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Published August 7, 2018 | Accepted Version + Supplemental Material
Journal Article Open

An Allosteric Inhibitor of KRas Identified Using a Barcoded Rapid Assay Microchip Platform

Abstract

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_(50)) of ∼24 μM.

Additional Information

© 2018 American Chemical Society. Received: February 11, 2018; Accepted: July 6, 2018; Published: July 6, 2018. Dr. Songming Peng is acknowledged for providing transformed E. coli cells used in the preparation of SAC protein and helpful discussions. We acknowledge Anvita Mishra and Rachel Ng for help in the preparation of some peptides and SynEps. We gratefully acknowledge the following agencies and foundations: The B-RAP technology was largely developed with support from the Institute for Collaborative Biotechnologies 6.2 program (J.R.H., # W911NF-09-D-0001). The KRas protein work was supported by the National Cancer Institute (J.R.H., 1U54 CA199090-01) and the Jean Perkins Foundation. The NSF is acknowledged for fellowship support (A.M.M., Grant DGE-1144469). The results in this paper were generated through the use of the Caltech Center for the Chemistry of Cellular Signaling, the Caltech Center for Catalysis and Chemical Synthesis, the Caltech Protein Expression and Purification Center, the Caltech CCE Multi-User Mass Spectrometry Laboratory, and the University of Washington mass spectrometry facility. Author Contributions: A.M.M. and J.K. contributed equally, developed the B-RAP assay platform, and executed all assays presented here, with assistance from A.K.M. R.K.H., J.E.H., E.W., and J.J.O. carried out the screens and initial validations of the anti-KRAS PCCs. J.L. and S.H. performed the additional HPLC and MALDI analysis on ligand L2 shown in Figure S23. A.M.M., J.K., R.K.H., and J.R.H. designed the experimental program. The manuscript was written primarily by A.M.M., J.K., and J.R.H. The authors declare the following competing financial interest(s): J.R.H. is a board member of InDi Molecular, which is a company seeking to commercialize the PCC technology.

Attached Files

Accepted Version - acs.analchem.8b00706

Accepted Version - nihms-1033309.pdf

Supplemental Material - ac8b00706_si_001.pdf

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August 19, 2023
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