The nonreceptor tyrosine kinase SRMS inhibits autophagy and promotes tumor growth by phosphorylating the scaffolding protein FKBP51.

Nutrient-responsive protein kinases control the balance between anabolic growth and catabolic processes such as autophagy. Aberrant regulation of these kinases is a major cause of human disease. We report here that the vertebrate nonreceptor tyrosine kinase Src-related kinase lacking C-terminal regulatory tyrosine and N-terminal myristylation sites (SRMS) inhibits autophagy and promotes growth in a nutrient-responsive manner. Under nutrient-replete conditions, SRMS phosphorylates the PHLPP scaffold FK506-binding protein 51 (FKBP51), disrupts the FKBP51-PHLPP complex, and promotes FKBP51 degradation through the ubiquitin-proteasome pathway. This prevents PHLPP-mediated dephosphorylation of AKT, causing sustained AKT activation that promotes growth and inhibits autophagy. SRMS is amplified and overexpressed in human cancers where it drives unrestrained AKT signaling in a kinase-dependent manner. SRMS kinase inhibition activates autophagy, inhibits cancer growth, and can be accomplished using the FDA-approved tyrosine kinase inhibitor ibrutinib. This illuminates SRMS as a targetable vulnerability in human cancers and as a new target for pharmacological induction of autophagy in vertebrates.

A highly selective inhibitor of interleukin-1 receptor-associated kinases 1/4 (IRAK-1/4) delineates the distinct signaling roles of IRAK-1/4 and the TAK1 kinase.

Interleukin-1 receptor-associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor ß-activated kinase 1 (TAK1), are protein kinases essential for transducing inflammatory signals from interleukin receptors. IRAK family proteins and TAK1 have high sequence identity within the ATP-binding pocket, limiting the development of highly selective IRAK-1/4 or TAK1 inhibitors. Beyond kinase activity, IRAKs and TAK1 act as molecular scaffolds along with other signaling proteins, complicating the interpretation of experiments involving knockin or knockout approaches. In contrast, pharmacological manipulation offers the promise of targeting catalysis-mediated signaling without grossly disrupting the cellular architecture. Recently, we reported the discovery of takinib, a potent and highly selective TAK1 inhibitor that has only marginal activity against IRAK-4. On the basis of the TAK1-takinib complex structure and the structure of IRAK-1/4, here we defined critical contact sites of the takinib scaffold within the nucleotide-binding sites of each respective kinase. Kinase activity testing of takinib analogs against IRAK-4 identified a highly potent IRAK-4 inhibitor (HS-243). In a kinome-wide screen of 468 protein kinases, HS-243 had exquisite selectivity toward both IRAK-1 (IC50 = 24 nm) and IRAK-4 (IC50 = 20 nm), with only minimal TAK1-inhibiting activity (IC50 = 0.5 µm). Using HS-243 and takinib, we evaluated the consequences of cytokine/chemokine responses after selective inhibition of IRAK-1/4 or TAK1 in response to lipopolysaccharide challenge in human rheumatoid arthritis fibroblast-like synoviocytes. Our results indicate that HS-243 specifically inhibits intracellular IRAKs without TAK1 inhibition and that these kinases have distinct, nonredundant signaling roles.

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.

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.

MicroRNA-218 functions as a tumor suppressor in lung cancer by targeting IL-6/STAT3 and negatively correlates with poor prognosis.

