Rima Al-Awar

SickKids, Toronto, Ontario, Canada

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Publications (19)133.76 Total impact

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    ABSTRACT: Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.
    Nature Communications 08/2014; 5:4202. · 10.74 Impact Factor
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    ABSTRACT: SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.
    Proceedings of the National Academy of Sciences 08/2014; · 9.81 Impact Factor
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    ABSTRACT: Glioblastoma (GBM) is a devastating disease and the most lethal of adult brain tumours. Despite standard surgery, radiation and chemotherapy, the median survival is 15 months, thought to be due, in part, to recurrence from a small reservoir of brain tumour initiating cells. Our laboratory discovered adult neural stem cells, now found to be present in the brains of all adult mammals, through the development of the clonal neurosphere assay. This assay has contributed to the identification of adult human brain tumour initiating cells (BTICs), which may represent a reservoir that leads to GBM recurrence and death. Building upon the identification of growth factors and cytokines that converge on the cytoplasmic signal transducer and activator of transcription 3 (STAT3) to maintain the adult neural stem cell undifferentiated state, and the fact that STAT3 is abnormally active in GBM and may be one of the causes of tumour growth and therapeutic resistance, targeting the janus kinase (JAK)/STAT3 signalling pathway has become a major research focus for our laboratory. Here, we report the GBM translational potential of R333, a JAK/SYK inhibitor in development for other indications by Rigel Pharmaceuticals.
    Neuro-oncology. 07/2014; 16 Suppl 3:iii35.
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    ABSTRACT: "Therapeutic Targeting of Glioblastoma" is a new pan-Canadian research team of the Terry Fox Research Institute and the Canadian Stem Cell Network funded to discover efficacious therapeutics for GBM. The team's goals are also to discover novel signaling pathways regulating GBM cell survival and genetic alterations that mediate drug resistance. As a platform, we use our collection of over 100 primary GBM tumor-initiating lines (BTIC) that are subjected to drug screening by over 1400 compounds, and to genetic and phosphoproteomic analysis.
    Neuro-oncology. 07/2014; 16 Suppl 3:iii20.
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    ABSTRACT: HER2+ breast cancer (BC) is currently treated with chemotherapy plus anti-HER2 inhibitors. Many patients do not respond or relapse with aggressive metastatic disease. As a result, there is an urgent need for new therapeutics that can target HER2+ BC and potentiate the effect of anti-HER2 inhibitors, in particular those that can target tumor-initiating cells (TICs). Here we show that MMTV-Her2/Neu mammary tumor cells cultured as non-adherent spheres or as adherent monolayer cells select for stabilizing mutations in p53 that "immortalize" the cultures, and that after serial passages, sphere conditions maintain TICs whereas monolayer cells gradually lose these tumorigenic cells. Using tumorsphere formation as surrogate for TICs, we screened p53-mutant Her2/Neu+ tumorsphere versus monolayer cells with a lenti-virus shRNA kinome library. We identified kinases such as MAPK and TGFβR protein family, previously implicated in HER2+ BC, as well as autophagy factor Atg1/Ulk1 and the non-canonical IκB kinase, TBK1, which have not been previously linked to HER2+ BC. Knock-down of TBK1 or pharmacological inhibition of TBK1 and the related protein, IKKε, suppressed growth of both mouse and human HER2+ BC cells. TBK1/IKKε inhibition promoted cellular senescence by suppressing p65-NFκB and inducing p16Ink4a. In addition, TBK1/IKKε inhibition cooperated with lapatinib, a HER2/EGFR1 targeted drug, to accelerate apoptosis and kill HER2+ BC cells both in culture and in xenografts. Our results suggest that patients with HER2+ BC may benefit from anti-TBK1/IKKε plus anti-HER2 combination therapies, and establish conditions that can be used to screen for additional TIC-specific inhibitors of HER2+ BC.
