KRAS Dimerization Impacts MEK Inhibitor Sensitivity and Oncogenic Activity of Mutant KRAS.

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD154Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers.

Modulation of Mitochondrial Metabolic Parameters and Antioxidant Enzymes in Healthy and Glaucomatous Trabecular Meshwork Cells with Hybrid Small Molecule SA-2.

Oxidative stress (OS)-induced mitochondrial damage is a risk factor for primary open-angle glaucoma (POAG). Mitochondria-targeted novel antioxidant therapies could unearth promising drug candidates for the management of POAG. Previously, our dual-acting hybrid molecule SA-2 with nitric oxide-donating and antioxidant activity reduced intraocular pressure and improved aqueous humor outflow in rodent eyes. Here, we examined the mechanistic role of SA-2 in trabecular meshwork (TM) cells in vitro and measured the activity of intracellular antioxidant enzymes during OS. Primary human TM cells isolated from normal (hNTM) or glaucomatous (hGTM) post-mortem donors and transformed glaucomatous TM cells (GTM-3) were used for in vitro assays. We examined the effect of SA-2 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in vitro using Seahorse Analyzer with or without the oxidant, tert-butyl hydroperoxide (TBHP) treatment. Concentrations of total antioxidant enzymes, catalase (CAT), malondialdehyde (MDA), and glutathione peroxidase (GPx) were measured. We observed significant protection of both hNTM and hGTM cells from TBHP-induced cell death by SA-2. Antioxidant enzymes were elevated in SA-2-treated cells compared to TBHP-treated cells. In addition, SA-2 demonstrated an increase in mitochondrial metabolic parameters. Altogether, SA-2 protected both normal and glaucomatous TM cells from OS via increasing mitochondrial energy parameters and the activity of antioxidant enzymes.

Acid-Catalyzed Synthesis of Isatoic Anhydride-8-Secondary Amides Enables IASA Transformations for Medicinal Chemistry.

Quinazolin-dione-N-3-alklyl derivatives are the core scaffolds for several categories of bioactive small molecules, but current synthetic methods are costly, involve environmental hazards, and are not uniformly scalable. Here, we report an inexpensive, flexible, and scalable method for the one-pot synthesis of substituted quinazolin-dione-N-3-alkyls (isomers of isatoic-8-secondary amides (IASAs)) from isatin that take advantage of in situ capture of imidic acid under acidic conditions. We further show that this method can be used for the synthesis of a wide variety of derivatives with medicinal uses.

Structural basis of the atypical activation mechanism of KRASV14I.

RAS regulation and signaling are largely accomplished by direct protein-protein interactions, making RAS protein dynamics a critical determinant of RAS function. Here, we report a crystal structure of GDP-bound KRASV14I, a mutated KRAS variant associated with the developmental RASopathy disorder Noonan syndrome (NS), at 1.5-1.6 Å resolution. The structure is notable for revealing a marked extension of switch 1 away from the G-domain and nucleotide-binding site of the KRAS protein. We found that this extension is associated with a loss of the magnesium ion and a tilt in the position of the guanine base because of the additional carbon introduced by the isoleucine substitution. Hydrogen-deuterium exchange MS analysis confirmed that this conformation occurs in solution, but also disclosed a difference in kinetics when compared with KRASA146T, another RAS mutant that displays a nearly identical conformation in previously reported crystal structures. This conformational change contributed to a high rate of guanine nucleotide-exchange factor (GEF)-dependent and -independent nucleotide exchange and to an increase in affinity for SOS Ras/Rac GEF 1 (SOS1), which appears to be the major mode of activation for this RAS variant. These results highlight a mechanistic connection between KRASA146T and KRASV14I that may have implications for the regulation of these variants and for the development of therapeutic strategies to manage KRAS variant-associated disorders.

KRAS Switch Mutants D33E and A59G Crystallize in the State 1 Conformation.

