RASopathy mutations open new insights into the mechanism of BRAF activation.

Spencer-Smith et al. (2022)1 investigate multiple functions of the BRAF cysteine-rich domain (CRD), finding distinct classes of RASopathy-associated BRAF mutations and unique features among RAF paralogs that may contribute to the spectrum of mutations observed in disease.

The evolution of protein kinase inhibitors from antagonists to agonists of cellular signaling.

Kinases are highly regulated enzymes with diverse mechanisms controlling their catalytic output. Over time, chemical discovery efforts for kinases have produced ATP-competitive compounds, allosteric regulators, irreversible binders, and highly specific inhibitors. These distinct classes of small molecules have revealed many novel aspects about kinase-mediated signaling, and some have progressed from simple tool compounds into clinically validated therapeutics. This review explores several small-molecule inhibitors for kinases highlighting elaborate mechanisms by which kinase function is modulated. A complete surprise of targeted kinase drug discovery has been the finding of ATP-competitive inhibitors that behave as agonists, rather than antagonists, of their direct kinase target. These studies hint at a connection between ATP-binding site occupancy and networks of communication that are independent of kinase catalysis. Indeed, kinase inhibitors that induce changes in protein localization, protein-protein interactions, and even enhancement of catalytic activity of the target kinase have been found. The relevance of these findings to the therapeutic efficacy of kinase inhibitors and to the future identification of new classes of drug targets is discussed.

Live-cell target engagement of allosteric MEKi on MEK-RAF/KSR-14-3-3 complexes.

The RAS-mitogen-activated protein kinase (MAPK) pathway includes KSR, RAF, MEK and the phospho-regulatory sensor 14-3-3. Specific assemblies among these components drive various diseases and likely dictate efficacy for numerous targeted therapies, including allosteric MEK inhibitors (MEKi). However, directly measuring drug interactions on physiological RAS-MAPK complexes in live cells has been inherently challenging to query and therefore remains poorly understood. Here we present a series of NanoBRET-based assays to quantify direct target engagement of MEKi on MEK1 and higher-order MEK1-bound complexes with ARAF, BRAF, CRAF, KSR1 and KSR2 in the presence and absence of 14-3-3 in living cells. We find distinct MEKi preferences among these complexes that can be compiled to generate inhibitor binding profiles. Further, these assays can report on the influence of the pathogenic BRAF-V600E mutant on MEKi binding. Taken together, these approaches can be used as a platform to screen for compounds intended to target specific complexes in the RAS-MAPK cascade.

Structural basis for the action of the drug trametinib at KSR-bound MEK.

The MAPK/ERK kinase MEK is a shared effector of the frequent cancer drivers KRAS and BRAF that has long been pursued as a drug target in oncology1, and more recently in immunotherapy2,3 and ageing4. However, many MEK inhibitors are limited owing to on-target toxicities5-7 and drug resistance8-10. Accordingly, a molecular understanding of the structure and function of MEK within physiological complexes could provide a template for the design of safer and more effective therapies. Here we report X-ray crystal structures of MEK bound to the scaffold KSR (kinase suppressor of RAS) with various MEK inhibitors, including the clinical drug trametinib. The structures reveal an unexpected mode of binding in which trametinib directly engages KSR at the MEK interface. In the bound complex, KSR remodels the prototypical allosteric pocket of the MEK inhibitor, thereby affecting binding and kinetics, including the drug-residence time. Moreover, trametinib binds KSR-MEK but disrupts the related RAF-MEK complex through a mechanism that exploits evolutionarily conserved interface residues that distinguish these sub-complexes. On the basis of these insights, we created trametiglue, which limits adaptive resistance to MEK inhibition by enhancing interfacial binding. Our results reveal the plasticity of an interface pocket within MEK sub-complexes and have implications for the design of next-generation drugs that target the RAS pathway.

Small molecule stabilization of the KSR inactive state antagonizes oncogenic Ras signalling.

Deregulation of the Ras-mitogen activated protein kinase (MAPK) pathway is an early event in many different cancers and a key driver of resistance to targeted therapies. Sustained signalling through this pathway is caused most often by mutations in K-Ras, which biochemically favours the stabilization of active RAF signalling complexes. Kinase suppressor of Ras (KSR) is a MAPK scaffold that is subject to allosteric regulation through dimerization with RAF. Direct targeting of KSR could have important therapeutic implications for cancer; however, testing this hypothesis has been difficult owing to a lack of small-molecule antagonists of KSR function. Guided by KSR mutations that selectively suppress oncogenic, but not wild-type, Ras signalling, we developed a class of compounds that stabilize a previously unrecognized inactive state of KSR. These compounds, exemplified by APS-2-79, modulate KSR-dependent MAPK signalling by antagonizing RAF heterodimerization as well as the conformational changes required for phosphorylation and activation of KSR-bound MEK (mitogen-activated protein kinase kinase). Furthermore, APS-2-79 increased the potency of several MEK inhibitors specifically within Ras-mutant cell lines by antagonizing release of negative feedback signalling, demonstrating the potential of targeting KSR to improve the efficacy of current MAPK inhibitors. These results reveal conformational switching in KSR as a druggable regulator of oncogenic Ras, and further suggest co-targeting of enzymatic and scaffolding activities within Ras-MAPK signalling complexes as a therapeutic strategy for overcoming Ras-driven cancers.

