Molecular Glue May Help CFTR Protein Work Better

Margarida Maia, PhD avatar

by Margarida Maia, PhD |

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A type of ring-shaped molecule called a macrocycle can help CFTR — the faulty protein in cystic fibrosis (CF) — go to the cell’s membrane, where it exerts its action, and work better in lab-grown cells carrying the most common CF-causing mutation, a study showed.

These benefits were associated with the macrocycle’s ability to stabilize the natural interaction between CFTR and 14-3-3, a protein known to facilitate CFTR’s trafficking to the cell’s membrane and boost its function.

Also, the macrocycle — called CY007424 — amplified the effect of certain approved CFTR modulators, which are medications designed to improve the functionality of the mutated CFTR.

“This macrocycle is a useful tool to study the CFTR/14-3-3 interaction and the potential of molecular glues in [CF] therapeutics,” the researchers wrote.

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The study, “Macrocycle-stabilization of its interaction with 14-3-3 increases plasma membrane localization and activity of CFTR, was published in Nature Communications.

CFTR is an ion channel shaped like a tube that goes across a cell’s membrane and controls how ions, such as chloride, get in and out of the cell. This movement is key to attract water into the various organs of the body such as the lungs, where this is essential for making the watery mucus that helps keep the airways clean.

In people with CF, the CFTR protein is either not made at all or folded into a wrong shape due to mutations in the CFTR gene. This prevents CFTR from traveling to the cell’s membrane, where it does its job, resulting in the buildup of thick and sticky mucus in the lungs and other organs, and ultimately leading to the symptoms of CF.

Previous studies have shown that members of a family of proteins called 14-3-3 can bind to a certain region of the CFTR, stabilizing it and helping it travel to the cell’s membrane to let chloride move through.

Also, researchers in the Netherlands and Canada found that fusicoccin A, a natural macrocycle made by a fungus, stabilizes the CFTR/14-3-3 interaction.

By doing so, fusicoccin A was shown to help F508del-mutated CFTR reach the cell’s membrane and allow chloride flow. The F508del mutation, the most common cause of CF, leads to the production of a nearly fully functional protein.

Since fusicoccin A’s structural complexity challenges its chemical optimization for medicinal purposes, the same research team set out to search for other macrocycles that could act as a molecular glue for the CFTR/14-3-3 complex.

They first screened a library of 5,760 macrocycle molecules to find which could make 14-3-3 bind the strongest to a shorter version of CFTR containing the region 14-3-3 is known to interact with.

The search narrowed the library down to a list of seven molecules, which inspired the researchers to design a second library of 480 macrocycles, of which eight additional molecules were found to significantly strengthen the CFTR/14-3-3 binding.

CY007424, one of the selected macrocycles, was found to stabilize the CFTR/14-3-3 complex by more than 300 times. When combining the two macrocycles, CY007424 plus fusicoccin A, the researchers found that CFTR/14-3-3 binding was stabilized by more than 800 times.

CY007424 worked by slightly changing the way 14-3-3 interacted with CFTR, analyses showed. It did so by taking advantage of the relatively flexible shape of the CFTR protein.

Out of 12 macrocycles tested, CY007424 also showed the strongest increase of F508del-mutated CFTR trafficking toward the cell’s membrane in lab-grown hamster cells.

In the presence of CY007424, not only more of F508del-mutated CFTR reached the cell’s membrane, but also it let more chloride ions pass through. This was not seen with any of the other macrocycles tested, however.

In addition, adding CY007424 to either lumacaftor — one of the medications in the CFTR modulator Orkambi — or Trikafta (elexacaftor/tezacaftor/ivacaftor), another approved CFTR modulator, resulted in an even greater chloride flow through the F508del-mutated CFTR channel in lab-grown cells derived from a CF patient.

Lumacaftor binds to the misshaped CFTR and helps it fold correctly, while the triple-combo therapy Trikafta not only improves protein folding, but also holds the ion channel open so that more chloride can pass through it.

These findings demonstrated that macrocycles such as CY007424 can stabilize CFTR/14-3-3 binding and ultimately improve CFTR’s function, while combinations with CFTR modulators “show an additive effect,” the researchers wrote.

Also, “since 14-3-3 proteins are positive regulators of CFTR that facilitate forward trafficking to the [cell] membrane and stabilize the functional fold of the channel, [macrocycle molecules] are useful tools to study the CFTR/14-3-3 interaction,” they said.