Physiological Factors Reshape How Drugs and Proteins Interact

A new Northwestern Medicine study has demonstrated that proteins studied in simplified laboratory conditions don’t behave the same way in the human body, according to the study published in Nature Structural and Molecular Biology.

Instead, the research shows that physiological factors such as temperature and calcium can dramatically alter how drugs interact with proteins, potentially reshaping the way scientists screen for and design new therapies.

“We found that temperature really matters,” said the study’s co-senior author Wei Lü, PhD, professor of Pharmacology and of Weinberg College of Arts and Sciences. “If you continue to use very simplified experimental conditions, you may miss opportunities to identify useful drug molecules, or even misunderstand how they work.”

In the study, Lü and his collaborators focused on TRPM4, an ion channel involved in processes ranging from heart rhythm and immune signaling to cancer progression.

For decades, scientists have studied protein structures at low temperatures, often between 4°C and 18°C, to stabilize samples for imaging. But proteins in the human body operate at 37°C, Lü said.

Using cryo-electron microscopy — a technique that determines protein structures at near-atomic resolution — the team compared how TRPM4 behaves under conventional lab conditions versus those mirroring normal physiological conditions.

They found that proteins adopt different shapes at body temperature, and drugs sometimes behave in unexpected ways.

One of the most surprising findings involved a compound called TPPO. Previously believed to be inactive against TRPM4, the molecule showed little effect when tested at room temperature. But at 37°C and in the presence of normal calcium levels, TPPO became an activator of the channel.

“This overturns what we thought about this compound,” Lü said. “It wasn’t inactive, it just needed the right physiological context.”

That context includes calcium, a tightly regulated ion inside cells. Even very low levels — around 100 nanomolars — significantly influenced how molecules interacted with the protein, according to the findings.

“Even though that’s a very low concentration, it has a major impact on how ligand binding happens,” Lü said.

The study also revealed that not all drugs respond to physiological conditions in the same way.

Another compound, Necrocide-1 (NC1), activated TRPM4 without calcium under lab conditions, but lost its effect when calcium levels rose. Meanwhile, two inhibitors, NBA and CBA, consistently blocked the channel regardless of temperature.

Together, the results show that drug behavior is not fixed and depends on the interplay between the molecule, the protein and the environment.

“This is not just for TRPM4,” said co-senior author Juan Du, PhD, professor of Molecular Biosciences at Weinberg. “All proteins function in the human body under physiological conditions — temperature, ions, lipids — and they can all be affected.”

The study suggests that relying on simplified laboratory conditions may lead scientists to overlook promising drug candidates or misclassify how compounds work.

“This has broader implications for drug discovery, pharmacology and biomedical studies,” Du said.

Lu and Du said the findings underscore a shift toward what they call an “environment-aware” approach to studying proteins, one that incorporates factors like temperature and cellular ions into experiments.

Such an approach could help scientists better mimic real biological conditions and uncover drug interactions that would otherwise go unnoticed.

Looking ahead, the team plans to expand their work by screening additional compounds against TRPM4 under physiological conditions and applying the same approach to other disease-related proteins.

They are also exploring other aspects of the cellular environment, such as membrane lipids, to further refine how proteins are studied in their native context.

“The bottom line is: temperature matters,” Lü said. “Physiological factors matter when you study your favorite protein.”

The study was supported by the National Institutes of Health under grants R01HL153219, R01NS112363 and R35GM138321, as well as a McKnight Scholar Award, a Klingenstein-Simon Scholar Award, a Sloan Research Fellowship in neuroscience, a Pew Scholar in the Biomedical Sciences award, and additional NIH funding (R01NS111031 and R01NS129804).