KRAS switch loop movements play a crucial role in regulating RAS signaling, and alteration of these sensitive dynamics is a principal mechanism through which disease-associated RAS mutations lead to aberrant RAS activation. Prior studies suggest that despite a high degree of sequence similarity, the switches in KRAS are more dynamic than those in HRAS. We determined X-ray crystal structures of the rare tumorigenic KRAS mutants KRASD33E, in switch 1 (SW1), and KRASA59G, in switch 2 (SW2), bound to GDP and found these adopt nearly identical, open SW1 conformations as well as altered SW2 conformations. KRASA59G bound to a GTP analogue crystallizes in the same conformation. This open conformation is consistent with the inactive "state 1" previously observed for HRAS bound to GTP. For KRASA59G, switch rearrangements may be regulated by increased flexibility in the 57DXXGQ61 motif at codon 59. However, loss of interactions between side chains at codons 33 and 35 in the SW1 33DPT35 motif drives changes for KRASD33E. The 33DPT35 motif is conserved for multiple members of the RAS subfamily but is not found in RAB, RHO, ARF, or Gα families, suggesting that dynamics mediated by this motif may be important for determining the selectivity of RAS-effector interactions. Biochemically, the consequence of altered switch dynamics is the same, showing impaired interaction with the guanine exchange factor SOS and loss of GAP-dependent GTPase activity. However, interactions with the RBD of RAF are preserved. Overall, these observations add to a body of evidence suggesting that HRAS and KRAS show meaningful differences in functionality stemming from differential protein dynamics independent of the hypervariable region.

Structural and Biochemical Analyses Reveal the Mechanism of Glutathione S-Transferase Pi 1 Inhibition by the Anti-cancer Compound Piperlongumine.

Glutathione S-transferase pi 1 (GSTP1) is frequently overexpressed in cancerous tumors and is a putative target of the plant compound piperlongumine (PL), which contains two reactive olefins and inhibits proliferation in cancer cells but not normal cells. PL exposure of cancer cells results in increased reactive oxygen species and decreased GSH. These data in tandem with other information led to the conclusion that PL inhibits GSTP1, which forms covalent bonds between GSH and various electrophilic compounds, through covalent adduct formation at the C7-C8 olefin of PL, whereas the C2-C3 olefin of PL was postulated to react with GSH. However, direct evidence for this mechanism has been lacking. To investigate, we solved the X-ray crystal structure of GSTP1 bound to PL and GSH at 1.1 Å resolution to rationalize previously reported structure activity relationship studies. Surprisingly, the structure showed that a hydrolysis product of PL (hPL) was conjugated to glutathione at the C7-C8 olefin, and this complex was bound to the active site of GSTP1; no covalent bond formation between hPL and GSTP1 was observed. Mass spectrometry (MS) analysis of the reactions between PL and GSTP1 confirmed that PL does not label GSTP1. Moreover, MS data also indicated that nucleophilic attack on PL at the C2-C3 olefin led to PL hydrolysis. Although hPL inhibits GSTP1 enzymatic activity in vitro, treatment of cells susceptible to PL with hPL did not have significant anti-proliferative effects, suggesting that hPL is not membrane-permeable. Altogether, our data suggest a model wherein PL is a prodrug whose intracellular hydrolysis initiates the formation of the hPL-GSH conjugate, which blocks the active site of and inhibits GSTP1 and thereby cancer cell proliferation.

Novel Thiol Containing Hybrid Antioxidant-Nitric Oxide Donor Small Molecules for Treatment of Glaucoma.

Oxidative stress induced death and dysregulation of trabecular meshwork (TM) cells contribute to the increased intraocular pressure (IOP) in primary open angle (POAG) glaucoma patients. POAG is one of the major causes of irreversible vision loss worldwide. Nitric oxide (NO), a small gas molecule, has demonstrated IOP lowering activity in glaucoma by increasing aqueous humor outflow and relaxing TM. Glaucomatous pathology is associated with decreased antioxidant enzyme levels in ocular tissues causing increased reactive oxygen species (ROS) production that reduce the bioavailability of NO. Here, we designed, synthesized, and conducted in vitro studies of novel second-generation sulfur containing hybrid NO donor-antioxidants SA-9 and its active metabolite SA-10 to scavenge broad-spectrum ROS as well as provide efficient protection from t-butyl hydrogen peroxide (TBHP) induced oxidative stress while maintaining NO bioavailability in TM cells. To allow a better drug delivery, a slow release nanosuspension SA-9 nanoparticles (SA-9 NPs) was prepared, characterized, and tested in dexamethasone induced ocular hypertensive (OHT) mice model for IOP lowering activity. A single topical eye drop of SA-9 NPs significantly lowered IOP (61%) at 3 h post-dose, with the effect lasting up to 72 h. This class of molecule has high potential to be useful for treatment of glaucoma.

GTP hydrolysis is modulated by Arg34 in the RASopathy-associated KRASP34R.

RAS proteins are commonly mutated in cancerous tumors, but germline RAS mutations are also found in RASopathy syndromes such as Noonan syndrome (NS) and cardiofaciocutaneous (CFC) syndrome. Activating RAS mutations can be subclassified based on their activating mechanisms. Understanding the structural basis for these mechanisms may provide clues for how to manage associated health conditions. We determined high-resolution X-ray structures of the RASopathy mutant KRASP34R seen in NS and CFCS. GTP and GDP-bound KRASP34R crystallized in multiple forms, with each lattice consisting of multiple protein conformations. In all GTP-bound conformations, the switch regions are not compatible with GAP binding, suggesting a structural mechanism for the GAP insensitivity of this RAS mutant. However, GTP-bound conformations are compatible with intrinsic nucleotide hydrolysis, including one that places R34 in a position analogous to the GAP arginine finger or intrinsic arginine finger found in heterotrimeric G proteins, which may support intrinsic GTP hydrolysis. We also note that the affinity between KRASP34R and RAF-RBD is decreased, suggesting another possible mechanism for dampening of RAS signaling. These results may provide a foothold for development of new mutation-specific strategies to address KRASP34R -driven diseases.

Chemical Biology Toolkit for DCLK1 Reveals Connection to RNA Processing.

Doublecortin-like kinase 1 (DCLK1) is critical for neurogenesis, but overexpression is also observed in multiple cancers and is associated with poor prognosis. Nevertheless, the function of DCLK1 in cancer, especially the context-dependent functions, are poorly understood. We present a "toolkit" that includes the DCLK1 inhibitor DCLK1-IN-1, a complementary DCLK1-IN-1-resistant mutation G532A, and kinase dead mutants D511N and D533N, which can be used to investigate signaling pathways regulated by DCLK1. Using a cancer cell line engineered to be DCLK1 dependent for growth and cell migration, we show that this toolkit can be used to discover associations between DCLK1 kinase activity and biological processes. In particular, we show an association between DCLK1 and RNA processing, including the identification of CDK11 as a potential substrate of DCLK1 using phosphoproteomics.

KRASQ61H Preferentially Signals through MAPK in a RAF Dimer-Dependent Manner in Non-Small Cell Lung Cancer.

Assembly of RAS molecules into complexes at the cell membrane is critical for RAS signaling. We previously showed that oncogenic KRAS codon 61 mutations increase its affinity for RAF, raising the possibility that KRASQ61H, the most common KRAS mutation at codon 61, upregulates RAS signaling through mechanisms at the level of RAS assemblies. We show here that KRASQ61H exhibits preferential binding to RAF relative to PI3K in cells, leading to enhanced MAPK signaling in in vitro models and human NSCLC tumors. X-ray crystallography of KRASQ61H:GTP revealed that a hyperdynamic switch 2 allows for a more stable interaction with switch 1, suggesting that enhanced RAF activity arises from a combination of absent intrinsic GTP hydrolysis activity and increased affinity for RAF. Disruption of KRASQ61H assemblies by the RAS oligomer-disrupting D154Q mutation impaired RAF dimerization and altered MAPK signaling but had little effect on PI3K signaling. However, KRASQ61H oligomers but not KRASG12D oligomers were disrupted by RAF mutations that disrupt RAF-RAF interactions. KRASQ61H cells show enhanced sensitivity to RAF and MEK inhibitors individually, whereas combined treatment elicited synergistic growth inhibition. Furthermore, KRASQ61H tumors in mice exhibited high vulnerability to MEK inhibitor, consistent with cooperativity between KRASQ61H and RAF oligomerization and dependence on MAPK signaling. These findings support the notion that KRASQ61H and functionally similar mutations may serve as predictive biomarkers for targeted therapies against the MAPK pathway. SIGNIFICANCE: These findings show that oncogenic KRASQ61H forms a cooperative RAS-RAF ternary complex, which renders RAS-driven tumors vulnerable to MEKi and RAFi, thus establishing a framework for evaluating RAS biomarker-driven targeted therapies.

