Total chemical synthesis and folding of all-L and all-D variants of oncogenic KRas(G12V) Journal Article

   

Authors:	Levinson, A. M.; McGee, J. H.; Roberts, A. G.; Creech, G. S.; Wang, T.; Peterson, M. T.; Hendrickson, R. C.; Verdine, G. L.; Danishefsky, S. J.
Article Title:	Total chemical synthesis and folding of all-L and all-D variants of oncogenic KRas(G12V)
Abstract:	The Ras proteins are essential GTPases involved in the regulation of cell proliferation and survival. Mutated oncogenic forms of Ras alter effector binding and innate GTPase activity, leading to deregulation of downstream signal transduction. Mutated forms of Ras are involved in approximately 30% of human cancers. Despite decades of effort to develop direct Ras inhibitors, Ras has long been considered "undruggable" due to its high affinity for GTP and its lack of hydrophobic binding pockets. Herein, we report a total chemical synthesis of all-l- and all-d-amino acid biotinylated variants of oncogenic mutant KRas(G12V). The protein is synthesized using Fmoc-based solid-phase peptide synthesis and assembled using combined native chemical ligation and isonitrile-mediated activation strategies. We demonstrate that both KRas(G12V) enantiomers can successfully fold and bind nucleotide substrates and binding partners with observable enantiodiscrimination. By demonstrating the functional competency of a mirror-image form of KRas bound to its corresponding enantiomeric nucleotide triphosphate, this study sets the stage for further biochemical studies with this material. In particular, this protein will enable mirror-image yeast surface display experiments to identify all-d peptide ligands for oncogenic KRas, providing a useful tool in the search for new therapeutics against this challenging disease target. © 2017 American Chemical Society.
Keywords:	signal transduction; mirrors; cell proliferation; proteins; biosynthesis; peptides; synthesis (chemical); native chemical ligation; biochemical studies; nucleotides; enantiomers; nucleotide triphosphates; solid phase peptide synthesis; bins; enantiodiscrimination; hydrophobic binding; oncogenic mutants; yeast surface displays
Journal Title:	Journal of the American Chemical Society
Volume:	139
Issue:	22
ISSN:	0002-7863
Publisher:	American Chemical Society
Date Published:	2017-06-07
Start Page:	7632
End Page:	7639
Language:	English
DOI:	10.1021/jacs.7b02988
PROVIDER:	scopus
PUBMED:	28448128
PMCID:	PMC5606205
DOI/URL:	https://www.scopus.com/inward/record.uri?eid=2-s2.0-85020414139&doi=10.1021%2fjacs.7b02988&partnerID=40&md5=2c18a8a0b1cff000068d3e1f6690edbf
Notes:	Article -- Export Date: 3 July 2017 -- Source: Scopus

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