    Cancer Research 01/2014; · 9.28 Impact Factor
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    ABSTRACT: The STAT3 gene is abnormally active in glioblastoma and is a critically important mediator of tumour growth and therapeutic resistance in GBM. Thus, for poorly treated brain cancers such as gliomas, astrocytomas and glioblastomas, which harbor constitutively activated STAT3, a STAT3-targeting therapeutic will be of significant im-portance. Herein, we report a most potent, small molecule, non-phosphorylated STAT3 inhibitor, 31 that displayed potent STAT3 binding affinity (KD = 300 nM). Inhibitor 31 potently kills glioblastoma brain cancer stem cells, effec-tively suppresses STAT3 phosphorylation and downstream transcriptional targets at low nM concentrations. In vivo studies with 31 in mice orthotopically xenografted with glioma and analyzed by immunohistochemical staining demonstrated that 31 exhibited blood brain barrier permeability, in vivo potency, on target anti-STAT3 activity and potent inhibition of tumour cell proliferation and increased apoptosis. This work demonstrates the clinical efficacy of a STAT3 inhibitor for clinical application in glioblastoma.
    ACS Medicinal Chemistry Letters 09/2013; · 3.31 Impact Factor
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    ABSTRACT: The reaction of 3-halo-4-aminopyridines with acyl chlorides and triethylamine is described. The pyridin-4-yl α-substituted acetamide products were obtained in moderate to high yields. The presented rearrangement reaction, in which the presumed N-acylated intermediate reacts intramolecularly via nucleophilic aromatic substitution, results in a formal two-carbon insertion.
    The Journal of Organic Chemistry 05/2013; · 4.56 Impact Factor
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    ABSTRACT: The WD40-repeat protein WDR5 plays a critical role in maintaining the integrity of MLL complexes and fully activating their methyltransferase function. MLL complexes, the trithorax-like family of SET1 methyltransferases, catalyze trimethylation of lysine 4 on histone 3, and they have been widely implicated in various cancers. Antagonism of WDR5 and MLL subunit interaction by small molecules has recently been presented as a practical way to inhibit activity of the MLL1 complex, and N-(2-(4-methylpiperazin-1-yl)-5-substituted-phenyl) benzamides were reported as potent and selective antagonists of such an interaction. Here, we describe the protein crystal structure guided optimization of prototypic compound 2 (K dis = 7 μM), leading to identification of more potent antagonist 47 (K dis = 0.3 μM).
    ACS Medicinal Chemistry Letters 03/2013; 4(3):353-7. · 3.31 Impact Factor
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    ABSTRACT: Chemical inhibition of proteins involved in chromatin-mediated signaling is an emerging strategy to control chromatin compaction with the aim to reprogram expression networks to alter disease states. Protein methyltransferases constitute one of the protein families that participate in epigenetic control of gene expression, and represent a novel therapeutic target class. Recruitment of the protein lysine methyltransferase DOT1L at aberrant loci is a frequent mechanism driving acute lymphoid and myeloid leukemias, particularly in infants, and pharmacological inhibition of DOT1L extends survival in a mouse model of mixed lineage leukemia. A better understanding of the structural chemistry of DOT1L inhibition would accelerate the development of improved compounds. Here, we report that the addition of a single halogen atom at a critical position in the cofactor product S-adenosylhomocysteine (SAH, an inhibitor of SAM-dependent methyltransferases) results in an 8-fold increase in potency against DOT1L, and reduced activities against other protein and non-protein methyltransferases. We solved the crystal structure of DOT1L in complex with Bromo-deaza-SAH and rationalized the observed effects. This discovery reveals a simple strategy to engineer selectivity and potency towards DOT1L into the adenosine scaffold of the cofactor shared by all methyltransferases, and can be exploited towards the development of clinical candidates against mixed lineage leukemia.
    Bioorganic & medicinal chemistry 01/2013; · 2.82 Impact Factor
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    ABSTRACT: Selective inhibition of protein methyltransferases is a promising new approach to drug discovery. An attractive strategy towards this goal is the development of compounds that selectively inhibit binding of the cofactor, S-adenosylmethionine, within specific protein methyltransferases. Here we report the three-dimensional structure of the protein methyltransferase DOT1L bound to EPZ004777, the first S-adenosylmethionine-competitive inhibitor of a protein methyltransferase with in vivo efficacy. This structure and those of four new analogues reveal remodelling of the catalytic site. EPZ004777 and a brominated analogue, SGC0946, inhibit DOT1L in vitro and selectively kill mixed lineage leukaemia cells, in which DOT1L is aberrantly localized via interaction with an oncogenic MLL fusion protein. These data provide important new insight into mechanisms of cell-active S-adenosylmethionine-competitive protein methyltransferase inhibitors, and establish a foundation for the further development of drug-like inhibitors of DOT1L for cancer therapy.
    Nature Communications 12/2012; 3:1288. · 10.74 Impact Factor
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    ABSTRACT: The WD40 protein, WDR5, is an essential component of the human trithorax-like family of SET1 methyltransferase complexes that carry out trimethylation of histone 3 lysine 4 (H3K4me3), play key roles in development, and are abnormally expressed in many cancers. Here we show that the interaction between WDR5 and peptides from the catalytic domain of MLL (KMT2) can be antagonized with a small molecule. Structural and biophysical analysis show that this antagonist binds in the WDR5 peptide binding pocket with a KD of 450 nM, and inhibits the catalytic activity of the MLL core complex in vitro. The degree of inhibition was enhanced at lower protein concentrations consistent with a role for WDR5 in directly stabilizing the MLL multiprotein complex. Our data demonstrate inhibition of an important protein-protein interaction and form the basis for further development of inhibitors of WDR5-dependent enzymes implicated in MLL-rearranged leukemias or other cancers.
    Biochemical Journal 09/2012; · 4.65 Impact Factor
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    ABSTRACT: PRMT3, a protein arginine methyltransferase, has been shown to influence ribosomal biosynthesis by catalyzing the dimethylation of the 40S ribosomal protein S2. Although PRMT3 has been reported to be a cytosolic protein, it has been shown to methylate histone H4 peptide (H4 1-24) in vitro. Here, we report the identification of a PRMT3 inhibitor (1-(benzo[d][1,2,3]thiadiazol-6-yl)-3-(2-cyclohexenylethyl)urea; compound 1) with IC50 value of 2.5 μM by screening a library of 16,000 compounds using H4 (1-24) peptide as a substrate. The crystal structure of PRMT3 in complex with compound 1 as well as kinetic analysis reveals an allosteric mechanism of inhibition. Mutating PRMT3 residues within the allosteric site or using compound 1 analogs that disrupt interactions with allosteric site residues both abrogated binding and inhibitory activity. These data demonstrate an allosteric mechanism for inhibition of protein arginine methyltransferases, an emerging class of therapeutic targets.
    Structure 07/2012; 20(8):1425-35. · 5.99 Impact Factor
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    ABSTRACT: The most common mutation in cystic fibrosis (CF) is a deletion of Phe at position 508 (ΔF508-CFTR). ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma membrane. To identify compounds and drugs that rescue this trafficking defect, we screened a kinase inhibitor library enriched for small molecules already in the clinic or in clinical trials for the treatment of cancer and inflammation, using our recently developed high-content screen technology (Trzcinska-Daneluti et al. Mol. Cell. Proteomics 8:780, 2009). The top hits of the screen were further validated by (1) biochemical analysis to demonstrate the presence of mature (Band C) ΔF508-CFTR, (2) flow cytometry to reveal the presence of ΔF508-CFTR at the cell surface, (3) short-circuit current (Isc) analysis in Ussing chambers to show restoration of function of the rescued ΔF508-CFTR in epithelial MDCK cells stably expressing this mutant (including EC(50) determinations), and importantly (4) Isc analysis of Human Bronchial Epithelial (HBE) cells harvested from homozygote ΔF508-CFTR transplant patients. Interestingly, several inhibitors of receptor Tyr kinases (RTKs), such as SU5402 and SU6668 (which target FGFRs, VEGFR, and PDGFR) exhibited strong rescue of ΔF508-CFTR, as did several inhibitors of the Ras/Raf/MEK/ERK or p38 pathways (e.g. (5Z)-7-oxozeaenol). Prominent rescue was also observed by inhibitors of GSK-3β (e.g. GSK-3β Inhibitor II and Kenpaullone). These results identify several kinase inhibitors that can rescue ΔF508-CFTR to various degrees, and suggest that use of compounds or drugs already in the clinic or in clinical trials for other diseases can expedite delivery of treatment for CF patients.
