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Scripps Team Develops Peptide Probes for Diagnosing, Monitoring Amyloidosis

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NEW YORK (GenomeWeb) – A team led by scientists at The Scripps Research Institute has developed peptide probes for isolating misfolded transthyretin (TTR) oligomers linked to amyloid diseases.

Detailed in a study published this week in Science Translational Medicine, the probes will allow researchers to extract and analyze oligomeric aggregates associated with various forms of amyloidosis and could potentially prove useful for early diagnosis or monitoring progression and response to treatment of such diseases, said Jeffrey Kelly, co-chair of the department of molecular medicine at Scripps and senior author on the paper.

Amyloidosis describes the build-up of amyloid fibrils in tissue leading to a variety of clinical outcomes, including, in some cases, organ failure and death. In their STM work, Kelly and his colleagues looked specifically at TTR hereditary amyloidosis where it is thought that misfolded TTR oligomers in plasma are key drivers of the condition.

The researchers wanted to analyze misfolded TTR aggregates specifically, as opposed to TTR amyloid fibrils, given past studies indicating that TTR aggregates may drive amyloidosis separate from amyloid fibril accumulation.

"To understand aggregate structure-proteotoxicity relationships, we need to expand our knowledge about the spectrum of aggregate structures that exists in patients by developing reliable probes for each structure," they wrote.

Analyzing TTR aggregates has proved challenging, Kelly said, due to a lack of good probes for extracting them out of plasma where they circulate.

Antibody-based probes do exist, but Kelly noted, there are questions around their performance due to their large size and high binding affinity.

"The concern with antibody-based probes is that because they are large molecules with very large binding sites that typically bind their antigens with quite high affinity, it has been demonstrated that they can change the conformation of [their targets upon binding]," he said. This, he noted, can make a low concentration of oligomers read as higher than it actually is.

"Of course, we all use [antibody-based probes], my lab included, but that is certainly a concern I think most everybody has," he added.

As an alternative, Kelly and his colleagues turned to peptides, which are smaller than antibodies and also have the advantage of being relatively inexpensive to produce.

A number of researchers and companies have explored peptides as diagnostic tools. For instance, Arizona State University spinout HealthTell is developing tests for autoimmune conditions using its peptide-based platform, which screens patient samples against arrays containing as many as 130,000 peptides to identify binding signatures specific to particular diseases.

The Scripps researchers took a more targeted approach to building their probes, using a set of 18 TTR peptides that they screened against the non-native TTR oligomers they hoped to detect. The study authors noted that the idea underlying this approach was that TTR peptides could integrate themselves into misfolded oligomeric TTR aggregates, which, due to their misfolded nature would be less tightly packed than native TTR or TTR amyloid fibrils.

Incubating the candidate peptides with non-native TTR oligomers, Kelly's team identified one peptide, derived from the B β-strand of TTR, that efficiently incorporated into these oligomers. Using this probe, which they named B-1, as well as a modified version containing a diazirine functional group and an alkyne handle, which they named B-2, the researchers looked at the presence of non-native TTR oligomers in patients with and without TTR hereditary amyloidosis, finding significantly higher levels of TTR oligomers in symptomatic carriers of mutations associated with amyloid polyneuropathy than healthy controls.

Notably, they did not detect TTR oligomers in patients with amyloid cardiomyopathy, suggesting that the structures of particular TTR oligomers are highly specific to the type of amyloidosis.

The researchers also profiled the other protein components that bound to the probes, finding that they pulled down a set of proteins whose signature clearly distinguished between familial amyloid polyneuropathy patients, asymptomatic patients, and healthy controls.

They also found that in a subset of FAP patients, a TTR fragment detected by the probes disappeared following treatment with tafamidis, a drug discovered by Kelly and developed by Pfizer for treating TTR hereditary amyloidosis.

Taken together, the results indicate the probes could prove useful for detecting early markers of TTR hereditary amyloidosis and potential gauging the effectiveness of therapy, Kelly said, though he noted that such applications remain far off.

"I would say that hopefully a biomarker could come out of such an analyses, but I'm not sure we've shown that yet," he said. "Biomarkers are tough. You need really good statistics for something to be used in a doctor's office, and I don't think we have that yet. We have a large database of human samples collected, and I think that hopefully as we keep improving these assays we'll see good correlation with clinical data, and only then can we say we have a response to therapy biomarker or an early diagnostic test."

In addition to establishing the utility of the probes for detecting biomarkers, the researchers will need to streamline their analysis to make it better suited to a clinical setting, Kelly said. In the STM paper, they used mass spec for reading out the probes, but, he said, they are currently at work developing a probe that could be read out using an ELISA format.

TTR amyloid diseases are relatively rare, with around 500,000 people worldwide affected, Kelly said, but, he noted, a similar strategy could be applicable to a disease like Alzheimer's, which also involved amyloid buildup. He cited work by University of California, San Francisco researcher Jason Gestwicki on developing peptide probes for the amyloid-β deposits characteristic of Alzheimer's, noting that while they have not yet expanded the work to human samples, "the in vitro data has looked very promising."

"We desperately need in this field early diagnostics and response to therapy biomarkers, so we're striving ultimately to make those," Kelly said.

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