Screening Test Raises Possibility of Therapeutic Approach for Alport Syndrome

Researchers have developed a high sensitivity, large-scale screening test that suggests a new molecular therapeutic approach for Alport syndrome.

The study, “A Split-Luciferase-Based Trimer Formation Assay as a High-throughput Screening Platform for Therapeutics in Alport Syndrome, “ was published in the journal Cell Chemical Biology.

Alport syndrome is a genetic condition characterized by progressive loss of kidney function. The disorder is caused by mutation in either the COL4A3, COL4A4 or COL4A5 genes. These genes encode type IV collagen chains, which form the backbone of the glomerular basement membrane, an extracellular matrix that contributes to the kidney’s filtration barrier.

Current treatment for patients with Alport syndrome typically uses blockers of the renin-angiotensin-aldosterone system (RAAS), which, although it may suppress progression of kidney inflammation, does not stop later development of end-stage renal disease.

Similar to cystic fibrosis and familial epilepsy, Alport also is classified as a misfolded protein disease, which describes disorders that include abnormal structure of proteins. Preclinical and clinical studies suggested that normalizing the alpha-345(IV) molecule (a trimer), which is formed by the gene products of COL4A3-5, may be a successful therapy for Alport patients. This also is indicated by the finding that even low amounts of alpha-5(IV) improves clinical manifestations in Alport. However, no current compounds are able to correct the alpha-345(IV) network.

One of the factors that may contribute to this lack of available treatments for altered alpha-345(IV) is the current insufficient knowledge of which molecules affect alpha-345(IV) inside the cells, the researchers observed. The lack of a screening assay for alpha-345(IV) formation that may be used in large-scale, automated testing, or high-throughput screening (HTS) is another limitation.

Aiming to address this gap, the research team developed a new test, or assay, that uses a system called Split Nanoluciferase binary technology, which produces light to enable detection of protein interaction.

With the new method, the scientists showed that some chemical chaperones, which are small molecules that promote the folding or structural stability of other proteins, improved the formation of alpha-345(IV) in alpha-5(IV) mutants in in vitro experiments..

Results also showed that different intracellular mutant alpha-5(IV) monomers, which are one of the three components of the alpha-345(IV) trimer, were stable and led to detectable alpha-3-5(IV) proteins.

Compared to normal proteins, these mutant alpha-5(IV) monomers also exhibited similar degradation mechanisms and cellular localization in the endoplasmic reticulum, the cell’s structure responsible for folding proteins.

The similar intracellular regulation between normal and mutant alpha-5(IV) indicates that the trafficking of alpha-5-(IV) may be a complicated target for therapy and that alpha-345(IV) formation may by a critical defect in mutants, the authors considered.

The scientists then used the new system to assess alpha-345(IV) formation in cells. They found that newly assembled alpha-345 complexes were detected with high sensitivity, including when scientists tested clinically relevant alpha-5(IV) mutants. The findings also revealed that “correcting the protein folding and promoting secretion is a better therapeutic focus than intracellular stabilization,” the researchers wrote.

Experiments that increased the production of a single chaperone at a time revealed that although these molecules are important for alpha-345(IV) formation, they could not improve its secretion. However, chemical chaperones were able to correct its formation.

“Our findings demonstrated a potential trimer [alpha-345(IV)]-based therapeutic strategy for [Alport syndrome],” the researchers concluded.

“The findings presented here are highly relevant to causal protein-based therapy for AS and may be applicable to other diseases,” they added.