    Molecular &amp Cellular Proteomics 06/2012; 11(9):745-57. · 7.25 Impact Factor
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    ABSTRACT: The fusion of mononucleated muscle progenitor cells (myoblasts) into multinucleated muscle fibers is a critical aspect of muscle development and regeneration. We identified the noncanonical nuclear factor κB (NF-κB) pathway as a signaling axis that drives the recruitment of myoblasts into new muscle fibers. Loss of cellular inhibitor of apoptosis 1 (cIAP1) protein led to constitutive activation of the noncanonical NF-κB pathway and an increase in the number of nuclei per myotube. Knockdown of essential mediators of NF-κB signaling, such as p100, RelB, inhibitor of κB kinase α, and NF-κB-inducing kinase, attenuated myoblast fusion in wild-type myoblasts. In contrast, the extent of myoblast fusion was increased when the activity of the noncanonical NF-κB pathway was enhanced by increasing the abundance of p52 and RelB or decreasing the abundance of tumor necrosis factor (TNF) receptor-associated factor 3, an inhibitor of this pathway. Low concentrations of the cytokine TNF-like weak inducer of apoptosis (TWEAK), which preferentially activates the noncanonical NF-κB pathway, also increased myoblast fusion, without causing atrophy or impairing myogenesis. These results identify roles for TWEAK, cIAP1, and noncanonical NF-κB signaling in the regulation of myoblast fusion and highlight a role for cytokine signaling during adult skeletal myogenesis.
    Science Signaling 01/2012; 5(246):ra75. · 7.65 Impact Factor
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    ABSTRACT: Excessive signaling from the Wnt pathway is associated with numerous human cancers. Using a high throughput screen designed to detect inhibitors of Wnt/β-catenin signaling, we identified a series of acyl hydrazones that act downstream of the β-catenin destruction complex to inhibit both Wnt-induced and cancer-associated constitutive Wnt signaling via destabilization of β-catenin. We found that these acyl hydrazones bind iron in vitro and in intact cells and that chelating activity is required to abrogate Wnt signaling and block the growth of colorectal cancer cell lines with constitutive Wnt signaling. In addition, we found that multiple iron chelators, desferrioxamine, deferasirox, and ciclopirox olamine similarly blocked Wnt signaling and cell growth. Moreover, in patients with AML administered ciclopirox olamine, we observed decreased expression of the Wnt target gene AXIN2 in leukemic cells. The novel class of acyl hydrazones would thus be prime candidates for further development as chemotherapeutic agents. Taken together, our results reveal a critical requirement for iron in Wnt signaling and they show that iron chelation serves as an effective mechanism to inhibit Wnt signaling in humans.
    Cancer Research 12/2011; 71(24):7628-39. · 9.28 Impact Factor
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    ABSTRACT: Gene regulatory networks that govern hematopoietic stem cells (HSCs) and leukemia-initiating cells (L-ICs) are deeply entangled. Thus, the discovery of compounds that target L-ICs while sparing HSC is an attractive but difficult endeavor. Presently, most screening approaches fail to counter-screen compounds against normal hematopoietic stem/progenitor cells (HSPCs). Here, we present a multistep in vitro and in vivo approach to identify compounds that can target L-ICs in acute myeloid leukemia (AML). A high-throughput screen of 4000 compounds on novel leukemia cell lines derived from human experimental leukemogenesis models yielded 80 hits, of which 10 were less toxic to HSPC. We characterized a single compound, kinetin riboside (KR), on AML L-ICs and HSPCs. KR demonstrated comparable efficacy to standard therapies against blast cells in 63 primary leukemias. In vitro, KR targeted the L-IC-enriched CD34(+)CD38(-) AML fraction, while sparing HSPC-enriched fractions, although these effects were mitigated on HSC assayed in vivo. KR eliminated L-ICs in 2 of 4 primary AML samples when assayed in vivo and highlights the importance of in vivo L-IC and HSC assays to measure function. Overall, we provide a novel approach to screen large drug libraries for the discovery of anti-L-IC compounds for human leukemias.
