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.
Biochemical profiling and subclassification of KRAS-driven cancers will enable the rational selection of therapies targeting specific KRAS isoforms or specific RAS effectors.
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.
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.
Bone defects above critical size do not heal completely by itself and thus represent major clinical challenge to reconstructive surgery. Numerous bone substitutes have already been used to promote ...bone regeneration, however their use, particularly for critical-sized bone defects along with their long term in vivo safety and efficacy remains a concern. The present study was designed to obtain a complete healing of critical-size defect made in the proximal tibia of New Zealand White rabbit, using nano-hydroxyapatite/gelatin and chemically carboxymethylated chitin (n-HA/gel/CMC) scaffold construct. The bone-implant interfaces and defect site healing was evaluated for a period up to 25 weeks using radiography, micro-computed tomography, fluorescence labeling, and histology and compared with respective SHAM (empty contra lateral control). The viscoelastic porous scaffold construct allows easy surgical insertion and post-operatively facilitate oxygenation and angiogenesis. Radiography of defect treated with scaffold construct suggested expedited healing at defect edges and within the defect site, unlike confined healing at edges of the SHAM sites. The architecture indices analyzed by micro-computed tomography showed a significant increase in percentage of bone volume fraction, resulted in reconciled cortico-trabecular bone formation at n-HA/gel/CMC constructs treated site (15.2% to 52.7%) when compared with respective SHAM (10.2% to 31.8%). Histological examination and fluorescence labeling revealed that the uniformly interconnected porous surface of scaffold construct enhanced osteoblasts' activity and mineralization. These preclinical data suggest that, n-HA/gel/CMC construct exhibit stimulation of bone's innate regenerative capacity, thus underscoring their use in guided bone regeneration.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background: c-Src was among the first oncogenes discovered. It encodes a non-receptor tyrosine kinase that regulates many cancer-related cellular processes including mitogenesis, ...angiogenesis, adhesion, invasion, migration, and survival. Src activity drives malignant phenotypes in hematologic and solid cancers including breast, prostate, lung, colorectal, and pancreatic cancer. However, no specific Src-directed targeted therapies are in clinical use. Various Src inhibitors such as dasatinib, bosutinib and others have been tested in human trials, but with variable outcomes including dose-limiting toxicity, which is due to the multi-targeted nature of these compounds, which also hit Src, SFKs, BCR-ABL, c-KIT, PDGFR, c-FMS, EPHA2 and others. Improved selective Src inhibitors may overcome these challenges to enable Src-directed therapies. Engineering selectivity into Src inhibitors is challenging because of the high degree of sequence homology between Src family members and other receptor tyrosine kinases. The selective, covalent Src inhibitor DGY-06-116, which targets a relatively unusual targetable cysteine near the active site in the p loop, was recently reported. Here, we further characterize DGY-06-116.
Methods: We characterized the inhibitory activity DGY-06-116 using an enzymatic assay and surface plasmon resonance (SPR). We also determined the co-crystal structure of DGY-06-116 bound to Src.
Results: DGY-06-116 showed a substantial improvement over both SM171 and bosutinib for inhibition of Src kinase activity: SM1-71 (IC50, 26.6 nM); bosutinib (IC50, 9.5 nM); DGY-06-116 (IC50, 2.59 nM). Determination of kinact using SPR showed a value of 5.7 x 10-7 (s-1) for DGY-06-116, slower than FDA-approved neratinib (2 x 10-3 s-1) and afatinib (1 x 10-3 s-1). A 2.15 Å resolution co-crystal structure of DGY-06-116 bound to Src (PDB ID: 6E6E) showed a covalent bond with Cys-280 and bending of the p-loop relative to other Src-inhibitor structures.
Conclusions: DGY-06-116 demonstrates a slow inactivation rate that likely reflects the need for repositioning of the p-loop for a covalent bond to occur. Src inhibitors based on DGY-06-116 may benefit from optimization of p-loop, inhibitor interactions.
Citation Format: Deepak Gurbani, Kenneth Westover, Asim Bera. Biochemical and biophysical characterization of a covalent inhibitor of Src kinase abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4017.
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 (1.13Å) 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.
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.
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.
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.
MiR-218 acts as a tumor suppressor in lung cancer via IL-6/STAT3 signaling pathway regulation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Titanium dioxide nanoparticles (TiO2 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 TiO2 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 TiO2 NPs occurs via the caspase-dependent pathway. This study systematically shows that TiO2 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.
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.
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