New study offers potential rational approach to treat cystic fibrosis

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Combining molecules may prove to be key

Dr. Gergely Lukacs (left) and Dr. Guido Veit

A new study conducted in the lab of Dr. Gergely Lukacs at McGill University’s Faculty of Medicine may offer a more effective avenue to explore the treatment of cystic fibrosis. The findings are published in Nature Medicine.

Cystic fibrosis (CF) mutations impair the function of the cystic fibrosis transmembrane conductance regulator (CFTR), a membrane protein encoded by the CF gene, which ensures proper salt and water content of fluids lining the gut and lung inner surfaces. CF mutations lead to lung infection and uncontrolled inflammation, the primary cause of premature death in CF patients.

The most common of these mutations, single amino acid deletion of phenylalanine at position 508 (ΔF508) in one of the five domains of CFTR, causes serious damage in the three dimensional architecture of the CFTR protein and, as a consequence, increases the stickiness of the surface liquid layer on epithelial cells that protect body tubes (e.g. airway and gut) from environmental insults (e.g. bacteria and virus particles) and the secretory portions of glands and their ducts.

While currently available drugs only modestly correct mutation-caused CFTR folding defects, in their study, the researchers showed that by selecting a combination of small molecules to target multiple, distinct structural defects they can potently rescue the mutant CFTR architecture and function in airway epithelia. “While individually these compounds marginally improve ΔF508-CFTR folding efficiency, plasma membrane function, and stability, combining them leads to approximately 50-100% of wild type correction in cell lines and primary cells isolated from the nose of CF patients, as well as in a mouse model of cystic fibrosis,” explains Dr. Guido Veit, a Research Associate in Dr. Lukacs’ lab and the study’s lead author. Remarkably, the small molecule cocktails also rescue the functional defects of some rare CF mutations resistant to available drugs.

The work builds on a 2012 study published in Cell by Dr. Lukacs’ lab that identified that ΔF508 mutation leads to at least two distinct structural defects in CFTR and that both need to be corrected. Since that time they had been trying to identify small molecule compounds that could serve as correctors with distinct mechanism of action. To achieve this goal they screened for compounds in the background of one corrector with a known mechanism of action in order to identify correctors with complementary mechanisms. The screening of 600,000 small molecules was performed in collaboration with the Genomics Institute of the Novartis Research Foundation (GNF). Subsequent mechanistic work led to the identification of a corrector combination with twice the efficacy of the current FDA-approved corrector therapy in cells isolated from homozygous ΔF508 patients.

The compounds have not yet been optimized for their pharmacological properties, a prerequisite for clinical trials but the researchers are working on this and are planning to connect with partners in order to develop the compounds into corrector drugs. “Our hope is that by increasing the correction efficacy of ΔF508-CFTR to about 50% of the wild type level, the clinical manifestations of cystic fibrosis in individuals that are homozygous for ΔF508, which comprises approximately 50% of all CF patients, can be completely alleviated through this therapy since heterozygous carriers lack disease symptoms,” explains Dr. Lukacs, who is a Professor in the Departments of Physiology and Biochemistry and Canada Research Chair in Cystic Fibrosis and Conformational Diseases at McGill. “Additionally, because of the similarity between the CFTR architecture and other members of the ABC transporter protein family, therapeutic approaches for other diseases such as neonatal respiratory distress syndrome, familial hyperinsulinism, and progressive familial intrahepatic cholestasis may also benefit from our findings down the line.”

CFTR consists of five building blocks (or domains): two membrane spanning domains (MSD1 and MSD2) which forms a pore that permits the movement of salt across the cell surface in a controlled manner and three additional cytosolic domains (nucleotide binding domains NBD1and NBD2 and a regulatory (R) domain, commanding the pore opening according to hormonal regulation). CFTR’s five domains are produced consecutively and connected together like pearls on a necklace, but in order to form a functioning protein the domains need to be connected like pieces of a puzzle (a). Unlike a puzzle, however, individual domains adopt their final shape, only when they are assembled together. We proposed earlier that many mutations, including the ΔF508, can perturb the shape of its own domain, which in turn impairs the shape of the other domains and their proper assembly (b). The mutant CFTR with scrambled domains is not functional, trapped inside the cell and tagged for degradation by the cellular quality control to preseve the cell integrity. We argued that targeting multiple domains’ structural deficiencies simultaneously can more effectively rescue the mutant architectural defect than a single corrector as the approved CF drug does. To achieve our goal we designed a simple screening assay to monitor the mutant CFTR accumulation at the cell surface. This assay allowed the screening of more than half a million compounds in an academic collaboration with researchers at GNF and identified several modestly effective corrector molecules. Using a variaty of assays, we grouped the identified hits into three classes: Type I correctors promoting formation of the NBD1-MSD1/2 interface, Type II correctors facilitatting folding of NBD2 and formation of the NBD1/NBD2 or NBD2/MSDs interface, and type III correctors which nudge the mutant NBD1 domain to adopt an improved fold (c). Combining the three mechanistically distinct correctors was sufficient to increase the biochemical rescue of ΔF508-CFTR to a level similar to the WT and to restore 50% of the channel function in airway cells.
Importantly, this strategy also worked for other point mutations in various domains that cause CFTR misfolding. This underscores the interdependent nature of CFTR domain folding/assembly (i.e. the puzzle pieces only adopt their final shape when they fit together) and the allosteric or long range conformational coupling of corrector molecules (i.e. a given corrector promotes folding of one domain, which in turn facilitates the spatial fitting of other pieces of the puzzle).

This work was performed in collaboration with Drs. Elie Matouk and Saul Frenkiel, McGill University Health Centre, Department of Respiratory Medicine and Department of Otolaryngology-Head and Neck Surgery, McGill, respectively, Drs. I. Sermet-Gaudelus, A Edelman and E Dreano, Necker Institute, Paris, and Dr. W Barnes group, GNF, San Diego.

The research was supported by Cystic Fibrosis Canada, Cystic Fibrosis Foundation Therpeautic Inc., CIHR and NIH.

“Structure-guided combination therapy to potently improve the function of mutant CFTRs” by Guido Veit et al. and Gergely Lukacs, is published in Nature Medicine.


October 11, 2018


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