Type II Binders Targeting the "GLR-Out" Conformation of the Pseudokinase STRADα.

Pseudokinases play important roles in signal transduction and cellular processes similar to those of catalytically competent kinases. However, pseudokinase pharmacological tractability and conformational space accessibility are poorly understood. Pseudokinases have only recently been suggested to adopt "inactive" conformations or interact with conformation-specific kinase inhibitors (e.g., type II compounds). In this work, the heavily substituted pseudokinase STRADα, which possesses a DFG → GLR substitution in the catalytic site that permits nucleotide binding while impairing divalent cation coordination, is used as a test case to demonstrate the potential applicability of conformation-specific, type II compounds to pseudokinase pharmacology. Integrated structural modeling is employed to generate a "GLR-out" conformational ensemble. Likely interacting type II compounds are identified through virtual screening against this ensemble model. Biophysical validation of compound binding is demonstrated through protein thermal stabilization and ATP competition. Localization of a top-performing compound through surface methylation strongly suggests that STRADα can adopt the "GLR-out" conformation and interact with compounds that comply with the standard type II pharmacophore. These results suggest that, despite a loss of catalytic function, some pseudokinases, including STRADα, may retain the conformational switching properties of conventional protein kinases.

A whole-animal platform to advance a clinical kinase inhibitor into new disease space.

Synthetic tailoring of approved drugs for new indications is often difficult, as the most appropriate targets may not be readily apparent, and therefore few roadmaps exist to guide chemistry. Here, we report a multidisciplinary approach for accessing novel target and chemical space starting from an FDA-approved kinase inhibitor. By combining chemical and genetic modifier screening with computational modeling, we identify distinct kinases that strongly enhance ('pro-targets') or limit ('anti-targets') whole-animal activity of the clinical kinase inhibitor sorafenib in a Drosophila medullary thyroid carcinoma (MTC) model. We demonstrate that RAF-the original intended sorafenib target-and MKNK kinases function as pharmacological liabilities because of inhibitor-induced transactivation and negative feedback, respectively. Through progressive synthetic refinement, we report a new class of 'tumor calibrated inhibitors' with unique polypharmacology and strongly improved therapeutic index in fly and human MTC xenograft models. This platform provides a rational approach to creating new high-efficacy and low-toxicity drugs.

Integrated computational and Drosophila cancer model platform captures previously unappreciated chemicals perturbing a kinase network.

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a 'hybrid' molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity.

Chemical genetic discovery of targets and anti-targets for cancer polypharmacology.

The complexity of cancer has led to recent interest in polypharmacological approaches for developing kinase-inhibitor drugs; however, optimal kinase-inhibition profiles remain difficult to predict. Using a Ret-kinase-driven Drosophila model of multiple endocrine neoplasia type 2 and kinome-wide drug profiling, here we identify that AD57 rescues oncogenic Ret-induced lethality, whereas related Ret inhibitors imparted reduced efficacy and enhanced toxicity. Drosophila genetics and compound profiling defined three pathways accounting for the mechanistic basis of efficacy and dose-limiting toxicity. Inhibition of Ret plus Raf, Src and S6K was required for optimal animal survival, whereas inhibition of the 'anti-target' Tor led to toxicity owing to release of negative feedback. Rational synthetic tailoring to eliminate Tor binding afforded AD80 and AD81, compounds featuring balanced pathway inhibition, improved efficacy and low toxicity in Drosophila and mammalian multiple endocrine neoplasia type 2 models. Combining kinase-focused chemistry, kinome-wide profiling and Drosophila genetics provides a powerful systems pharmacology approach towards developing compounds with a maximal therapeutic index.

A Raf-induced allosteric transition of KSR stimulates phosphorylation of MEK.

