Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output.

K-Ras is targeted to the plasma membrane by a C-terminal membrane anchor that comprises a farnesyl-cysteine-methyl-ester and a polybasic domain. We used quantitative spatial imaging and atomistic molecular dynamics simulations to examine molecular details of K-Ras plasma membrane binding. We found that the K-Ras anchor binds selected plasma membrane anionic lipids with defined head groups and lipid side chains. The precise amino acid sequence and prenyl group define a combinatorial code for lipid binding that extends beyond simple electrostatics; within this code lysine and arginine residues are non-equivalent and prenyl chain length modifies nascent polybasic domain lipid preferences. The code is realized by distinct dynamic tertiary structures of the anchor on the plasma membrane that govern amino acid side-chain-lipid interactions. An important consequence of this specificity is the ability of such anchors when aggregated to sort subsets of phospholipids into nanoclusters with defined lipid compositions that determine K-Ras signaling output.

Constitutive formation of caveolae in a bacterium.

Caveolin plays an essential role in the formation of characteristic surface pits, caveolae, which cover the surface of many animal cells. The fundamental principles of caveola formation are only slowly emerging. Here we show that caveolin expression in a prokaryotic host lacking any intracellular membrane system drives the formation of cytoplasmic vesicles containing polymeric caveolin. Vesicle formation is induced by expression of wild-type caveolins, but not caveolin mutants defective in caveola formation in mammalian systems. In addition, cryoelectron tomography shows that the induced membrane domains are equivalent in size and caveolin density to native caveolae and reveals a possible polyhedral arrangement of caveolin oligomers. The caveolin-induced vesicles or heterologous caveolae (h-caveolae) form by budding in from the cytoplasmic membrane, generating a membrane domain with distinct lipid composition. Periplasmic solutes are encapsulated in the budding h-caveola, and purified h-caveolae can be tailored to be targeted to specific cells of interest.

Biophysical and Biochemical Characterization of Structurally Diverse Small Molecule Hits for KRAS Inhibition.

We describe six compounds as early hits for the development of direct inhibitors of KRAS, an important anticancer drug target. We show that these compounds bind to KRAS with affinities in the low micromolar range and exert different effects on its interactions with binding partners. Some of the compounds exhibit selective binding to the activated form of KRAS and inhibit signal transduction through both the MAPK or the phosphatidylinositide 3-kinase PI3K-protein kinase B (AKT) pathway in cells expressing mutant KRAS. Most inhibit intrinsic and/or SOS-mediated KRAS activation while others inhibit RAS-effector interaction. We propose these compounds as starting points for the development of non-covalent allosteric KRAS inhibitors.

Acylpeptide hydrolase is a novel regulator of KRAS plasma membrane localization and function.

The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic.

Depletion of phosphatidylinositol 4-phosphate at the Golgi translocates K-Ras to mitochondria.

Ras proteins are small GTPases localized to the plasma membrane (PM), which regulate cellular proliferation, apoptosis and differentiation. After a series of post-translational modifications, H-Ras and N-Ras traffic to the PM from the Golgi via the classical exocytic pathway, but the exact mechanism of K-Ras trafficking to the PM from the ER is not fully characterized. ATP5G1 (also known as ATP5MC1) is one of the three proteins that comprise subunit c of the F0 complex of the mitochondrial ATP synthase. In this study, we show that overexpression of the mitochondrial targeting sequence of ATP5G1 perturbs glucose metabolism, inhibits oncogenic K-Ras signaling, and redistributes phosphatidylserine (PtdSer) to mitochondria and other endomembranes, resulting in K-Ras translocation to mitochondria. Also, it depletes phosphatidylinositol 4-phosphate (PI4P) at the Golgi. Glucose supplementation restores PtdSer and K-Ras PM localization and PI4P at the Golgi. We further show that inhibition of the Golgi-localized PI4-kinases (PI4Ks) translocates K-Ras, and PtdSer to mitochondria and endomembranes, respectively. We conclude that PI4P at the Golgi regulates the PM localization of PtdSer and K-Ras.This article has an associated First Person interview with the first author of the paper.

