Research reveals molecular origins of how a key drug target works

Through an international collaboration, scientists at St. Jude Children’s Research Hospital leveraged data science, pharmacology and structural information to conduct an atomic-level investigation into how each amino acid in the receptor that binds adrenaline contributes to the activity of the receptor in the presence of this natural amino acid. ligand.

They discovered precisely which amino acids control key pharmacological properties of the ligand. The adrenaline receptor being studied is a member of the G protein-coupled receptor (GPCR) family, and this family is the target of a third of all drugs approved by the Food and Drug Administration (FDA). Thus, understanding how GPCRs respond to natural or therapeutic ligands is essential for developing new therapies with precise effects on receptor activity.

The article is published in the journal Science.

To understand how a watch works, simply take it apart piece by piece and study the role played by each component in its chronometric function. Similarly, in a protein such as a GPCR, each amino acid could play a different role in how the protein responds to an external signal.

St. Jude researchers, in collaboration with scientists from Stanford University, the University of Montreal, the MRC Molecular Biology Laboratory and the University of Cambridge, studied the β2-adrenergic receptor (β2AR) by substituting one amino acid at a time to understand the contribution of each amino acid of that receptor to mediate a signaling response.

“Scientists are learning how genes contribute to the functioning of cells by disrupting them one by one. We asked: ‘Why not go further in this area?’ Let’s understand how each amino acid contributes to the function of a receptor by mutating it, one amino acid at a time,” said co-corresponding author M. Madan Babu, Ph.D., of the Department of Structural Biology at St. Jude, director of the Center of Excellence for Data-Driven Discovery and the George J. Pedersen Chair in Biological Data Science.

“Over the course of evolution, each amino acid in the receptor has been sculpted in one way or another to ensure that it binds to the natural ligand, in this case adrenaline, and elicits the appropriate physiological response .”

Search function in the form

GPCRs are proteins that cross the cell membrane and connect the exterior of the cell to its internal environment by transmitting external signals inside the cell. In the case of β2AR, adrenaline binds to the GPCR outside the cell, inducing a response inside the cell.

When a ligand binds, it causes changes in the shape of the receptor, particularly in the intracellular region of the receptor where a G protein binds. The ligand and G protein binding sites are on opposite sides of the receptor. the protein but connect via a complex network of amino acid contacts that span the entire protein. Conformational (shape) changes within the GPCR activate the G protein to trigger a downstream signaling response within the cell. Through its effects on multiple tissues and GPCRs, including β2AR, adrenaline can trigger the fight-or-flight response, similar to an adrenaline rush.

To understand the role of each amino acid in a GPCR, Franziska Heydenreich, Ph.D., of Philipps University Marburg, lead author and co-correspondent of this project, mutated each of the 412 amino acids of the β2AR. She then assessed each mutant’s response to the ligand adrenaline and determined classical pharmacological properties of efficacy and potency. Efficiency measures the maximum response a ligand can elicit, and potency measures the amount of ligand needed to elicit half the maximum response. The goal was to reveal, at the atomic scale, how each amino acid contributes to these pharmacological properties.

“Surprisingly, only about 80 of the more than 400 amino acids contributed to these pharmacological properties. Of these pharmacologically relevant amino acids, only a third were located in regions where the ligand or G protein bound to the receptor,” he said. Heydenreich said.

“It was fascinating to observe that there are certain amino acids that control effectiveness, others that control potency, and others that affect both,” Babu said. “This means that if you want to make a more potent or effective drug, you now know that there are specific residues that the new ligand must influence.” The researchers also noted that each residue’s individual contribution to efficacy and potency was not equal, implying even more opportunities to fine-tune drug responses while designing new therapeutic ligands.

“The efficiency and potency of many ligand-receptor signaling systems have been measured for several decades. We can now understand how specific amino acids in a protein’s sequence can influence these pharmacological properties,” Babu explained.

“A fascinating aspect of the results is that potency and efficacy may be regulated independently of each other by distinct mechanisms. This provides a basis for understanding how genetic variation influences drug responses in individuals,” Michel Bouvier, Ph.D., co-correspondent,” added the author from the Department of Biochemistry and Molecular Medicine and Director General of the Institute for Research in Immunology and Cancer at the University of Montreal.

A beautiful network

Previous research has illustrated the structure of the active and inactive states of β2AR. Armed with this knowledge, the researchers embarked on a new investigation. They explored whether the two-thirds of pharmacologically relevant amino acids previously shown to be not involved in ligand or G protein binding could play a role in the transition between the active and inactive states of the receptor.

“We systematically started looking at each single residue contact in the active state,” Heydenreich said, “to understand whether all amino acids that make contact with the active state are important.”

Researchers developed a data science framework to systematically integrate pharmacological and structural data and revealed the first comprehensive picture of GPCR signaling. “When we mapped the pharmacological data onto the structure, it formed a beautiful network,” Babu said.

“This provided new insights into the allosteric network connecting the ligand binding pocket to the G protein binding site that governs efficiency and potency,” added Brian Kobilka, co-corresponding author and Nobel laureate. in Chemistry 2012 from Stanford University School of Medicine.

By understanding GPCR signaling at the atomic level, researchers are optimistic that they can begin to probe even deeper, to see transient substates between active and inactive conformations and to explore the conformational landscape of proteins.

“We now know which mutants to look for, those that only affect efficiency, potency, or both,” Heydenreich said.

“Now we can perform molecular dynamics calculations and single-molecule experiments on these mutants to reveal the exact mechanisms by which the allosteric network influences the efficiency and potency to mediate a signaling response. This is one direction which we are continuing through a St. Jude GPCR Collaborative that includes PIs from multiple institutions, Babu explained.

In addition to these “conductive” residues involved in mediating state-specific active contacts and affecting pharmacology when mutated, Babu and colleagues intend to explore other key findings revealed by this work. They aim to study “passenger” amino acids which, despite making contacts in the active state, do not affect efficiency or potency when mutated.

They are also interested in “modulatory” residues that do not mediate state-specific active contacts, but alter pharmacology when mutated. Their data science approach, integrating structural information and pharmacological measurements, is not limited to β2AR. It can be extended to any GPCR to improve our understanding of the mechanisms governing this crucial class of drug targets.