Exploring Targeted Degradation Strategy for Oncogenic KRASG12C.

KRAS is the most frequently mutated oncogene found in pancreatic, colorectal, and lung cancers. Although it has been challenging to identify targeted therapies for cancers harboring KRAS mutations, KRASG12C can be targeted by small-molecule inhibitors that form covalent bonds with cysteine 12 (C12). Here, we designed a library of C12-directed covalent degrader molecules (PROTACs) and subjected them to a rigorous evaluation process to rapidly identify a lead compound. Our lead degrader successfully engaged CRBN in cells, bound KRASG12Cin vitro, induced CRBN/KRASG12C dimerization, and degraded GFP-KRASG12C in reporter cells in a CRBN-dependent manner. However, it failed to degrade endogenous KRASG12C in pancreatic and lung cancer cells. Our data suggest that inability of the lead degrader to effectively poly-ubiquitinate endogenous KRASG12C underlies the lack of activity. We discuss challenges for achieving targeted KRASG12C degradation and proposed several possible solutions which may lead to efficient degradation of endogenous KRASG12C.

Publisher Correction: Green Synthesis of Substituted Anilines and Quinazolines from Isatoic Anhydride-8-amide.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Green Synthesis of Substituted Anilines and Quinazolines from Isatoic Anhydride-8-amide.

Synthetic methods used to generate substituted anilines and quinazolines, both privileged pharmacological structures, are cumbersome, hazardous or, in some cases, unavailable. We developed a straightforward method for making isatoic anhydride-8-amide from isatin-7-carboxylic acid as a tool to easily produce a range of quinazoline and substituted aniline derivatives using adaptable pH-sensitive cyclization chemistry. The approaches are inexpensive, simple, fast, efficient at room temperature and scalable, enabling the synthesis of both established and new quinazolines and also highly substituted anilines including cyano derivatives.

Leveraging Compound Promiscuity to Identify Targetable Cysteines within the Kinome.

Covalent kinase inhibitors, which typically target cysteine residues, represent an important class of clinically relevant compounds. Approximately 215 kinases are known to have potentially targetable cysteines distributed across 18 spatially distinct locations proximal to the ATP-binding pocket. However, only 40 kinases have been covalently targeted, with certain cysteine sites being the primary focus. To address this disparity, we have developed a strategy that combines the use of a multi-targeted acrylamide-modified inhibitor, SM1-71, with a suite of complementary chemoproteomic and cellular approaches to identify additional targetable cysteines. Using this single multi-targeted compound, we successfully identified 23 kinases that are amenable to covalent inhibition including MKNK2, MAP2K1/2/3/4/6/7, GAK, AAK1, BMP2K, MAP3K7, MAPKAPK5, GSK3A/B, MAPK1/3, SRC, YES1, FGFR1, ZAK (MLTK), MAP3K1, LIMK1, and RSK2. The identification of nine of these kinases previously not targeted by a covalent inhibitor increases the number of targetable kinases and highlights opportunities for covalent kinase inhibitor development.

Tissue-Specific Oncogenic Activity of KRASA146T.