HRAS-driven cancer cells are vulnerable to TRPML1 inhibition.

By serving as intermediaries between cellular metabolism and the bioenergetic demands of proliferation, endolysosomes allow cancer cells to thrive under normally detrimental conditions. Here, we show that an endolysosomal TRP channel, TRPML1, is necessary for the proliferation of cancer cells that bear activating mutations in HRAS Expression of MCOLN1, which encodes TRPML1, is significantly elevated in HRAS-positive tumors and inversely correlated with patient prognosis. Concordantly, MCOLN1 knockdown or TRPML1 inhibition selectively reduces the proliferation of cancer cells that express oncogenic, but not wild-type, HRAS Mechanistically, TRPML1 maintains oncogenic HRAS in signaling-competent nanoclusters at the plasma membrane by mediating cholesterol de-esterification and transport. TRPML1 inhibition disrupts the distribution and levels of cholesterol and thereby attenuates HRAS nanoclustering and plasma membrane abundance, ERK phosphorylation, and cell proliferation. These findings reveal a selective vulnerability of HRAS-driven cancers to TRPML1 inhibition, which may be leveraged as an actionable therapeutic strategy.

Lipidomic atlas of mammalian cell membranes reveals hierarchical variation induced by culture conditions, subcellular membranes, and cell lineages.

Lipid membranes are ubiquitous biological organizers, required for structural and functional compartmentalization of the cell and sub-cellular organelles. Membranes in living cells are compositionally complex, comprising hundreds of dynamically regulated, distinct lipid species. Cellular physiology requires tight regulation of these lipidomic profiles to achieve proper membrane functionality. While some general features of tissue- and organelle-specific lipid complements have been identified, less is known about detailed lipidomic variations caused by cell-intrinsic or extrinsic factors. Here, we use shotgun lipidomics to report detailed, comprehensive lipidomes of a variety of cultured and primary mammalian membrane preparations to identify trends and sources of variation. Unbiased principle component analysis (PCA) shows clear separation between cultured and primary cells, with primary erythrocytes, synaptic membranes, and other mammalian tissue lipidomes sharply diverging from all cultured cell lines and also from one other. Most broadly, cultured cell membrane preparations were distinguished by their paucity of polyunsaturated lipids. Cultured mammalian cell lines were comparatively similar to one another, although we detected clear, highly reproducible lipidomic signatures of individual cell lines and plasma membrane (PM) isolations thereof. These measurements begin to establish a comprehensive lipidomic atlas of mammalian cells and tissues, identifying some major sources of variation. These observations will allow investigation of the regulation and functional significance of mammalian lipidomes in various contexts.

Chalcones bearing a 3,4,5-trimethoxyphenyl motif are capable of selectively inhibiting oncogenic K-Ras signaling.

Ras proteins are small GTPases which regulate cellular proliferation, differentiation, and apoptosis. Constitutively active mutant Ras are expressed in ~15-20% human cancers, and K-Ras mutations account for ~85% of all Ras mutations. Despite the significance of Ras proteins in refractory cancers, there is no anti-Ras drug available in clinic. Since K-Ras must interact with the plasma membrane (PM) for biological activity, inhibition of the K-Ras/PM interaction is a tractable approach to block oncogenic K-Ras activity. Here, we discovered chalcones 1 and 8 exhibit anti-K-Ras activity, and show that the compounds mislocalize K-Ras from the PM and block oncogenic K-Ras signal output. Also, 1 inhibits the growth of K-Ras-driven human cancer cells. Our data suggest that 1 could be a promising starting point for developing anti-K-Ras cancer drug.

An oxanthroquinone derivative that disrupts RAS plasma membrane localization inhibits cancer cell growth.

Oncogenic RAS proteins are commonly expressed in human cancer. To be functional, RAS proteins must undergo post-translational modification and localize to the plasma membrane (PM). Therefore, compounds that prevent RAS PM targeting have potential as putative RAS inhibitors. Here we examine the mechanism of action of oxanthroquinone G01 (G01), a recently described inhibitor of KRAS PM localization. We show that G01 mislocalizes HRAS and KRAS from the PM with similar potency and disrupts the spatial organization of RAS proteins remaining on the PM. G01 also inhibited recycling of epidermal growth factor receptor and transferrin receptor, but did not impair internalization of cholera toxin, indicating suppression of recycling endosome function. In searching for the mechanism of impaired endosomal recycling we observed that G01 also enhanced cellular sphingomyelin (SM) and ceramide levels and disrupted the localization of several lipid and cholesterol reporters, suggesting that the G01 molecular target may involve SM metabolism. Indeed, G01 exhibited potent synergy with other compounds that target SM metabolism in KRAS localization assays. Furthermore, G01 significantly abrogated RAS-RAF-MAPK signaling in Madin-Darby canine kidney (MDCK) cells expressing constitutively activated, oncogenic mutant RASG12V. G01 also inhibited the proliferation of RAS-less mouse embryo fibroblasts expressing oncogenic mutant KRASG12V or KRASG12D but not RAS-less mouse embryo fibroblasts expressing oncogenic mutant BRAFV600E. Consistent with these effects, G01 selectively inhibited the proliferation of KRAS-transformed pancreatic, colon, and endometrial cancer cells. Taken together, these results suggest that G01 should undergo further evaluation as a potential anti-RAS therapeutic.

Cell-Penetrating Peptide-Patchy Deformable Polymeric Nanovehicles with Enhanced Cellular Uptake and Transdermal Delivery.

We herein propose a polymeric nanovehicle system that has the ability to remarkably improve cellular uptake and transdermal delivery. Cell-penetrating peptide-patchy deformable polymeric nanovehicles were fabricated by tailored coassembly of amphiphilic poly(ethylene oxide)- block-poly(ε-caprolactone) (PEO- b-PCL), mannosylerythritol lipid (MEL), and YGRKKRRQRRR-cysteamine (TAT)-linked MEL. Using X-ray diffraction, differential scanning calorimetry, and nuclear magnetic resonance analyses, we revealed that the incorporation of MEL having an asymmetric alkyl chain configuration was responsible for the deformable phase property of the vehicles. We also discovered that the nanovehicles were mutually attracted, exhibiting a gel-like fluid characteristic due to the dipole-dipole interaction between the hydroxyl group of MEL and the methoxy group of PEO- b-PCL. Coassembly of TAT-linked MEL with the deformable nanovehicles significantly enhanced cellular uptake due to macropinocytosis and caveolae-/lipid raft-mediated endocytosis. Furthermore, the in vivo skin penetration test revealed that our TAT-patchy deformable nanovehicles remarkably improved transdermal delivery efficiency.

Spatiotemporal Analysis of K-Ras Plasma Membrane Interactions Reveals Multiple High Order Homo-oligomeric Complexes.

Self-assembly of plasma membrane-associated Ras GTPases has major implications to the regulation of cell signaling. However, the structural basis of homo-oligomerization and the fractional distribution of oligomeric states remained undetermined. We have addressed these issues by deciphering the distribution of dimers and higher-order oligomers of K-Ras4B, the most frequently mutated Ras isoform in human cancers. We focused on the constitutively active G12V K-Ras and two of its variants, K101E and K101C/E107C, which respectively destabilize and stabilize oligomers. Using raster image correlation spectroscopy and number and brightness analysis combined with fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and electron microscopy in live cells, we show that G12V K-Ras exists as a mixture of monomers, dimers and larger oligomers, while the K101E mutant is predominantly monomeric and K101C/E107C is dominated by oligomers. This observation demonstrates the ability of K-Ras to exist in multiple oligomeric states whose population can be altered by interfacial mutations. Using molecular modeling and simulations we further show that K-Ras uses two partially overlapping interfaces to form compositionally and topologically diverse oligomers. Our results thus provide the first detailed insight into the multiplicity, structure, and membrane organization of K-Ras homomers.

