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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. Mol Cancer Res; 13(9); 1325–35. ©2015 AACR.

Supplementary Figures S1 and S2. Figure S1. -GTP hydrolysis rate is stimulated by P120GAP. Figure S2. - Model of Intrinsic GTP hydrolysis.

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 A 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.

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., This study describes the discovery and characterization of a nutrient-sensitive signaling pathway that drives growth and inhibits autophagy in mammalian cells. This pathway, which involves the non-receptor tyrosine kinase SRMS and the PHLPP scaffold protein FKBP51, promotes tumor growth and is amenable to pharmacological inhibition.

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.

Interleukin-1 receptor–associated kinase-1 (IRAK-1) and IRAK-4, as well as transforming growth factor β–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 (IC(50) = 24 nm) and IRAK-4 (IC(50) = 20 nm), with only minimal TAK1-inhibiting activity (IC(50) = 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.

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 KRAS(V14I), 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 KRAS(A146T), 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 KRAS(A146T) and KRAS(V14I) that may have implications for the regulation of these variants and for the development of therapeutic strategies to manage KRAS variant-associated disorders.

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.

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.

Summary 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.

Tumor necrosis factor alpha (TNFα) administration has shown limited therapeutic utility in cancer and inflammatory disorders due to dose‐limiting side effects. We propose an alternative approach to targeting TNFα‐mediated diseases by selectively inducing the TNFα apoptotic response in TNFα‐rich microenvironments. TAK1 acts as a key mediator between survival and cell death in TNFα signaling. We report a novel potent and exquisitely selective TAK1 inhibitor (EDHS‐206, IC50 9.5nM) that induces apoptosis in a TNFα‐dependent manner in models of metastatic breast cancer and rheumatoid arthritis. Co‐crystallization studies demonstrated that EDHS‐206 inhibits the kinase in a DFG‐in conformation. Kinetic analysis of EDHS‐206‐TAK1 interactions revealed a substrate‐like intermolecular autophosphorylation mechanism for TAK1 activation, during which EDHS‐206 delays the proceedings of the rate‐limiting step. EDHS‐206 represents an attractive starting point for the development of a novel generation of inhibitors that greatly sensitize cells to TNFα‐induced cell death, potentially broadening the therapeutic efficacy of TNFα.

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.

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.

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.

The release of particulate pollutants into the air through burning of coal, crude oil, diesel, coal tar, etc. raises concerns of potential health hazards to the exposed human population. Polycyclic aromatic hydrocarbons (PAHs) are major toxic constituents of particulate matter (PM), which upon ingestion get metabolized to even more toxic metabolites such as quinones. The PAHs levels were assessed in both respirable particulate matter (RSPM,10μM size) and suspended particulate matter (SPM,10μM size) of urban ambient air (UAA) and that of major contributors viz. diesel exhaust particles (DEPs) and coal tar combustions emissions (CTCE). Seven US Environmental Protection Agency (USEPA) prioritized PAHs in RSPM and 10 in SPM were detected in UAA. Ten and 15 prioritized PAHs, respectively, were also detected in diesel exhaust particles (DEP) and coal tar combustion emission (CTCE) evidencing their release in the air. These PM associated PAHs for UAA, DEP and CTCE showed significant increase (p0.05) in mutagenicity and mammalian genotoxicity in the order CTCEDEPUAA. Human lung alveolar (A549) and bronchiolar (BEAS-2B) cells when treated with PAH-metabolites viz. 1,4-benzoquinone (1,4-BQ), hydroquinone (HQ), 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ) and 9,10-phenanthroquinone (9,10-PQ) showed metabolic modulation in these cell lines with significant depletion of principal cellular metabolites viz. NADP, uracil, asparagines, glutamine, and histidine and accumulation of di-methyl amine and beta-hydroxybutyrate, identified using (1)H NMR spectroscopy. These results suggest that PAH-quinones induce genotoxic effects by modulating the metabolic machinery inside the cells by a combined effect of oxidative stress and energy depletion. Our data for metabolic profiling of human lung cells could also help in understanding the mechanism of toxicity of other xenobiotics.

TAK1 (transforming growth factor-β-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.

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.

The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K(d) values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events.

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. Mol Cancer Res; 13(9); 1325–35. ©2015 AACR.

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.

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.

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 (p0.05) 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.

Metal oxide nanoparticles such as TiO2 (100 nm) are used in lotions and sunscreens. Toxicity studies so far have reported the use of TiO2 NPs with size100 nm as determined by dynamic light scattering (DLS) technique. We used non-reactive chemical agents such as propylene glycol (PG), glycerol (G), ethylene glycol (EG) to prevent aggregation and maintain NPs in monodispersed state closer to the reported TEM size. The lowest concentration of PG (0.1%) along with complete culture media (RPMI 1640) showed mean hydrodynamic diameter of TiO2 NPs as 33.71 nm measured by DLS. The present study demonstrates a method to prepare stable TiO2 NPs in culture media for toxicity assessment. This is a significant contribution as the biological properties of NPs vary considerably with decrease in size.

