Bifunctional Small‐Molecule Ligands of K‐Ras Induce Its Association with Immunophilin Proteins

Abstract

Here we report the design, synthesis and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Rasdriven cancers.

Exploring Targeted Degradation Strategy for Oncogenic KRASG12C

“…KRAS G12C have raised hopes of directly inhibiting aberrant KRAS G12C function (Canon et al, 2019;Christensen et al, 2019;Janes et al, 2018). Availability of potent and selective KRAS G12C inhibitors, such as ARS-1620, AMG-510, and MRTX-849, has opened opportunities to develop next-generation KRAS targeting strategies, including developing bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins (Zhang and Shokat, 2019) or degrader molecules. Small-molecule degraders have several key advantages over the classical small-molecule inhibitors.…”

Bifunctional modalities for repurposing protein function

“…Used initially to tag a protein with ubiquitin for degradation, there are now bifunctional molecules that recruit the lysosomal degradation pathway, the autophagy pathway, ribonucleases, kinases, phosphatases, glycosyltransferases, and glycosylases to desired targets. Beyond PTMs, Shokat and colleagues created bifunctional compounds that recruit a non-native protein binder (FKBP12 or cyclophilin A) to Ras, which ultimately inhibits Ras's interactions with its native substrates (Zhang and Shokat, 2019). These examples demonstrate that the possibility of redirecting enzymes or proteins to perform non-native actions on substrates can reveal other biological applications.…”

Inhibition of Nonfunctional Ras

“…In addition, despite searches, deep pockets for small-molecule binding have not been identified (Spiegel et al, 2014). The diverse strategies of potential Ras inhibitors have been reviewed extensively in the literature (Chatani and Yang, 2020;Cox et al, 2014Cox et al, , 2015Dang et al, 2017;Gentile et al, 2017;Gorfe and Cho, 2019;Gupta et al, 2019;Khan et al, 2020a;Liu et al, 2010Liu et al, , 2019Lu et al, 2016aLu et al, , 2016cMattingly, 2013;McCormick, 2018;Mullard, 2019;O'Bryan, 2019;Papke and Der, 2017;Papke et al, 2016;Patricelli et al, 2016;Sakamoto et al, 2017;Sheridan, 2020;Singh et al, 2015;Spencer-Smith and O'Bryan, 2019;Spiegel et al, 2014;Zhang and Shokat, 2019;Zimmermann et al, 2013). Among those that have been explored are agents blocking Ras dimerization (e.g., NS1 and K13) (Spencer- Smith et al, 2017) and inhibition of farnesyl transferase (FTase), thus blocking cysteine farnesylation at the C terminus, translocation from the ER to the plasma membrane, and anchorage (e.g., tipifarnib, deltasonamide) (Baranyi et al, 2020;Cheng et al, 2020;Martin-Gago et al, 2017;Reid and Beese, 2004).…”