Andrographolide derivatives inhibit guanine nucleotide exchange and abrogate oncogenic Ras function.

Aberrant signaling by oncogenic mutant rat sarcoma (Ras) proteins occurs in ∼15% of all human tumors, yet direct inhibition of Ras by small molecules has remained elusive. Recently, several small-molecule ligands have been discovered that directly bind Ras and inhibit its function by interfering with exchange factor binding. However, it is unclear whether, or how, these ligands could lead to drugs that act against constitutively active oncogenic mutant Ras. Using a dynamics-based pocket identification scheme, ensemble docking, and innovative cell-based assays, here we show that andrographolide (AGP)--a bicyclic diterpenoid lactone isolated from Andrographis paniculata--and its benzylidene derivatives bind to transient pockets on Kirsten-Ras (K-Ras) and inhibit GDP-GTP exchange. As expected for inhibitors of exchange factor binding, AGP derivatives reduced GTP loading of wild-type K-Ras in response to acute EGF stimulation with a concomitant reduction in MAPK activation. Remarkably, however, prolonged treatment with AGP derivatives also reduced GTP loading of, and signal transmission by, oncogenic mutant K-RasG12V. In sum, the combined analysis of our computational and cell biology results show that AGP derivatives directly bind Ras, block GDP-GTP exchange, and inhibit both wild-type and oncogenic K-Ras signaling. Importantly, our findings not only show that nucleotide exchange factors are required for oncogenic Ras signaling but also demonstrate that inhibiting nucleotide exchange is a valid approach to abrogating the function of oncogenic mutant Ras.

Rare Streptomyces sp. polyketides as modulators of K-Ras localisation.

Chemical investigations of a soil-derived Streptomyces sp. led to the isolation of five new polyketides, (+)-oxanthromicin, (±)-hemi-oxanthromicins A/B, (±)-spiro-oxanthromicin A and oxanthroquinone, and the known alkaloid staurosporine, and the detection of four new metastable analogues, (±)-spiro-oxanthromicins B1/B2/C1/C2. Among the compounds tested, SAR investigations established that the synthetic oxanthroquinone ethyl ester and 3-O-methyl-oxanthroquinone ethyl ester were optimal at mislocalising oncogenic mutant K-Ras from the plasma membrane of intact Madin-Darby canine kidney (MDCK) cells (IC50 4.6 and 1.2 µM), while a sub-EC50 dose of (±)-spiro-oxanthromicin A was optimal at potentiating (750%) the K-Ras inhibitory activity of staurosporine (IC50 60 pM). These studies demonstrate that a rare class of Streptomyces polyketide modulates K-Ras plasma membrane localisation, with implications for the future treatment of K-Ras dependent cancers.

Network pharmacology study to explore the multiple molecular mechanism of SH003 in the treatment of non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of human death worldwide. Herbal prescription SH003 has been developed to treat several cancers including NSCLC. Due to the multi-component nature of SH003 with multiple targets and pathways, a network pharmacology study was conducted to analyze its active compounds, potential targets, and pathways for the treatment of NSCLC. METHODS: We systematically identified oral active compounds within SH003, employing ADME criteria-based screening from TM-MC, OASIS, and TCMSP databases. Concurrently, SH003-related and NSCLC-associated targets were amalgamated from various databases. Overlapping targets were deemed anti-NSCLC entities of SH003. Protein-protein interaction networks were constructed using the STRING database, allowing the identification of pivotal proteins through node centrality measures. Empirical validation was pursued through LC-MS analysis of active compounds. Additionally, in vitro experiments, such as MTT cell viability assays and western blot analyses, were conducted to corroborate network pharmacology findings. RESULTS: We discerned 20 oral active compounds within SH003 and identified 239 core targets shared between SH003 and NSCLC-related genes. Network analyses spotlighted 79 hub genes, including TP53, JUN, AKT1, STAT3, and MAPK3, crucial in NSCLC treatment. GO and KEGG analyses underscored SH003's multifaceted anti-NSCLC effects from a genetic perspective. Experimental validations verified SH003's impact on NSCLC cell viability and the downregulation of hub genes. LC-MS analysis confirmed the presence of four active compounds, namely hispidulin, luteolin, baicalein, and chrysoeriol, among the eight compounds with a median of > 10 degrees in the herb-compounds-targets network in SH003. Previously unidentified targets like CASP9, MAPK9, and MCL1 were unveiled, supported by existing NSCLC literature, enhancing the pivotal role of empirical validation in network pharmacology. CONCLUSION: Our study pioneers the harmonization of theoretical predictions with practical validations. Empirical validation illuminates specific SH003 compounds within NSCLC, simultaneously uncovering novel targets for NSCLC treatment. This integrated strategy, accentuating empirical validation, establishes a paradigm for in-depth herbal medicine exploration. Furthermore, our network pharmacology study unveils fresh insights into SH003's multifaceted molecular mechanisms combating NSCLC. Through this approach, we delineate active compounds of SH003 and target pathways, reshaping our understanding of its therapeutic mechanisms in NSCLC treatment.