In metazoans, the Ras-Raf-MEK (mitogen-activated protein-kinase kinase)-ERK (extracellular signal-regulated kinase) signalling pathway relays extracellular stimuli to elicit changes in cellular function and gene expression. Aberrant activation of this pathway through oncogenic mutations is responsible for a large proportion of human cancer. Kinase suppressor of Ras (KSR) functions as an essential scaffolding protein to coordinate the assembly of Raf-MEK-ERK complexes. Here we integrate structural and biochemical studies to understand how KSR promotes stimulatory Raf phosphorylation of MEK (refs 6, 7). We show, from the crystal structure of the kinase domain of human KSR2 (KSR2(KD)) in complex with rabbit MEK1, that interactions between KSR2(KD) and MEK1 are mediated by their respective activation segments and C-lobe αG helices. Analogous to BRAF (refs 8, 9), KSR2 self-associates through a side-to-side interface involving Arg 718, a residue identified in a genetic screen as a suppressor of Ras signalling. ATP is bound to the KSR2(KD) catalytic site, and we demonstrate KSR2 kinase activity towards MEK1 by in vitro assays and chemical genetics. In the KSR2(KD)-MEK1 complex, the activation segments of both kinases are mutually constrained, and KSR2 adopts an inactive conformation. BRAF allosterically stimulates the kinase activity of KSR2, which is dependent on formation of a side-to-side KSR2-BRAF heterodimer. Furthermore, KSR2-BRAF heterodimerization results in an increase of BRAF-induced MEK phosphorylation via the KSR2-mediated relay of a signal from BRAF to release the activation segment of MEK for phosphorylation. We propose that KSR interacts with a regulatory Raf molecule in cis to induce a conformational switch of MEK, facilitating MEK's phosphorylation by a separate catalytic Raf molecule in trans.

The Pan-RAF-MEK Nondegrading Molecular Glue NST-628 Is a Potent and Brain-Penetrant Inhibitor of the RAS-MAPK Pathway with Activity across Diverse RAS- and RAF-Driven Cancers.

Alterations in the RAS-MAPK signaling cascade are common across multiple solid tumor types and are a driver for many cancers. NST-628 is a potent pan-RAF-MEK molecular glue that prevents the phosphorylation and activation of MEK by RAF, overcoming the limitations of traditional RAS-MAPK inhibitors and leading to deep durable inhibition of the pathway. Cellular, biochemical, and structural analyses of RAF-MEK complexes show that NST-628 engages all isoforms of RAF and prevents the formation of BRAF-CRAF heterodimers, a differentiated mechanism from all current RAF inhibitors. With a potent and durable inhibition of the RAF-MEK signaling complex as well as high intrinsic permeability into the brain, NST-628 demonstrates broad efficacy in cellular and patient-derived tumor models harboring diverse MAPK pathway alterations, including orthotopic intracranial models. Given its functional and pharmacokinetic mechanisms that are differentiated from previous therapies, NST-628 is positioned to make an impact clinically in areas of unmet patient need. Significance: This study introduces NST-628, a molecular glue having differentiated mechanism and drug-like properties. NST-628 treatment leads to broad efficacy with high tolerability and central nervous system activity across multiple RAS- and RAF-driven tumor models. NST-628 has the potential to provide transformative clinical benefits as both monotherapy and vertical combination anchor.

A pickup in pseudokinase activity.

Kinases catalyse the phosphorylation of target substrates on hydroxy group-containing residues as a means to nucleate multi-component complexes or to stabilize unique conformational states. Through this biochemical activity, kinases play critical roles in many signal transduction and disease pathways. Pseudokinases constitute a subclass of these enzymes that were originally predicted as inactive on the basis of mutations of key conserved active-site residues. However, recent biochemical and structural analyses have revealed several enzymatically active pseudokinases, suggesting either that novel mechanisms of phosphorylation are at play or that the constraints for highly conserved active-site residues are looser than originally anticipated. The purpose of the present review is to summarize several of the active pseudokinases, and one in particular termed KSR (kinase suppressor of Ras), which was recently found to possess a kinase activity that can become accelerated through an allosteric mechanism. Utilization of catalytic activity or structural features of the kinase fold may be key to the function of many pseudokinases.

WNTinib is a multi-kinase inhibitor with specificity against ß-catenin mutant hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. ß-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant ß-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

Understanding and drugging RAS: 40†years to break the tip of the iceberg.

Several cancers and rare genetic diseases are caused by dysregulation in the RAS signaling pathway. RAS proteins serve as molecular switches that regulate pathways involved in cellular growth, differentiation and survival. These pathways have been an intense area of investigation for four decades, since the initial identification of somatic RAS mutations linked to human cancers. In the past few years, inhibitors against several RAS effectors, as well as direct inhibitors of the K-RAS mutant G12C, have been developed. This Special Issue in DMM includes original Research articles on RAS-driven cancers and RASopathies. The articles provide insights into mechanisms and biomarkers, and evaluate therapeutic targets. Several articles also present new disease models, whereas others describe technologies or approaches to evaluate the function of RAS in vivo. The collection also includes a series of Review articles on RAS biology and translational aspects of defining and treating RAS-driven diseases. In this Editorial, we summarize this collection and discuss the potential impact of the articles within this evolving area of research. We also identify areas of growth and possible future developments.

