These findings support the further development of BBM as an alternative treatment for FAP.
The study, “Repurposing Benzbromarone for Familial Amyloid Polyneuropathy: A New Transthyretin Tetramer Stabilizer,” was published in the International Journal of Molecular Sciences.
Mutations in the TTR gene — the underlying cause of FAP — destabilize the encoded transthyretin protein such that it accumulates as fibers, called amyloid deposits. These deposits are seen in multiple tissues, including the nerves, heart, kidneys, and eyes, leading to damage and disease symptoms.
Vyndaqel (tafamidis) is an oral medication approved in Europe and under investigation in the U.S. to treat FAP. It works by stabilizing the TTR protein, thus preventing the formation of amyloid deposits. Tolcapone (SOM0226) is another investigational FAP therapy that also stabilizes TTR.
Both Vyndaqel and tolcapone share a similar molecular structure and work by binding directly to the transthyretin (TTR) protein in the same area as the natural substrate (thyroxine), called the T4 binding pocket.
BBM is a medication used for more than 30 years to treat hyperuricemia, or high levels of urinary uric acid, and gout — arthritis caused by hyperuricemia — and shares many of the same molecular features as Vyndaqel and tolcapone.
As such, researchers based at the Institute for Advanced Chemistry of Catalonia, in Spain, along with colleagues at the Institute of Molecular and Cell Biology, in Portugal, investigated the potential of BBM to stabilize TTR protein as an alternative to Vyndaqel and tolcapone.
First, purified TTR protein was incubated with urea, a chemical that destabilizes proteins, causing them to unfold (denature). Other samples added BBM, as well as iododiflunisal (IDIF) — a potent TTR stabilizer used here as a positive control — and sulfaquinoxaline, a compound that does not stabilize TTR. Here, sulfaquinoxaline was used as a negative control.
Compared with TTR alone, or the negative control, the addition of BBR stabilized the protein from urea denaturation to the same extent as IDIF.
Next, the team showed that BBM stabilized TTR by binding to the T4 binding pocket as it completely displaced the natural substrate thyroxine. A more detailed test demonstrated that BBM (and IDIF) bound to the T4 pocket with a similar affinity, or binding strength, as thyroxine.
The specific strength of binding was then measured by a technique called isothermal titration calorimetry (ITC), which generates a Kd value — the concentration at which half of the target proteins are bound to the molecule. A lower Kd means stronger binding affinity.
The Kd for BBM with purified TTR protein was 60 nanomolar (nM), compared with 200 nM for Vyndaqel and 270 nM for tolcapone.
“These ITC results confirm that BBM is a potent binder of TTR,” the researchers wrote.
The binding of BBM directly in the T4 binding pocket within the TTR protein was then confirmed by a method that determines the structural arrangement of all BBM atoms within the protein.
Finally, the impact of BBM on TTR amyloid fiber formation was measured using a mutated form of TTR protein prone to destabilization. BBM prevented fiber formation at the same concentration as IDIF, but also Vyndaqel and tolcapone.
“On the basis of the obtained results, we can conclude that the uricosuric drug benzbromarone (BBM) presents an interesting scaffold in the quest to design of new and improved TTR stabilizers,” the investigators concluded. “Further studies are in progress to evaluate if this drug can be repurposed for FAP.”
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