Arundhati Nag

In situ click chemistry: from small molecule discovery to synthetic antibodies

Advances in the fields of proteomics, molecular imaging, and therapeutics are closely linked to the availability of affinity reagents that selectively recognize their biological targets. Here we present a review of Iterative Peptide In Situ Click Chemistry (IPISC), a novel screening technology for designing peptide multiligands with high affinity and specificity. This technology builds upon in situ click chemistry, a kinetic target-guided synthesis approach where the protein target catalyzes the conjugation of two small molecules, typically through the azide-alkyne Huisgen cycloaddition. Integrating this methodology with solid phase peptide libraries enables the assembly of linear and branched peptide multiligands we refer to as Protein Catalyzed Capture Agents (PCC Agents). The resulting structures can be thought of as analogous to the antigen recognition site of antibodies and serve as antibody replacements in biochemical and cell-based applications. In this review, we discuss the recent progress in ligand design through IPISC and related approaches, focusing on the improvements in affinity and specificity as multiligands are assembled by target-catalyzed peptide conjugation. We compare the IPISC process to small molecule in situ click chemistry with particular emphasis on the advantages and technical challenges of constructing antibody-like PCC Agents.

A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

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.

Degradation of Akt using protein-catalyzed capture agents.

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein‐catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein‐based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti‐Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non‐traditional drugging moieties to inhibit challenging targets. Copyright

Abstract 2172: Circle Akt in: Epitope catalyzed assembly of macrocyclic therapeutics against phosphorylated Akt

An approach to drugging ‘undruggable’ target proteins using macrocyclic, epitope targeted peptide based affinity reagents is demonstrated. An unbiased comprehensive Cu-catalyzed Azide Alkyne Cycloaddition cyclized peptide macrocyclic library is synthesized and screened against the phoshoS474 containing Hydrophobic Motif (HM) peptide epitope of Akt2. The best macrocyclic ligand, which exhibits specificity at the peptide and protein levels, is further extended through an in situ click screen to yield bivalent macrocyclic reagent with high affinity and specificity. The bivalent peptide, targeted against the phospho-S474 region of Akt, inhibits the Akt kinase in in vitro kinase assays. This ligand is being optimized, through systematic changes in its composition, to increase its cell permeability characteristics, so that its effects can be studied in Akt overexpressing carcinoma cell lines. The optimized macrocyclic peptide can eventually be used as an in vivo imaging probe and as a peptide inhibitor drug. Citation Format: Arundhati Nag. Circle Akt in: Epitope catalyzed assembly of macrocyclic therapeutics against phosphorylated Akt. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2172.