The rockefeller university

The best experimental approach begins with the question you need to answer—not with the technique you happen to have available.

Modern drug discovery offers many ways to study a biological system. The same question may be
approached through biochemical assays, cellular measurements, screening, biophysical methods,
imaging, computational analysis, or combinations of these approaches.

Choosing among them requires more than asking which technology can produce a measurement.
The important question is which experiment will generate evidence appropriate to the scientific
decision that needs to be made.

Define the Question First

Before choosing a method, define as precisely as possible what you want to learn.

Are you trying to determine whether two molecules interact? Identify compounds that alter an
enzyme or pathway? Measure binding affinity? Understand a cellular phenotype? Establish target
engagement? Compare the potency of related compounds? Determine why an unexpected result
occurred?

These are different questions, even when they involve the same biological target.

A clearly defined question makes it easier to determine what should be measured, which controls
are needed, and what result would support—or challenge—the working hypothesis.

Consider the Decision the Experiment Needs to Support

Experiments are most useful when the possible outcomes lead to decisions.

An early feasibility experiment may only need to establish whether a measurable biological
effect exists. A screening assay must be sufficiently robust to distinguish activity across
many compounds. A medicinal chemistry assay must measure potency precisely enough to compare
analogs. A mechanistic experiment may need to distinguish among competing explanations for
observed activity.

The required level of precision, throughput, biological complexity, and validation therefore
depends on what will be done with the result.

Design the experiment around the decision—not simply around the measurement.

Start with What Is Known—and What Is Not

The most informative next experiment depends on the evidence that already exists.

If a compound produces a cellular phenotype but its molecular target is uncertain, another
phenotypic experiment may add less information than a study addressing mechanism or target
engagement.

If biochemical activity has been established but direct binding has not, a complementary
biophysical measurement may address an important gap.

If a purified protein behaves inconsistently, additional sample characterization may be more
useful than immediately attempting an affinity measurement or screening campaign.

The next experiment should reduce an important uncertainty.

Let the Reagents Help Define What Is Possible

Experimental strategy is constrained by the biological material available.

The amount, concentration, purity, activity, stability, and format of proteins, cells, antibodies,
substrates, compounds, and other reagents can determine which approaches are practical and which
require additional development.

A technique that is theoretically ideal may not be the best first experiment if it requires
quantities or qualities of material that are not yet available.

Conversely, existing reagents may allow a relatively simple pilot experiment to answer an
important question before more complex materials are produced.

Match the Method to the Information You Need

Different experimental approaches provide different kinds of evidence.

A biochemical assay can measure activity in a controlled system. A cellular assay can provide
greater biological context. A binding experiment can establish direct molecular interaction.
Kinetic measurements can reveal association and dissociation behavior. Imaging can capture
spatial or phenotypic changes. High-throughput screening can search broadly for chemical matter
with a desired activity.

These approaches are complementary rather than interchangeable.

The fact that an instrument can measure a sample does not necessarily mean that the measurement
will answer the question of interest.

Balance Biological Relevance and Experimental Control

Increasing biological complexity can make an experiment more physiologically relevant, but it
can also make interpretation more difficult.

Purified biochemical systems allow individual components to be controlled precisely, but they
may not reproduce the environment in which those molecules function in a cell. Cellular assays
preserve more biological context, but observed activity may result from multiple targets,
pathways, transport processes, metabolism, toxicity, or other effects.

Neither approach is inherently better. The appropriate level of complexity depends on the
question.

In many projects, confidence develops by moving between controlled molecular experiments and
more complex biological systems.

Use More Than One Measurement When the Question Requires It

Important conclusions often benefit from independent lines of evidence.

A functional assay may show that a compound changes biological activity, while a biophysical
measurement can test whether it interacts directly with the proposed target. An orthogonal assay
can determine whether activity persists when the detection technology changes. A cellular
experiment can test whether activity observed with purified components survives in a more
complex biological system.

Agreement among experiments based on different principles can greatly strengthen an
interpretation.

Disagreement can be equally informative because it identifies assumptions that need to be
examined.

Start with the Simplest Experiment That Can Answer the Question

The most sophisticated experiment is not necessarily the most informative first experiment.

A small pilot study can often establish feasibility, define useful concentration ranges, reveal
problems with reagents, or distinguish among several possible approaches before substantial
time and material are committed.

If the pilot answers the question, additional complexity may not be necessary. If it raises a
new question, the next experiment can be designed using what has been learned.

Complexity should be added because the scientific question requires it, not because the technology is available.

Expect the Strategy to Evolve

Drug discovery rarely follows a perfectly linear sequence.

A screening result may lead back to assay development. A biophysical measurement may reveal a
sample problem. A cellular result may suggest an unexpected mechanism. Medicinal chemistry may
produce compounds whose behavior requires new assays or different analytical methods.

Experimental strategy should therefore evolve as evidence accumulates.

The result of one experiment becomes part of the design of the next.

Know When Not to Do the Experiment

Sometimes the most useful conclusion from an initial discussion is that the proposed experiment
should not yet be performed.

The necessary controls may be missing. A reagent may require additional characterization.
The expected signal may be below the useful range of the proposed method. Another experiment
may answer the question more directly, with less material or fewer assumptions.

Recognizing these issues early can save considerable time and resources and often leads to a
better experiment.

When Should You Talk to the DDRC?

You do not need to know which technique you need before contacting us.

Bring the scientific question, what is already known, the reagents and data you have available,
and the decision you are trying to make. We can help evaluate possible approaches, identify
important controls or missing information, and determine what experiment—or sequence of
experiments—is most likely to move the project forward.

Sometimes that leads to assay development, screening, biophysical characterization, data
analysis, or a combination of approaches. Sometimes it identifies work that should happen
before any of those begin.

The right experimental approach is the one that gives you evidence you can use to decide what to do next.