2 research outputs found
Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform
Protein
catalyzed capture agents (PCCs) are synthetic antibody
surrogates that can target a wide variety of biologically relevant
proteins. As a step toward developing a high-throughput PCC pipeline,
we report on the preparation of a barcoded rapid assay platform for
the analysis of hits from PCC library screens. The platform is constructed
by first surface patterning a micrometer scale barcode composed of
orthogonal ssDNA strands onto a glass slide. The slide is then partitioned
into microwells, each of which contains multiple copies of the full
barcode. Biotinylated candidate PCCs from a click screen are assembled
onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified
streptavidin library. This platform was employed to evaluate candidate
PCC ligands identified from an epitope targeted in situ click screen
against the two conserved allosteric switch regions of the Kirsten
rat sarcoma (KRas) protein. A single microchip was utilized for the
simultaneous evaluation of 15 PCC candidate fractions under more than
a dozen different assay conditions. The platform also permitted more
than a 10-fold savings in time and a more than 100-fold reduction
in biological and chemical reagents relative to traditional multiwell
plate assays. The best ligand was shown to exhibit an in vitro inhibition
constant (IC<sub>50</sub>) of ∼24 μM
Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1
We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties