Ligands that can selectively bind to proteins with single amino acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wildtype. However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical, synthetic epitope-targeting strategy which we used to discover a 5-mer peptide with selectivity for the E17K transforming point mutation in the Pleckstrin Homology Domain of the Akt1 oncoprotein. A fragment of Akt1 containing the E17K mutation and a I19[Propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that covalently clicked onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to wildtype, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 substrate.

We present an iterative in situ click chemistry approach to sequentially assemble peptide ligands that can selectively bind to and inhibit a transforming point mutation (E17K) found in the Pleckstrin Homology Domain (PHD) of the Akt1 kinase. The Akt1 kinase plays a critical role in the PI3K signaling pathway - the activation of which is closely linked to tumor development and cancer cell survival. It has recently been shown that the E17K mutation in the PHD of Akt1 results in an increased affinity of the PHD for the PIP3 substrate. Consequently, deregulated recruitment of Akt1 to the cell membrane causes constitutive activation of the PI3K pathway, which has been shown to induce leukemia in mice. We previously showed that peptide affinity agents that mimic the performance of antibodies can be created through the use of iterative in situ click chemistry. These affinity agents have more recently been shown to penetrate cells and function as enzyme inhibitors or activators. Because the selective inhibition of this E17K mutant would be a potentially powerful therapeutic agent, herein a similar in situ click technique was used to create a capture agent targeting the mutant PHD. To selectively target this E17K point mutation, we employed an epitope targeting strategy that began with a 33mer peptide “epitope” fragment from Akt1-PHD containing the mutation (residues 1-32). An alkyne-containing amino acid was substituted into the sequence near the site of the mutation to facilitate an in situ click reaction near that point. An azide-equipped comprehensive One-Bead-One-Compound (OBOC) peptide library was screened for the positive in situ click product with the epitope fragment. The resulting hit “anchors” were sequenced and their affinity and selectivity to the E17K mutant target were validated. Those anchor peptides were further expanded into biligands by repeating the in situ click screening steps, but this time against the full length PHD. Early results with the identified biligand show that the affinity and selectivity for the mutant protein is significantly enhanced compared to the anchor peptide. Preliminary inhibition studies also show that the biligand can partially inhibit the binding of the mutant PHD to the PIP3 substrate. Further studies are currently being conducted to characterize the identified ligands and to evaluate their efficacy as a potential therapeutic. The epitope targeting approach combined with the iterative in situ click screening represents a unique methodology to develop capture agents targeting protein mutations. The resultant selective ligands hold highly anticipated potential value in the field of cancer treatment and/or diagnosis. Citation Format: Kaycie M. Deyle, Ying Qiao Hee, Steven Millward, James Heath. Use of iterative in situ click chemistry to develop a capture agent specific for a transforming point mutation in the pleckstrin homology domain of Akt1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-4. doi:10.1158/1538-7445.AM2013-LB-4