Myotubularin-related proteins regulate KRAS function by controlling plasma membrane levels of polyphosphoinositides and phosphatidylserine.

KRAS is a small GTPase, ubiquitously expressed in mammalian cells, that functions as a molecular switch to regulate cell proliferation and differentiation. Oncogenic mutations that render KRAS constitutively active occur frequently in human cancers. KRAS must localize to the plasma membrane (PM) for biological activity. KRAS PM binding is mediated by interactions of the KRAS membrane anchor with phosphatidylserine (PtdSer), therefore, depleting PM PtdSer content abrogates KRAS PM binding and oncogenic function. From a genome-wide siRNA screen to search for genes that regulate KRAS PM localization, we identified a set of phosphatidylinositol (PI) 3-phosphatase family members: myotubularin-related (MTMR) proteins 2, 3, 4 and 7. Here we show that knockdown of MTMR 2/3/4/7 expression disrupts KRAS PM interactions. The molecular mechanism involves depletion of PM PI 4-phosphate (PI4P) levels, which in turn disrupts the subcellular localization and operation of oxysterol-binding protein related protein (ORP) 5, a PtdSer lipid transfer protein that maintains PM PtdSer content. Concomitantly, silencing MTMR 2/3/4/7 expression elevates PM levels of PI3P and reduces PM and total cellular levels of PtdSer. In summary we propose that the PI 3-phosphatase activity provided by MTMR proteins is required to generate PM PI for the synthesis of PM PI4P, which in turn, promotes the PM localization of PtdSer and KRAS.

Nonsteroidal anti-inflammatory drugs alter the spatiotemporal organization of Ras proteins on the plasma membrane.

Ras proteins on the inner leaflet of the plasma membrane signal from transient nanoscale proteolipid assemblies called nanoclusters. Interactions between the Ras lipid anchors and plasma membrane phospholipids, cholesterol, and actin cytoskeleton contribute to the formation, stability, and dynamics of Ras nanoclusters. Many small biological molecules are amphiphilic and capable of intercalating into membranes and altering lipid immiscibility. In this study we systematically examined whether amphiphiles such as indomethacin influence Ras protein nanoclustering in intact plasma membrane. We found that indomethacin, a nonsteroidal anti-inflammatory drug, induced profound and complex effects on Ras spatial organization, all likely related to liquid-ordered domain stabilization. Indomethacin enhanced the clustering of H-Ras.GDP and N-Ras.GTP in cholesterol-dependent nanoclusters. Indomethacin also abrogated efficient GTP-dependent lateral segregation of H- and N-Ras between cholesterol-dependent and cholesterol-independent clusters, resulting in mixed heterotypic clusters of Ras proteins that normally are separated spatially. These heterotypic Ras nanoclusters showed impaired Raf recruitment and kinase activation resulting in significantly compromised MAPK signaling. All of the amphiphilic anti-inflammatory agents we tested had similar effects on Ras nanoclustering and signaling. The potency of these effects correlated with the membrane partition coefficients of the individual agents and was independent of COX inhibition. This study shows that biological amphiphiles have wide-ranging effects on plasma membrane heterogeneity and protein nanoclustering, revealing a novel mechanism of drug action that has important consequences for cell signaling.

