Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:6.2.1.1 (ACS)
 78,556 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

One-third of all human cancers harbor somatic RAS mutations. This leads to aberrant activation of downstream signaling pathways involving the RAF kinases. Current ATP-competitive RAF inhibitors are active in cancers with somatic RAF mutations, such as BRAF(V600) mutant melanomas. However, they paradoxically promote the growth of RAS mutant tumors, partly due to the complex interplay between different homo- and heterodimers of A-RAF, B-RAF, and C-RAF. Based on pathway analysis and structure-guided compound identification, we describe the natural product cotylenin-A (CN-A) as stabilizer of the physical interaction of C-RAF with 14-3-3 proteins. CN-A binds to inhibitory 14-3-3 interaction sites of C-RAF, pSer233, and pSer259, but not to the activating interaction site, pSer621. While CN-A alone is inactive in RAS mutant cancer models, combined treatment with CN-A and an anti-EGFR antibody synergistically suppresses tumor growth in vitro and in vivo. This defines a novel pharmacologic strategy for treatment of RAS mutant cancers.
ACS Chem Biol 2013 Sep 20
PMID:Stabilization of physical RAF/14-3-3 interaction by cotylenin A as treatment strategy for RAS mutant cancers. 2380 90

Light-dependent dimerization is the basis for recently developed noninvasive optogenetic tools. Here we present a novel tool combining optogenetics with the control of protein kinase activity to investigate signal transduction pathways. Mediated by Arabidopsis thaliana photoreceptor cryptochrome 2, we activated the protein kinase C-RAF by blue light-dependent dimerization, allowing for decoupling from upstream signaling events induced by surface receptors. The activation by light is fast, reversible, and not only time but also dose dependent as monitored by phosphorylation of ERK1/2. Additionally, light-activated C-RAF controls serum response factor-mediated gene expression. Light-induced heterodimerization of C-RAF with a kinase-dead mutant of B-RAF demonstrates the enhancing role of B-RAF as a scaffold for C-RAF activity, which leads to the paradoxical activation of C-RAF found in human cancers. This optogenetic tool enables reversible control of protein kinase activity in signal duration and strength. These properties can help to shed light onto downstream signaling processes of protein kinases in living cells.
ACS Synth Biol 2014 May 16
PMID:Optogenetic control of protein kinase activity in mammalian cells. 2409 Apr 49

Benzimidazole reverse amides were designed and synthesized as Pan RAF kinase inhibitors. Investigation of the structure-activity relationship of the compounds revealed that they were potent in vitro and exhibited desirable in vivo properties.
ACS Med Chem Lett 2014 Sep 11
PMID:Design and Synthesis of Orally Bioavailable Benzimidazole Reverse Amides as Pan RAF Kinase Inhibitors. 2551 17

The BRAF kinase, within the mitogen activated protein kinase (MAPK) signaling pathway, harbors activating mutations in about half of melanomas and to a significant extent in many other cancers. A single valine to glutamic acid substitution at residue 600 (BRAFV600E) accounts for about 90% of these activating mutations. While BRAFV600E-selective small molecule inhibitors, such as debrafenib and vemurafenib, have shown therapeutic benefit, almost all patients develop resistance. Resistance often arises through reactivation of the MAPK pathway, typically through mutation of upstream RAS, downstream MEK, or splicing variants. RAF kinases signal as homo- and heterodimers, and another complication associated with small molecule BRAFV600E inhibition is drug-induced allosteric activation of a wild-type RAF subunit (BRAF or CRAF) of the kinase dimer, a process called "transactivation" or "paradoxical activation." Here, we used BRAFV600E and vemurafenib as a model system to develop chemically linked kinase inhibitors to lock RAF dimers in an inactive conformation that cannot undergo transactivation. This structure-based design effort resulted in the development of Vem-BisAmide-2, a compound containing two vemurafenib molecules connected by a bis amide linker. We show that Vem-BisAmide-2 has comparable inhibitory potency as vemurafenib to BRAFV600E both in vitro and in cells but promotes an inactive dimeric BRAFV600E conformation unable to undergo transactivation. The crystal structure of a BRAFV600E/Vem-BisAmide-2 complex and associated biochemical studies reveal the molecular basis for how Vem-BisAmide-2 mediates selectivity for an inactive over an active dimeric BRAFV600E conformation. These studies have implications for targeting BRAFV600E/RAF heterodimers and other kinase dimers for therapy.
ACS Chem Biol 2016 10 21
PMID:Chemically Linked Vemurafenib Inhibitors Promote an Inactive BRAFV600E Conformation. 2757 13

ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK. Importantly, most resistance mechanisms still rely on activation of the downstream effector kinase ERK, making it a promising target for drug development efforts. Here, we report the design and structural/functional characterization of a set of bivalent ERK inhibitors that combine a small molecule inhibitor that binds to the ATP-binding pocket with a peptide that selectively binds to an ERK protein interaction surface, the D-site recruitment site (DRS). Our studies show that the lead bivalent inhibitor, SBP3, has markedly improved potency compared to the small molecule inhibitor alone. Unexpectedly, we found that SBP3 also binds to several ERK-related kinases that contain a DRS, highlighting the importance of experimentally verifying the predicted specificity of bivalent inhibitors. However, SBP3 does not target any other kinases belonging to the same CMGC branch of the kinome. Additionally, our modular click chemistry inhibitor design facilitates the generation of different combinations of small molecule inhibitors with ERK-targeting peptides.
ACS Med Chem Lett 2017 Jul 13
PMID:Structure-Guided Strategy for the Development of Potent Bivalent ERK Inhibitors. 2874 Jun 6

