In the U.S. alone, millions of people develop amyloid cardiomyopathy, a fatal but frequently unrecognized disease. This amyloidosis occurs when the serum protein transthyretin falls apart from its normal tetramer, clumps into amyloid fibrils, and weakens heart muscle. Currently, there are no approved medications to treat the disorder, but several promising drug candidates are in the works. In the August 24 Science Translational Medicine, researchers led by Isabella Graef at Stanford University, Palo Alto, California, report the results of a high-throughput screen for transthyretin stabilizers. The scientists turned up several novel drug candidates that look quite promising in initial testing. In addition, the method they used may have broad applicability for finding stabilizers for other amyloid diseases, other scientists say.

Amyloid cardiomyopathy has both a familial and a sporadic form. About 3 to 4 percent of African-Americans carry a valine to isoleucine transthyretin mutation (V122I) that destabilizes the protein and leads to predominantly cardiac deposits. Transthyretin amyloidosis can also arise spontaneously in older adults. Cardiac amyloid deposits are found in about 10 to 15 percent of adults over 65 at autopsy (see Rapezzi et al., 2010). This means perhaps six million Americans, and many more people worldwide, could benefit from an effective treatment.

Most transthyretin mutations lead to a systemic amyloidosis called familial amyloid polyneuropathy (FAP). For years, scientists have been developing transthyretin stabilizers to treat this form of the disease, using rational drug design to create molecules that fit into the binding pocket of the transthyretin tetramer and, in effect, glue the molecule together. One such drug, tafamidis, is now poised for approval for FAP in Europe (see ARF related news story), and is expected to begin clinical trials for amyloid cardiomyopathy soon. Another compound, diflunisal, a non-steroidal anti-inflammatory drug (NSAID), is currently in clinical trials for FAP. NSAIDs are contra-indicated for cardiac patients, however, because they inhibit cyclooxygenase (COX) enzymes and can cause serious heart problems. Graef notes that many rationally designed transthyretin binders resemble NSAIDs and may, therefore, inhibit COX. Co-author Jeff Kelly at the Scripps Research Institute, La Jolla, California, who also designed tafamidis, told ARF that tests show tafamidis does not inhibit COX.

Graef and colleagues wanted to find a broader spectrum of transthyretin stabilizers. To this end, first authors Mamoun Alhamadsheh at Stanford and Stephen Connelly at the Scripps Research Institute performed a high-throughput screen of about 130,000 small molecules. They first developed a rapid, robust fluorescence polarization assay, in which they could detect the binding of fluorescently labeled diflunisal to transthyretin because the bound molecule tumbled less in solution and gave off a stronger fluorescent signal. The researchers screened their small-molecule library for compounds that could displace diflunisal from transthyretin and lower fluorescence. They found 33 candidates with strong activity, including several compounds not previously known to bind transthyretin.

The authors showed that these compounds inhibited transthyretin amyloid formation both in vitro and in cardiac myocyte cultures. They also tested the compounds’ ability to inhibit amyloidosis in human serum; eight of the candidates stabilized the protein more effectively than diflunisal does. Importantly, most of the candidates had low levels of COX inhibition; three had virtually none. In addition, the researchers extensively characterized each candidate, measuring binding energy and kinetics as well as determining the crystal structure of the bound complexes.

The next step, Graef told ARF, is to test the compounds’ ability to stabilize transthyretin in serum from patients with the V122I mutation. They are collaborating with the Stanford Cardiovascular Institute to do those experiments. The authors also want to look for toxicity in animal models, as well as test for non-specific binding to other human proteins. Graef said her hope is that some of these compounds will advance to clinical trials, and if successful, the drugs will provide a greater choice of treatments for people with amyloid cardiomyopathy. Because of genetic variations in the population, the same drug is rarely right for all people, she said.

“This is the first completely unbiased screen for transthyretin binders,” Graef noted. She said one of the exciting findings is that “There is a much greater variety than originally anticipated of chemical scaffolds that can bind to this pocket and can stabilize transthyretin.”

“This is a fantastic strategy, and I think what is most important is that they proved this strategy will work,” said Nikolay Dokholyan at the University of North Carolina, Chapel Hill. He believes the method shows promise for other protein aggregation diseases, and noted that his group is pursuing a similar approach for finding drugs that inhibit superoxide dismutase aggregation, the culprit in some familial forms of amyotrophic lateral sclerosis. Dokholyan added that the paper also sets a high standard for characterization of the compounds, and represents an important step forward for tackling amyloid diseases.—Madolyn Bowman Rogers


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News Citations

  1. Amyloid-Blocking Drug Poised for Approval for Rare Disease

Paper Citations

  1. . Transthyretin-related amyloidoses and the heart: a clinical overview. Nat Rev Cardiol. 2010 Jul;7(7):398-408. PubMed.

Further Reading

Primary Papers

  1. . Potent kinetic stabilizers that prevent transthyretin-mediated cardiomyocyte proteotoxicity. Sci Transl Med. 2011 Aug 24;3(97):97ra81. PubMed.