Staurosporines disrupt phosphatidylserine trafficking and mislocalize Ras proteins.

Oncogenic mutant Ras is frequently expressed in human cancers, but no anti-Ras drugs have been developed. Since membrane association is essential for Ras biological activity, we developed a high content assay for inhibitors of Ras plasma membrane localization. We discovered that staurosporine and analogs potently inhibit Ras plasma membrane binding by blocking endosomal recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine from plasma membrane to endomembrane. Staurosporines are more active against K-Ras than H-Ras. K-Ras is displaced to endosomes and undergoes proteasomal-independent degradation, whereas H-Ras redistributes to the Golgi and is not degraded. K-Ras nanoclustering on the plasma membrane is also inhibited. Ras mislocalization does not correlate with protein kinase C inhibition or induction of apoptosis. Staurosporines selectively abrogate K-Ras signaling and proliferation of K-Ras-transformed cells. These results identify staurosporines as novel inhibitors of phosphatidylserine trafficking, yield new insights into the role of phosphatidylserine and electrostatics in Ras plasma membrane targeting, and validate a new target for anti-Ras therapeutics.

A new ferrocene derivative blocks KRAS localization and function by oxidative modification at His95.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers (NSCLCs) harboring oncogenic mutant K-Ras. Here, we developed and tested a novel ferrocene derivative on the growth of human pancreatic ductal adenocarcinoma (PDAC) and NSCLC. Our compound inhibited the growth of K-Ras-dependent PDAC and NSCLC and abrogated the PM binding and signaling of K-Ras, but not other Ras isoforms. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified K-Ras His95 residue in the G-domain as being involved in the ferrocene-induced K-Ras PM dissociation via oxidative modification. Together, our studies demonstrate that the redox system directly regulates K-Ras PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced metastasis in K-Ras-driven lung cancers.

A new ferrocene derivative blocks K-Ras localization and function by oxidative modification at His95.

Ras proteins are membrane-bound GTPases that regulate essential cellular processes at the plasma membrane (PM). Constitutively active mutations of K-Ras, one of the three Ras isoforms in mammalian cells, are frequently found in human cancers. Ferrocene derivatives, which elevate cellular reactive oxygen species (ROS), have shown to block the growth of non-small cell lung cancers harboring oncogenic mutant K-Ras. Here, we tested a novel ferrocene derivative on the growth of pancreatic ductal adenocarcinoma and non-small cell lung cancer. Our compound, which elevated cellular ROS levels, inhibited the growth of K-Ras-driven cancers, and abrogated the PM binding and signaling of K-Ras in an isoform-specific manner. These effects were reversed upon antioxidant supplementation, suggesting a ROS-mediated mechanism. We further identified that K-Ras His95 residue plays an important role in this process, and it is putatively oxidized by cellular ROS. Together, our study demonstrates that the redox system directly regulates K-Ras/PM binding and signaling via oxidative modification at the His95, and proposes a role of oncogenic mutant K-Ras in the recently described antioxidant-induced growth and metastasis of K-Ras-driven cancers.

Approaches to inhibiting oncogenic K-Ras.

Activating somatic K-Ras mutations are associated with >15% all human tumors and up to 90% of specific tumor types such as pancreatic cancer. Successfully inhibiting abnormal K-Ras signaling would therefore be a game changer in cancer therapy. However, K-Ras has long been considered an undruggable target for various reasons. This view is now changing by the discovery of allosteric inhibitors that directly target K-Ras and inhibit its functions, and by the identification of new mechanisms to dislodge it from the plasma membrane and thereby abrogate its cellular activities. In this review, we will discuss recent progresses and challenges to inhibiting aberrant K-Ras functions by these two approaches. We will also provide a broad overview of other approaches such as inhibition of K-Ras effectors, and offer a brief perspective on the way forward.