Monotherapy clinical trials with mutation-targeted kinase inhibitors, despite some success in other cancers, have yet to impact glioblastoma (GBM). Besides insufficient blood-brain barrier penetration, combinations are key to overcoming obstacles such as intratumoral heterogeneity, adaptive resistance, and the epistatic nature of tumor genomics that cause mutation-targeted therapies to fail. With now hundreds of potential drugs, exploring the combination space clinically and preclinically is daunting. We are building a simulation-based approach that integrates patient-specific data with a mechanistic computational model of pan-cancer driver pathways (receptor tyrosine kinases, RAS/RAF/ERK, PI3K/AKT/mTOR, cell cycle, apoptosis, and DNA damage) to prioritize drug combinations by their simulated effects on tumor cell proliferation and death. Here we illustrate a first step, tailoring the model to 14 GBM patients from The Cancer Genome Atlas defined by an mRNA-seq transcriptome, and then simulating responses to three promiscuous FDA-approved kinase inhibitors (bosutinib, ibrutinib, and cabozantinib) with evidence for blood-brain barrier penetration. The model captures binding of the drug to primary targets and off-targets based on published affinity data and simulates responses of 100 heterogeneous tumor cells within a patient. Single drugs are marginally effective or even counterproductive. Common copy number alterations (PTEN loss, EGFR amplification, and NF1 loss) have a negligible correlation with single-drug or combination efficacy, reinforcing the importance of postgenetic approaches that account for kinase inhibitor promiscuity to match drugs to patients. Drug combinations tend to be either cytostatic or cytotoxic, but seldom both, highlighting the need for considering targeted and nontargeted therapy. Although we focus on GBM, the approach is generally applicable.
ACS Chem Neurosci 2018 01 17
PMID:Integrating Transcriptomic Data with Mechanistic Systems Pharmacology Models for Virtual Drug Combination Trials. 2895 62

BRAF is the most frequently mutated kinase in human cancers and is one of the major effectors of oncogenic RAS, making BRAF a target of considerable interest for anticancer drug development. Wild-type BRAF and a variety of oncogenic BRAF mutants are dependent on dimerization of the kinase domain, which also emerges as a culprit of drug resistance and side effects of current BRAF therapies. Thus, allosteric BRAF inhibitors capable of disrupting BRAF dimers could abrogate hyperactivated MAPK (mitogen-activated protein kinase) signaling driven by oncogenic BRAF or RAS and overcome the major limitations of current BRAF inhibitors. To establish this, we applied an in silico approach to design a series of peptide inhibitors targeting the dimer interface of BRAF. One resulting inhibitor was found to potently inhibit the kinase activity of BRAF homo- and heterodimers, including oncogenic BRAFG469A mutant. Moreover, this inhibitor synergizes with FDA-approved, ATP-competitive BRAF inhibitors against dimeric BRAF, suggesting that allosteric BRAF inhibitors have great potential to extend the application of current BRAF therapies. Additionally, targeting the dimer interface of BRAF kinase leads to protein degradation of both RAF and MEK, uncovering a novel scaffolding function of RAF in protecting large MAPK complexes from protein degradation. In conclusion, we have developed a potent lead peptide inhibitor for targeting the dimer interface of BRAF in cancer cells. The dual function of this peptide inhibitor validates the strategy for developing allosteric BRAF inhibitors that specifically dissociate RAF dimers and destabilize the MAPK signaling complex.
ACS Chem Biol 2019 07 19
PMID:Development of Allosteric BRAF Peptide Inhibitors Targeting the Dimer Interface of BRAF. 3124 62

One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAFV600E-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.
ACS Med Chem Lett 2019 Jul 11
PMID:Rigidification Dramatically Improves Inhibitor Selectivity for RAF Kinases. 3131 11

Protein-protein interactions (PPIs) are critical for organizing molecules in a cell and mediating signaling pathways. Dysregulation of PPIs is often a key driver of disease. To better understand the biophysical basis of such disease processes-and to potentially target them-it is critical to understand the molecular determinants of PPIs. Deep mutational scanning (DMS) facilitates the acquisition of large amounts of biochemical data by coupling selection with high throughput sequencing (HTS). The challenging and labor-intensive design and optimization of a relevant selection platform for DMS, however, limits the use of powerful directed evolution and selection approaches. To address this limitation, we designed a versatile new phage-assisted continuous selection (PACS) system using our previously reported proximity-dependent split RNA polymerase (RNAP) biosensors, with the aim of greatly simplifying and streamlining the design of a new selection platform for PPIs. After characterization and validation using the model KRAS/RAF PPI, we generated a library of RAF variants and subjected them to PACS and DMS. Our HTS data revealed positions along the binding interface that are both tolerant and intolerant to mutations, as well as which substitutions are tolerated at each position. Critically, the "functional scores" obtained from enrichment data through continuous selection for individual variants correlated with KD values measured in vitro, indicating that biochemical data can be extrapolated from sequencing using our new system. Due to the plug and play nature of RNAP biosensors, this method can likely be extended to a variety of other PPIs. More broadly, this, and other methods under development support the continued development of evolutionary and high-throughput approaches to address biochemical problems, moving toward a more comprehensive understanding of sequence-function relationships in proteins.
ACS Chem Biol 2019 12 20
PMID:A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein-Protein Interactions. 3180 66