BACKGROUND: Aberrant expression of microRNAs in different human cancer types has been widely reported. MiR-218 acts as a tumor suppressor in diverse human cancer types impacting regulation of multiple genes in oncogenic pathways. Here, we evaluated the expression and function of miR-218 in human lung cancer and ALDH positive lung cancer cells to understand the potential mechanisms responsible for disease pathology. Also, the association between its host genes and the target genes could be useful towards the better understanding of prognosis in clinical settings. METHODS: Publicly-available data from The Cancer Genome Atlas (TCGA) was mined to compare the levels of miR-218 and its host gene SLIT2/3 between lung cancer tissues and normal lung tissues. Transfection of miR-218 to investigate its function in lung cancer cells was done and in vivo effects were determined using miR-218 expressing lentiviruses. Aldefluor assay and Flow cytometry was used to quantify and enrich ALDH positive lung cancer cells. Levels of miR-218, IL-6R, JAK3 and phosphorylated STAT3 were compared in ALDH1A1 positive and ALDH1A1 negative cells. Overexpression of miR-218 in ALDH positive cells was carried to test the survival by tumorsphere culture. Finally, utilizing TCGA data we studied the association of target genes of miR-218 with the prognosis of lung cancer. RESULTS: We observed that the expression of miR-218 was significantly down-regulated in lung cancer tissues compared to normal lung tissues. Overexpression of miR-218 decreased cell proliferation, invasion, colony formation, and tumor sphere formation in vitro and repressed tumor growth in vivo. We further found that miR-218 negatively regulated IL-6 receptor and JAK3 gene expression by directly targeting the 3'-UTR of their mRNAs. In addition, the levels of both miR-218 host genes and the components of IL-6/STAT3 pathway correlated with prognosis of lung cancer patients. CONCLUSIONS: MiR-218 acts as a tumor suppressor in lung cancer via IL-6/STAT3 signaling pathway regulation.

Structure-guided development of covalent TAK1 inhibitors.

TAK1 (transforming growth factor-ß-activated kinase 1) is an essential intracellular mediator of cytokine and growth factor signaling and a potential therapeutic target for the treatment of immune diseases and cancer. Herein we report development of a series of 2,4-disubstituted pyrimidine covalent TAK1 inhibitors that target Cys174, a residue immediately adjacent to the 'DFG-motif' of the kinase activation loop. Co-crystal structures of TAK1 with candidate compounds enabled iterative rounds of structure-based design and biological testing to arrive at optimized compounds. Lead compounds such as 2 and 10 showed greater than 10-fold biochemical selectivity for TAK1 over the closely related kinases MEK1 and ERK1 which possess an equivalently positioned cysteine residue. These compounds are smaller, more easily synthesized, and exhibit a different spectrum of kinase selectivity relative to previously reported macrocyclic natural product TAK1 inhibitors such as 5Z-7-oxozeanol.

Studies of TAK1-centered polypharmacology with novel covalent TAK1 inhibitors.

Targeted polypharmacology provides an efficient method of treating diseases such as cancer with complex, multigenic causes provided that compounds with advantageous activity profiles can be discovered. Novel covalent TAK1 inhibitors were validated in cellular contexts for their ability to inhibit the TAK1 kinase and for their polypharmacology. Several inhibitors phenocopied reported TAK1 inhibitor 5Z-7-oxozaenol with comparable efficacy and complementary kinase selectivity profiles. Compound 5 exhibited the greatest potency in RAS-mutated and wild-type RAS cell lines from various cancer types. A biotinylated derivative of 5, 27, was used to verify TAK1 binding in cells. The newly described inhibitors constitute useful tools for further development of multi-targeting TAK1-centered inhibitors for cancer and other diseases.

In situ selectivity profiling and crystal structure of SML-8-73-1, an active site inhibitor of oncogenic K-Ras G12C.

Directly targeting oncogenic V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (K-Ras) with small-molecule inhibitors has historically been considered prohibitively challenging. Recent reports of compounds that bind directly to the K-Ras G12C mutant suggest avenues to overcome key obstacles that stand in the way of developing such compounds. We aim to target the guanine nucleotide (GN)-binding pocket because the natural contents of this pocket dictate the signaling state of K-Ras. Here, we characterize the irreversible inhibitor SML-8-73-1 (SML), which targets the GN-binding pocket of K-Ras G12C. We report a high-resolution X-ray crystal structure of G12C K-Ras bound to SML, revealing that the compound binds in a manner similar to GDP, forming a covalent linkage with Cys-12. The resulting conformation renders K-Ras in the open, inactive conformation, which is not predicted to associate productively with or activate downstream effectors. Conservation analysis of the Ras family GN-binding pocket reveals variability in the side chains surrounding the active site and adjacent regions, especially in the switch I region. This variability may enable building specificity into new iterations of Ras and other GTPase inhibitors. High-resolution in situ chemical proteomic profiling of SML confirms that SML effectively discriminates between K-Ras G12C and other cellular GTP-binding proteins. A biochemical assay provides additional evidence that SML is able to compete with millimolar concentrations of GTP and GDP for the GN-binding site.