Background: Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is serine/threonine kinase belonging to mitogen-activated protein kinase kinase family that can promote tumor cell survival by modulation of the tumor microenvironment, mediation of stress responses and suppression of pro-apoptotic signaling. Moreover, TAK1 has been identified as essential for the survival of certain KRAS-dependent cancer cells and has therefore been studied as a therapeutic target. Several TAK1 inhibitors including LYTAK1, NG25 and fungal isolate 5Z-7-oxozeanol (5Z-7) have been reported, but these compounds are relatively non-selective as illustrated by 5Z-7 which inhibits numerous other kinases including PKD2, IKKα, Mnk2, Flt3, Flt4, KDR, Trk, PDGFRα, MKK4, NLK, MEK at modest concentrations. Furthermore, 5Z-7 is a resorcyclic lactone with a complex cyclic structure, making it difficult to synthesize derivatives that might have with better selectivity and potency. In an effort to develop new TAK1 inhibitors with excellent selectivity and synthetic accessibility, we studied a series of pyrimidine-based covalent inhibitors which target CYS-174, a cysteine adjacent to the ATP binding site of TAK1. Methods: Structure-guided compound evolution was used to guide design of compounds. Candidate covalent compounds were evaluated for relative binding affinity to TAK1, and the ability to covalently label recombinant TAK1 protein. Co-crystal x-ray structures of selected compounds were additionally solved to guide iterations of compound design. Compounds were evaluated for anti-proliferative activity in TAK1-dependent cell lines from 3 distinct cancer types. Results: Co-crystal structures of TAK1 in complex with the pyrrolopyrimidine-based compound CPT1691 showed an unexpected binding mode leading to the hypothesis that substitution of the pyrrolopyrimidine for a simpler pyrimidine would provide synthetically simpler routes to compounds which covalently bind to Cys-174. This substitution was tolerated, yielding potent TAK1-binding compounds which were further evolved to optimize differential selectivity between TAK1 and other highly related kinases such as MEK1, ERK2 and FLT3. Additional co-crystal structures of these compounds were solved. These inhibitors showed anti-proliferative activity in various TAK1-dependent colon cancer cells (LoVo, SW620, SK-CO-1), pancreatic cancer cells (PANC-1, AsPc-1 and Colo357FG cell lines) and renal cancer cells. Conclusions: Covalent pyrimidine-based TAK1 probes provide an effective means of TAK1 inhibition that may have value as therapeutic agents and scientific tools. Citation Format: Deepak Gurbani, Li Tan, Scott Ficarro, John C. Hunter, William Singer, Faviola B. Vazquez, Ting Xie, Sang Min Lim, Jarrod Marto, Nathanael S. Gray, Kenneth D. Westover. Structure guided development of irreversible inhibitors for TAK1. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A178.

As one of the first identified and most commonly activated oncogenes, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) has been a focus of cancer research for many decades. Despite progress in our understanding of RAS biology, significant questions remain unanswered regarding observed differences in clinical outcomes between tumors harboring different KRAS mutations. In an attempt to understand these clinical differences we characterized the pertinent biochemical properties of the most commonly observed KRAS mutants including, nucleotide exchange rate, intrinsic and GTPase activating protein (GAP)-stimulated GTP hydrolysis rate and affinity for one of the enzyme's primary downstream effectors, Raf kinase. We additionally solved high resolution crystal structures of four of these mutants and attempt to explain the observed biochemical differences between the mutants in the context of these structures. Based on the individual biochemical properties of each KRAS mutant, we propose a classification scheme to predict the propensity of the various mutants to activate Raf-kinase and respond to therapies targeting this downstream signaling pathway. Citation Format: John C. Hunter, Deepak Gurbani, Martin Carrasco, Anuj Manandhar, Sudershan Gondi, Kenneth Westover. Biochemical profiling of cancer-associated KRAS mutants: clues towards an understanding of differential clinical outcomes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-031. doi:10.1158/1538-7445.AM2015-LB-031

C60-fullerene has promising biological applications, such as drug delivery, biosensors, diagnosis and theraupetics. Despite of these applications, several in vitro studies have also reported the DNA damaging potential of this nanomaterial. Though, very little is known about the mechanism involved behind the fullerene mediated DNA damage. Our study was aimed at identifying the binding site of fullerene in the ATP binding domain of human topoisomerase II alpha, a major enzyme involved in maintaining DNA topology. In silico studies of fullerene with the enzyme demonstrated that it can interact with the active site residues of this enzyme through hydrophobic, pi-stacking and van der Waals interactions and could inhibit the activity of this enzyme.

Activating K-Ras mutations are common oncogenic events in many cancers but effective direct-acting inhibitors have not been developed. Targeting the nucleotide binding site of GTPases like K-Ras has long been dismissed as a possibility because of the high affinity of these proteins for their natural ligands GTP and GDP. We previously reported the synthesis of a GDP analogue, SML-8-73-1, and a prodrug derivative, SML-10-70-1, which are selective, direct-acting covalent inhibitors of the K-Ras G12C mutant relative to wild-type Ras. Biochemical and biophysical measurements suggested that modification of K-Ras with SML-8-73-1 renders the protein in an inactive state. Here we report a crystal structure of K-Ras G12C labeled with SML-8-73-1. The structure reveals that when bound to SML-8-73-1, K-Ras presents in an inactive conformation. This first-in-class covalent K-Ras inhibitor suggests that irreversible targeting of the K-Ras guanine-nucleotide binding site is a viable therapeutic strategy for inhibition of Ras signaling. This abstract is also presented as Poster B54. Citation Format: John Hunter, Sang Min Lim, Scott B. Ficcarro, Hwan Geun Choi, Deepak Gurbani, Ting Xie, Jarrod A. Marto, Nathanael S. Gray, Ken Westover. Crystal structure of K-Ras G12C bound to an active site inhibitor. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr PR07. doi: 10.1158/1557-3125.RASONC14-PR07