KRAS is the most frequently mutated oncogene. The incidence of specific KRAS alleles varies between cancers from different sites, but it is unclear whether allelic selection results from biological selection for specific mutant KRAS proteins. We used a cross-disciplinary approach to compare KRASG12D, a common mutant form, and KRASA146T, a mutant that occurs only in selected cancers. Biochemical and structural studies demonstrated that KRASA146T exhibits a marked extension of switch 1 away from the protein body and nucleotide binding site, which activates KRAS by promoting a high rate of intrinsic and guanine nucleotide exchange factor-induced nucleotide exchange. Using mice genetically engineered to express either allele, we found that KRASG12D and KRASA146T exhibit distinct tissue-specific effects on homeostasis that mirror mutational frequencies in human cancers. These tissue-specific phenotypes result from allele-specific signaling properties, demonstrating that context-dependent variations in signaling downstream of different KRAS mutants drive the KRAS mutational pattern seen in cancer. SIGNIFICANCE: Although epidemiologic and clinical studies have suggested allele-specific behaviors for KRAS, experimental evidence for allele-specific biological properties is limited. We combined structural biology, mass spectrometry, and mouse modeling to demonstrate that the selection for specific KRAS mutants in human cancers from different tissues is due to their distinct signaling properties.See related commentary by Hobbs and Der, p. 696.This article is highlighted in the In This Issue feature, p. 681.

Temperature-regulated activity of responsive polymer-protein conjugates prepared by grafting-from via RAFT polymerization.

A facile route to well-defined "smart" polymer-protein conjugates with tunable bioactivity is reported. Protein modification with a reversible addition-fragmentation chain transfer (RAFT) agent and subsequent room temperature polymerization in aqueous media led to conjugates of poly(N-isopropylacrylamide) and a model protein. Representing the first example of polymer-protein conjugation with RAFT agent immobilization via the "R-group" approach, high molecular weight and reductively stable conjugates were accessible without extensive purification or adverse effects on the protein structure. An increase in molecular weight with conversion was observed for the chains grafted from the protein surface, confirming the controlled nature of the polymerization. The responsive behavior of the immobilized polymer facilitated conjugate isolation and also allowed environmental modulation of bioactivity.

Folate-conjugated thermoresponsive block copolymers: highly efficient conjugation and solution self-assembly.

A combination of controlled radical polymerization and azide-alkyne click chemistry was employed to prepare temperature-responsive block copolymer micelles conjugated with biological ligands with potential for active targeting of cancer tissues. Block copolymers of N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA) were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization with an azido chain transfer agent (CTA). Pseudo-first-order kinetics and linear molecular weight dependence on conversion were observed for the RAFT polymerizations. CuI-catalyzed coupling with propargyl folate resulted in folic acid residues being efficiently conjugated to the alpha-azido chain ends of the homo and block copolymers. Temperature-induced self-assembly resulted in aggregates capable of controlled release of a model hydrophobic drug. CuI-catalyzed azide-alkyne cycloaddition has proven superior to conventional methods for conjugation of biological ligands to macromolecules, and the general strategy presented herein can potentially be extended to the preparation of folate-functionalized assemblies with other stimuli susceptibility (e.g., pH) for therapeutic and imaging applications.

KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry.

KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

Covalent Guanosine Mimetic Inhibitors of G12C KRAS.

Ras proteins are members of a large family of GTPase enzymes that are commonly mutated in cancer where they act as dominant oncogenes. We previously developed an irreversible guanosine-derived inhibitor, SML-8-73-1, of mutant G12C RAS that forms a covalent bond with cysteine 12. Here we report exploration of the structure-activity relationships (SAR) of hydrolytically stable analogues of SML-8-73-1 as covalent G12C KRAS inhibitors. We report the discovery of difluoromethylene bisphosphonate analogues such as compound 11, which, despite exhibiting reduced efficiency as covalent G12C KRAS inhibitors, remove the liability of the hydrolytic instability of the diphosphate moiety present in SML-8-73-1 and provide the foundation for development of prodrugs to facilitate cellular uptake. The SAR and crystallographic results reaffirm the exquisite molecular recognition that exists in the diphosphate region of RAS for guanosine nucleotides which must be considered in the design of nucleotide-competitive inhibitors.

Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching.

Structural dynamics of Ras proteins contributes to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on the movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras-targeting strategies, experimental methods to measure Ras dynamics are required. Here, we demonstrate the utility of hydrogen-deuterium exchange (HDX) mass spectrometry (MS) to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main-chain movement. HDX provides a means of evaluating Ras protein dynamics, which may be useful for understanding the mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.