Pharmacological targeting of the pseudokinase Her3.

Her3 (also known as ErbB3) belongs to the epidermal growth factor receptor tyrosine kinases and is well credentialed as an anti-cancer target but is thought to be 'undruggable' using ATP-competitive small molecules because it lacks appreciable kinase activity. Here we report what is to our knowledge the first selective Her3 ligand, TX1-85-1, that forms a covalent bond with Cys721 located in the ATP-binding site of Her3. We demonstrate that covalent modification of Her3 inhibits Her3 signaling but not proliferation in some Her3-dependent cancer cell lines. Subsequent derivatization with a hydrophobic adamantane moiety demonstrates that the resultant bivalent ligand (TX2-121-1) enhances inhibition of Her3-dependent signaling. Treatment of cells with TX2-121-1 results in partial degradation of Her3 and serendipitously interferes with productive heterodimerization between Her3 with either Her2 or c-Met. These results suggest that small molecules will be capable of perturbing the biological function of Her3 and ∼60 other pseudokinases found in human cells.

Development of small molecules targeting the pseudokinase Her3.

Her3 is a member of the human epidermal growth factor receptor (EGFR) tyrosine kinase family, and it is often either overexpressed or deregulated in many types of human cancer. Her3 has not been the subject of small-molecule inhibitor development because it is a pseudokinase and does not possess appreciable kinase activity. We recently reported on the development of the first selective irreversible Her3 ligand (TX1-85-1) that forms a covalent bond with cysteine 721 which is unique to Her3 among all kinases. We also developed a bi-functional compound (TX2-121-1) containing a hydrophobic adamantane moiety and the same warhead of TX1-85-1 that is capable of inhibiting Her3-dependent signaling and growth. Here we report on the structure-based medicinal chemistry effort that resulted in the discovery of these two compounds.

Development of a Covalent Inhibitor of c-Jun N-Terminal Protein Kinase (JNK) 2/3 with Selectivity over JNK1.

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) family, which includes JNK1-JNK3. Interestingly, JNK1 and JNK2 show opposing functions, with JNK2 activity favoring cell survival and JNK1 stimulating apoptosis. Isoform-selective small molecule inhibitors of JNK1 or JNK2 would be useful as pharmacological probes but have been difficult to develop due to the similarity of their ATP binding pockets. Here, we describe the discovery of a covalent inhibitor YL5084, the first such inhibitor that displays selectivity for JNK2 over JNK1. We demonstrated that YL5084 forms a covalent bond with Cys116 of JNK2, exhibits a 20-fold higher Kinact/KI compared to that of JNK1, and engages JNK2 in cells. However, YL5084 exhibited JNK2-independent antiproliferative effects in multiple myeloma cells, suggesting the existence of additional targets relevant in this context. Thus, although not fully optimized, YL5084 represents a useful chemical starting point for the future development of JNK2-selective chemical probes.

Structure and Characterization of a Covalent Inhibitor of Src Kinase.

Unregulated Src activity promotes malignant processes in cancer, but no Src-directed targeted therapies are used clinically, possibly because early Src inhibitors produce off-target effects leading to toxicity. Improved selective Src inhibitors may enable Src-directed therapies. Previously, we reported an irreversible Src inhibitor, DGY-06-116, based on the hybridization of dasatinib and a promiscuous covalent kinase probe SM1-71. Here, we report biochemical and biophysical characterization of this compound. An x-ray co-crystal structure of DGY-06-116: Src shows a covalent interaction with the kinase p-loop and occupancy of the back hydrophobic kinase pocket, explaining its high potency, and selectivity. However, a reversible analog also shows similar potency. Kinetic analysis shows a slow inactivation rate compared to other clinically approved covalent kinase inhibitors, consistent with a need for p-loop movement prior to covalent bond formation. Overall, these results suggest that a strong reversible interaction is required to allow sufficient time for the covalent reaction to occur. Further optimization of the covalent linker may improve the kinetics of covalent bond formation.

Structure-Based Design of a Potent and Selective Covalent Inhibitor for SRC Kinase That Targets a P-Loop Cysteine.

SRC is a major regulator of many signaling pathways and contributes to cancer development. However, development of a selective SRC inhibitor has been challenging, and FDA-approved SRC inhibitors, dasatinib and bosutinib, are multitargeted kinase inhibitors. Here, we describe our efforts to develop a selective SRC covalent inhibitor by targeting cysteine 277 on the P-loop of SRC. Using a promiscuous covalent kinase inhibitor (CKI) SM1-71 as a starting point, we developed covalent inhibitor 15a, which discriminates SRC from other covalent targets of SM1-71 including TAK1 and FGFR1. As an irreversible covalent inhibitor, compound 15a exhibited sustained inhibition of SRC signaling both in vitro and in vivo. Moreover, 15a exhibited potent antiproliferative effects in nonsmall cell lung cancer cell lines harboring SRC activation, thus providing evidence that this approach may be promising for further drug development efforts.

In silico studies with human DNA topoisomerase-II alpha to unravel the mechanism of in vitro genotoxicity of benzene and its metabolites.

Exposure of humans to benzene present in environment may lead to adverse chronic effects-even at the genetic level. However, the mechanism of its genotoxicity is not well understood. In the present study, in vitro genotoxicity of benzene (BZ) and its major metabolites [p-benzoquinone (BQ), hydroquinone (HQ), catechol (CT), 1,2,4-benzenetriol (BT) and trans-trans muconic acid (MA)] at concentrations 0.5-50 microM, was assessed in Chinese hamster ovary (CHO) cells employing the alkaline Comet assay, cytokinesis blocked micronucleus (CBMN) assay, flow cytometric analysis of micronucleus (flow MN) and chromosome aberration (CA) test. The data revealed significant (P<0.05) concentration-dependent response in all end points. HQ was found to be the most potent DNA damaging metabolite in the Comet assay followed by BQ>BT>CT>BZ>MA. Both CBMN and flow MN assays revealed a good correlation in their results, where BQ and MA exhibited maximum and minimum micronucleus induction respectively. Significant chromosomal aberrations were induced mainly by BQ, BT and HQ, with moderate response shown by CT and BZ and least by MA. The results demonstrated the utility of sensitive techniques like Comet assay and flow cytometry for determination of MN, to quantify in vitro genotoxicity at low levels and also suggested that partly non-repaired DNA damage could cause adverse health effects in human population exposed to benzene. In silico studies using different endpoints of genotoxicity and molecular docking studies with human topoisomerase-II alpha, a major DNA repair enzyme were also conducted. These corroborated the results obtained from the in vitro data, pointing to a direct relationship of the observed genotoxicity with the structural properties and various interactions of metabolites with the enzyme. This comprehensive study demonstrated that genotoxicity of benzene in mammalian cells is mainly due to the inhibition of topoisomerase by the metabolites.

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.

Takinib, a Selective TAK1 Inhibitor, Broadens the Therapeutic Efficacy of TNF-α Inhibition for Cancer and Autoimmune Disease.

Tumor necrosis factor alpha (TNF-α) has both positive and negative roles in human disease. In certain cancers, TNF-α is infused locally to promote tumor regression, but dose-limiting inflammatory effects limit broader utility. In autoimmune disease, anti-TNF-α antibodies control inflammation in most patients, but these benefits are offset during chronic treatment. TAK1 acts as a key mediator between survival and cell death in TNF-α-mediated signaling. Here, we describe Takinib, a potent and selective TAK1 inhibitor that induces apoptosis following TNF-α stimulation in cell models of rheumatoid arthritis and metastatic breast cancer. We demonstrate that Takinib is an inhibitor of autophosphorylated and non-phosphorylated TAK1 that binds within the ATP-binding pocket and inhibits by slowing down the rate-limiting step of TAK1 activation. Overall, Takinib is an attractive starting point for the development of inhibitors that sensitize cells to TNF-α-induced cell death, with general implications for cancer and autoimmune disease treatment.

Structural dataset for the fast-exchanging KRAS G13D.

Cancers bearing the KRAS G13D mutation are notable for their distinct clinical behavior relative to other oncogenic KRAS mutations. We hypothesized that primary biochemical or biophysical properties of KRAS G13D might contribute to these clinical observations and as part of our study undertook structural studies using x-ray crystallography. In this data article we discuss several x-ray diffraction datasets that yielded structures of oncogenic KRAS mutants including a high resolution (1.13 Å) structure of KRAS G13D. The datasets are typical for high resolution x-ray diffraction data and allow the construction of atomic resolution, three dimensional structural models with high confidence. This data can be correlated with biochemical information such as defects in substrate binding kinetics, GTPase activities and interactions with the RAS effector RAF kinase.

Biochemical and Structural Analysis of Common Cancer-Associated KRAS Mutations.

UNLABELLED: KRAS mutations are the most common genetic abnormalities in cancer, but the distribution of specific mutations across cancers and the differential responses of patients with specific KRAS mutations in therapeutic clinical trials suggest that different KRAS mutations have unique biochemical behaviors. To further explain these high-level clinical differences and to explore potential therapeutic strategies for specific KRAS isoforms, we characterized the most common KRAS mutants biochemically for substrate binding kinetics, intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities, and interactions with the RAS effector, RAF kinase. Of note, KRAS G13D shows rapid nucleotide exchange kinetics compared with other mutants analyzed. This property can be explained by changes in the electrostatic charge distribution of the active site induced by the G13D mutation as shown by X-ray crystallography. High-resolution X-ray structures are also provided for the GDP-bound forms of KRAS G12V, G12R, and Q61L and reveal additional insight. Overall, the structural data and measurements, obtained herein, indicate that measurable biochemical properties provide clues for identifying KRAS-driven tumors that preferentially signal through RAF. IMPLICATIONS: Biochemical profiling and subclassification of KRAS-driven cancers will enable the rational selection of therapies targeting specific KRAS isoforms or specific RAS effectors.

Discovery of type II inhibitors of TGFß-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase kinase kinase 2 (MAP4K2).

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure-activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.

TiO(2) nanoparticles induce oxidative DNA damage and apoptosis in human liver cells.

Titanium dioxide nanoparticles (TiO(2) NPs), widely used in consumer products, paints, pharmaceutical preparations and so on, have been shown to induce cytotoxicity, genotoxicity and carcinogenic responses in vitro and in vivo. The present study revealed that TiO(2) NPs induce significant (p < 0.05) oxidative DNA damage by the Fpg-Comet assay even at 1 µg/ml concentration. A corresponding increase in the micronucleus frequency was also observed. This could be attributed to the reduced glutathione levels with concomitant increase in lipid peroxidation and reactive oxygen species generation. Furthermore, immunoblot analysis revealed an increased expression of p53, BAX, Cyto-c, Apaf-1, caspase-9 and caspase-3 and decreased the level of Bcl-2 thereby indicating that apoptosis induced by TiO(2) NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO(2) NPs induce DNA damage and cause apoptosis in HepG2 cells even at very low concentrations. Hence the use of such nanoparticles should be carefully monitored.