BRAF-inhibitor associated MEK mutations increase RAF-dependent and -independent enzymatic activity

Alterations in MEK1/2 occur in cancers, both in the treatment-naïve state and following targeted therapies, most notably BRAF and MEK inhibitors in BRAF-V600E–mutant melanoma and colorectal cancer. Efforts were undertaken to understand the effects of these mutations, based upon protein structural location, and MEK1/2 activity. Two categories of MEK1/2 alterations were evaluated, those associated with either the allosteric pocket or helix-A. Clinically, MEK1/2 alterations of the allosteric pocket are rare and we demonstrate that they confer resistance to MEK inhibitors, while retaining sensitivity to BRAF inhibition. Most mutations described in patients fall within, or are associated with, helix-A. Mutations in this region reduce sensitivity to both BRAF and MEK inhibition and display elevated phospho-ERK1/2 levels, independent from increases in phospho-MEK1/2. Biochemical experiments with a representative helix-A variant, MEK1-Q56P, reveal both increased catalytic efficiency of the activated enzyme, and phosphorylation-independent activity relative to wild-type MEK1. Consistent with these findings, MEK1/2 alterations in helix A retain sensitivity to downstream antagonism via pharmacologic inhibition of ERK1/2. This work highlights the importance of classifying mutations based on structural and phenotypic consequences, both in terms of pathway signaling output and response to pharmacologic inhibition

EndoBind - Real-time detection of endogenous protein-protein interactions

We present two methods to detect the interaction of ectopically expressed (RT-Bind) or endogenously tagged (EndoBind) proteins of interest. Both approaches provide temporal evaluation of dimer over an extended duration. Using examples of the NRF2-KEAP1 and the CRAF-KRAS_G12V interaction, we demonstrate that our method allows for the detection of signal for more than 2 days after substrate addition, allowing for continuous monitoring of the protein-protein interaction in real time

Design and Discovery of N-(3-(2-(2-hydroxyethoxy)-6-morpholinopyridin-4-yl)-4-methylphenyl)-2-(trifluoromethyl)isonicotinamide (LXH254), A selective, efficacious, well-tolerated RAF inhibitor targeting RAS mutant cancers: The path to the clinic

RAS and BRAF oncogenes are mutated in more than one-third of human cancers and exquisite dependency on CRAF, MEK1/2 and ERK1/2 has been demonstrated in preclinical models of RAS mutant cancer. Direct pharmacological inhibition of RAS has remained elusive and efforts to target CRAF have been challenging due to the nature of the RAF signaling complex downstream of activated RAS and the poor overall kinase selectivity profile of putative RAF inhibitors such as sorafenib and RAF265. Herein, we describe 15 (LXH254), a selective B/C RAF inhibitor, which has been developed through a hypothesis-driven approach focusing on drug-like properties. We have previously disclosed the discovery of 3 (RAF709), a preclinical tool compound which was potent, selective, efficacious, and well-tolerated in preclinical models, but the high intrinsic clearance [HLM Cl(int) = 94] precluded further development.X The high clearance of 3 by HLM prompted the medicinal chemistry team to further investigate close analogs as well as novel scaffolds. While keeping drug-like properties in mind, the team identified multiple cell-potent scaffolds with low-to-moderate human clearance and progressed them into in-vivo pharmacology studies. Unexpectedly, the majority of novel scaffolds caused significant body weight loss in mice for unknown reasons, with the 2-pyridine series emerging as the only scaffold which was not plagued by this problem. A structure-based approach led to the realization that an alcohol side-chain in the 2-position of the pyridine could interact with the DFG loop and significantly improve cell potency. Further mitigation of human intrinsic clearance and time-dependent inhibition of CYP3A4 (TDI) led to the discovery of 15, which had favorable PK and proved to be efficacious in multiple xenograft models such as Calu-6 (KRASQ61K),with a favorable therapeutic index. Due to its excellent in vitro/ in vivo properties, it has progressed through preclinical toxicology studies and now being tested as a single agent and as a combination partner in phase 1 clinical trial

Structure-based design of selective LONP1 inhibitors for probing in vitro biology

LONP1 is an AAA+ protease that maintains mitochondrial homeostasis by removing damaged or misfolded proteins. Elevated activity and expression of LONP1 promotes cancer cell proliferation and resistance to apoptosis-inducing reagents. Despite the importance of LONP1 in human biology and disease, very few LONP1 inhibitors have been described in the literature. Herein, we report the development of selective boronic acid-based LONP1 inhibitors using structure based drug design as well as the first structures of human LONP1 bound to various inhibitors. Our efforts led to several nanomolar LONP1 inhibitors with little to no activity against the 20S proteasome that serve as tool compounds to investigate LONP1 biology