Conformational control and regulation of the pseudokinase KSR via small molecule binding interactions.

Pseudokinases often operate through functionally related enzymes and receptors. A prime example is the pseudokinase KSR (Kinase Suppressor of RAS), which can act as both an amplifier and inhibitor of members in the RAS-MAPK (Mitogen Activated Protein Kinase) signaling pathway. KSR is structurally related to the active RAF kinases over multiple domains; moreover, the pseudokinase domain of KSR forms physical and regulatory complexes with both RAF and MEK through distinct interfaces. Characterization of small molecule interactions on KSR has been used to uncover novel chemical tools and understand the mechanism of action of clinical drugs. Here, we elaborate on assays and structural methods for measuring binding at orthosteric and interfacial binding sites on KSR. These distinct small molecule pockets provide therapeutic paths for targeting KSR1 and KSR2 pseudokinases in disease, including in RAS and RAF mutant cancers.

Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR.

Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2-specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulates monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations.

Protein kinase PKR mutants resistant to the poxvirus pseudosubstrate K3L protein.

As part of the mammalian cell innate immune response, the double-stranded RNA activated protein kinase PKR phosphorylates the translation initiation factor eIF2alpha to inhibit protein synthesis and thus block viral replication. Poxviruses including vaccinia and smallpox viruses express PKR inhibitors such as the vaccinia virus K3L protein that resembles the N-terminal substrate-targeting domain of eIF2alpha. Whereas high-level expression of human PKR was toxic in yeast, this growth inhibition was suppressed by coexpression of the K3L protein. We used this yeast assay to screen for PKR mutants that are resistant to K3L inhibition, and we identified 12 mutations mapping to the C-terminal lobe of the PKR kinase domain. The PKR mutations specifically conferred resistance to the K3L protein both in yeast and in vitro. Consistently, the PKR-D486V mutation led to nearly a 15-fold decrease in K3L binding affinity yet did not impair eIF2alpha phosphorylation. Our results support the identification of the eIF2alpha-binding site on an extensive face of the C-terminal lobe of the kinase domain, and they indicate that subtle changes to the PKR kinase domain can drastically impact pseudosubstrate inhibition while leaving substrate phosphorylation intact. We propose that these paradoxical effects of the PKR mutations on pseudosubstrate vs. substrate interactions reflect differences between the rigid K3L protein and the plastic nature of eIF2alpha around the Ser-51 phosphorylation site.

Ploidy Leads a Molecular Motor to Walk Different Paths to Drug Resistance.

In this issue of Cell Chemical Biology, Pisa et al. (2020) find that haploid and diploid cells differentially develop resistance to the CENP-E inhibitor GSK923295. The results highlight the power of tumor cells to evade growth inhibition and potentially inform the design of next-generation CENP-E drugs to overcome resistance.

Small molecule recognition of c-Src via the Imatinib-binding conformation.

The cancer drug, Imatinib, is a selective Abl kinase inhibitor that does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last 15 years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation, effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the DFG-out conformation of c-Src with high affinity. We report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavorable DFG-out conformation in c-Src. Structural comparison between c-Src in complex with these inhibitors allows us to speculate on the differential selectivity of Imatinib for c-Src and Abl.

Phenotype-Based Screens with Conformation-Specific Inhibitors Reveal p38 Gamma and Delta as Targets for HCC Polypharmacology.

The approved kinase inhibitors for hepatocellular carcinoma (HCC) are not matched to specific mutations within tumors. This has presented a daunting challenge; without a clear target or mechanism, no straightforward path has existed to guide the development of improved therapies for HCC. Here, we combine phenotypic screens with a class of conformation-specific kinase inhibitors termed type II to identify a multikinase inhibitor, AD80, with antitumoral activity across a variety of HCC preclinical models, including mouse xenografts. Mass spectrometry profiling found a number of kinases as putative targets for AD80, including several receptor and cytoplasmic protein kinases. Among these, we found p38 gamma and delta as direct targets of AD80. Notably, a closely related analog of AD80 lacking p38δ/γ activity, but retaining several other off-target kinases, lost significant activity in several HCC models. Moreover, forced and sustained MKK6 → p38→ATF2 signaling led to a significant reduction of AD80 activity within HCC cell lines. Together with HCC survival data in The Cancer Genome Atlas and RNA-seq analysis, we suggest p38 delta and gamma as therapeutic targets in HCC and an "AD80 inhibition signature" as identifying those patients with best clinical outcomes.