throughput Screening

What if you do not know the target?

Not all targets are known 1. Compound X extracted from Australian Jumping Fungus kills cancer cells. What is its mechanism?

Not all targets are known 1. Compound X extracted from Australian Jumping Fungus kills cancer cells. What is its mechanism? 2. A collection of leads has been designed for a particular target, do they have any off-target effects?

High Content Screening (image from

Questions 1. Can we profile the biological activities of a library of compounds efficiently? 2. Can we use simple probes of the cell cycle to obtain high-resolution biological information? 3. How do we draw conclusions from the massive amounts of data generated?

The Screening Library 6,547 Compounds 58% natural products 21% known bioactives 21% structural diversity

The Setup Add Hela cells to wells in 384 well plates Add 20 μM compound Incubate 20 hrs Fix, stain

The Fluorescent Probes Hoescht Labels DNA (DAPI) G1/G2 Immunostain Labels Histone H3 Phospho-Ser10 Mitotis Click Chemistry Labeled nucleotide pulse DNA Replication

High-throughput Image Analysis #FeatureDescriptionValue 1AreaCh1Nuclear Area X 1 2PerimCh1Nuclear Perimeter X 2 36 IntenCoocASMCh3 EdU Texture X 36 Image 500 cells/well Analyze 36 features from 3-channel fluorescence images

Factor Analysis Assume measured variables are linear functions of common, underlying factors. Reduce dimensionality of data to factors that explain common sample variance

Factors represent meaningful phenotypes

Compounds can be ranked by activity For each factor: (treated – untreated)^2

Compounds can be ranked by activity For each factor: (treated – untreated)^2 Six Factors

Compounds can be ranked by activity For each factor: (treated – untreated)^2 Six Factors A.J. Hanson/Indiana Univ.

Compounds can be ranked by activity For each factor: (treated – untreated)^2 Six Factors

Compounds can be clustered by factor scores

Compounds cluster into distinct phenotypic groups

Phenotypic similarity correlates with structural similarity Phenotype vector Euclidian Distance 2D Fingerprint (ECFP_4) Tanimoto coefficient

Structural similarity does not always correlate with phenotypic similarity

A: D2 receptor antagonist B: Binds many GPCRs including D2 dopamine receptor

Compounds cluster into distinct phenotypic groups

Phenotypic similarity captures fine details of activity

Cytotoxic macrocylic hexadepsipeptides

Phenotypic similarity captures fine details of activity Cytotoxic macrocylic hexadepsipeptides Cytotoxic macrocyclic nonpeptides

Clusters can represent mechanisms of action G1/G0 Arrest Phenotype

Clusters can represent mechanisms of action Cardiac glycosides inhibit Na/K pumps G1/G0 Arrest Phenotype Can inhibit translation at high levels

Clusters can represent mechanisms of action G1/G0 Arrest Phenotype Steroid Hormones

Clusters can represent mechanisms of action Cardiac glycosides inhibit Na/K pumps G1/G0 Arrest Phenotype Can inhibit translation at high levels

Clusters can represent mechanisms of action Cardiac glycosides inhibit Na/K pumps G1/G0 Arrest Phenotype Can inhibit translation at high levels Translation inhibitor

Conclusions Factor analysis can be applied to reduce a huge amount of microscopy data to interpretable biological phenotypes A small number of probes is capable of detecting fine distinctions in compound activity

Caveats Could not connect compounds to individual targets

Caveats Could not connect compounds to individual targets Is there even a single target?

Acknowledgements Faculty Coaches Brian Shoichet Jack Taunton Jim Wells Student Coaches Noah Ollikainen David Booth Heather Newman “One useless compound is called a placebo, two useless compounds are called a controlled experiment, and three or more become a screening library!”

Acknowledgements Faculty Coaches Brian Shoichet Jack Taunton Jim Wells Student Coaches Noah Ollikainen David Booth Heather Newman “One useless compound is called a placebo, two useless compounds are called a controlled experiment, and three or more become a screening library!”

All Image Analysis Variables

384-well Optical Plates 2000 HeLa Cells/well Grow overnight Add 20 μM compound Incubate 37 o 20hr EdU pulse, Fix The Setup (image from

Factor Analysis Assume measured variables are linear functions of common, underlying factors. Select factors that explain common variance

Different compounds vary in potency Phenotypic Response Metric Distance in factor space from control, untreated cells Select “hits” as compounds with top 5% phenotypic response in both replicates 211 compounds (3%)

Phenotype-structure correlation is statistically significant

Clusters can represent mechanisms of action Cardiac glycosides inhibit Na/K pumps G1/G0 Arrest Phenotype Can inhibit translation at high levels Translation Inhibitor Another translation inhibitor!

“One useless compound is called a placebo, two useless compounds are called a controlled experiment, and three or more become a screening library!” High Content Screening and You