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Researchers Demonstrate Potential of Indi's PCC Agents to Target 'Undruggable' Proteins

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NEW YORK(GenomeWeb) – A team led by California Institute of Technology researcher James Heath has demonstrated the ability of Indi Molecular's PCC (protein catalyzed capture) agents to target single point mutations not associated with the binding pockets on the target protein.

The work, which was detailed in a study published this week in Nature Chemistry, is an important proof-of-principle of the PCC agents' capabilities, particularly with regard to their potential as pharmaceutical agents, Heath, who is a co-founder of Indi Molecular and the developer of the PCC technology, told GenomeWeb.

PCCs use click chemistry combined with pairs of random peptide libraries — one containing acetylene functionalities and the other containing azide groups — to create affinity reagents to given proteins. Target proteins are screened against these libraries to find peptides that bind them, and when these peptides bind, the protein epitope acts as a catalytic point for the acetylene- and azide-containing peptides, which then link together via click chemistry, forming multi-peptide, protein-binding constructs that can then be pulled down and identified.

According to Indi, the PCC agents offer several potential advantages over conventional affinity reagents like monoclonal antibodies, including better specificity as well as the ability to target regions of proteins not accessible to monoclonals.

A spinoff of proteomics firm Integrated Diagnostics, Indi Molecular launched as a separate entity in September 2013 with $1.8 million in seed funding. The company planned initially to focus on developing PCCs for use as therapeutics, but funding difficulties forced it to concentrate more on establishing the molecules as potential affinity reagents for biological research and in vitro diagnostics.

More recently, though, Indi has shifted back toward its original drug development aims, and the Nature Chemistry paper provides a look at where it hopes to take the technology.

As the study authors noted, there is great demand for drugs capable of selectively inhibiting proteins with disease-related mutations while leaving wildtype versions of the protein alone. A key challenge to this approach is the fact that the relevant mutation may not be a part of any of the target protein's binding pockets, which is typically required for targeting by traditional small molecule agents.

Unlike small molecules, antibodies can be developed to target point mutations outside a proteins' binding pocket. However, it is difficult to introduce antibodies into living cells, meaning, Heath noted, that antibody-based drugs aren't typically able to target intracellular proteins.

PCCs, on the other hand, are capable of both targeting point mutations outside a protein's binding pocket and entering living cells to target intracellular proteins.

"This ability of PCC agents to target undruggable sites is a pretty big deal," Heath said. "Think of targets in cancer that are considered undruggable – like KRAS – because of the lack of suitable binding pockets for small molecule inhibitors."

Heath and his colleagues demonstrated the PCCs' ability to go after such targets in the Nature Chemistry work by developing agents that selectively targeted the E17K point mutation of Akt1, which lies outside the protein's binding pocket.

To develop the agents, they synthesized an Akt1 peptide representing the E17K point mutation of interest and incorporated near the mutation site an alkyne presenting feature capable of binding to a complementary azide-containing feature via standard click-chemistry. They then screened azide-containing PCCs against this synthetic peptide, working under the assumption that PCCs that bound to the target epitope would also activate the azide-alkyne click-chemistry. By looking for hits that caused the click-chemistry anchors to bind, the researchers were able to distinguish PCCs targeting the E17K point mutation from those targeting other parts of the peptide.

From a screen of some 1.5 million agents, Heath and his colleagues identified 21 hits, one of which – the yleaf ligand – showed a significantly higher affinity for the E17K point mutation compared to the wildtype protein.

The researchers then confirmed that this yleaf ligand in fact bound to the E17K point mutation using a variety of methods including ELISA, directed labelling followed by MALDI TOF/TOF mass spec, and cell imaging.

While the yleaf ligand bound to the target mutation with selectivity of 10:1 compared to wildtype protein, it did not inhibit the mutated protein's activity. In particular, Heath and his team aimed to inhibit the mutated Akt1 protein's binding to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). The mutated Akt1 has increased affinity for PIP3, which has been implicated in leukemia in mice.

To develop agents capable of inhibiting protein activity, Heath and his team performed an additional two rounds of screening to develop a bi-ligand and tri-ligand based on the original yleaf ligand. Ultimately, Heath said, the tri-ligand PCC demonstrated the ability to block Akt1-PIP3 interaction with selectivity of 1000:1 for the mutated Akt1.

The larger agent still needs to be reengineered to make it capable of penetrating a living cell, however, he noted.

"I think at this stage you would call the performance of this inhibitor extremely promising," Heath said. "This is the stage where a pharma company might begin doing what are known as medicinal chemistry iterations, which are small tweaks to the structure of the molecule to improve performance and pharmacokinetics."

Nonetheless, Heath characterized the Nature Chemistry work as primarily proof-of-principle, noting that the mutated Akt1 "is a bit of a rare oncogene to really make this a viable drug."

Indi Molecular is engaged in similar work targeting other oncogenes, he said.

"Those oncogenes are much more common, and so they comprise excellent drug targets from all points of view," he added. "We are building PCC agent therapeutics against a couple and are in negotiations with a couple of pharma companies right now as we look to further develop those PCCs."

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