    Blood 12/2011; 119(5):1200-7. · 9.78 Impact Factor
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    ABSTRACT: To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.
    Cancer cell 10/2011; 20(4):443-56. · 25.29 Impact Factor
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    ABSTRACT: Neuroblastoma (NB) is an often fatal pediatric tumor of neural crest origin. We previously isolated NB tumor-initiating cells (NB TIC) from bone marrow metastases that resemble cancer stem cells and form metastatic NB in immunodeficient animals with as few as ten cells. To identify signaling pathways important for the survival and self-renewal of NB TICs and potential therapeutic targets, we screened a small molecule library of 143 protein kinase inhibitors, including 33 in clinical trials. Cytostatic or cytotoxic drugs were identified that targeted PI3K (phosphoinositide 3-kinase)/Akt, PKC (protein kinase C), Aurora, ErbB2, Trk, and Polo-like kinase 1 (PLK1). Treatment with PLK1 siRNA or low nanomolar concentrations of BI 2536 or BI 6727, PLK1 inhibitors in clinical trials for adult malignancies, were cytotoxic to TICs whereas only micromolar concentrations of the inhibitors were cytotoxic for normal pediatric neural stem cells. Furthermore, BI 2536 significantly inhibited TIC tumor growth in a therapeutic xenograft model, both as a single agent and in combination with irinotecan, an active agent for relapsed NB. Our findings identify candidate kinases that regulate TIC growth and survival and suggest that PLK1 inhibitors are an attractive candidate therapy for metastatic NB.
    Cancer Research 02/2011; 71(4):1385-95. · 9.28 Impact Factor
  • Barry Press, Rima Al-Awar, Ahmed Aman
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    ABSTRACT: Incorporating higher throughput analytical methods and enhancing the ability for data mining of in vitro ADME samples can greatly benefit drug discovery teams during lead optimization. Standard bioanalytical techniques generally rely on triple quadrupole mass spectrometers using the technique of multiple reaction monitoring (MRM) for quantification (QUAN). Although this conventional MRM approach can provide high specificity and sensitivity, this practice is also time-consuming since it is a two step process and requires the optimization of multiple parameters (cone voltage, collision energy, and Q1/Q3 m/z values) for each and every compound being analyzed. Furthermore, MRM techniques only detect the initial compound of interest and data mining for other, qualitative (QUAL) information (such as metabolite formation) is not possible. The use of a sensitive hybrid Q-Tof instrument in a full scan acquisition mode and subsequent narrow window mass eliminates the need for compound optimization while also providing both qualitative and quantitative (QUAL/QUAN) data to assist chemistry efforts in structure-property-relationships (SPR). The Xevo-Q-TOF mass spectrometer is a high resolution accurate mass instrument which possesses the speed, sensitivity, linearity, and wide mass range needed to screen in vitro ADME samples. The high resolution capabilities of Q-TOF-MS, combined with mass defect filtering and monitoring of isotopic peak patterns, allows for the discrimination against background noise when performing narrow window mass extraction from a full scan analysis. Samples were generated from multiple ADME assays including cellular (Caco-2) permeability, microsomal and hepatocyte stability, plasma stability, protein binding, and CYP450 inhibition of (known) metabolite formation. Chromatographic separations were obtained utilizing an Acquity UPLC and generic gradient conditions. Quantitative results obtained by narrow m/z windows were compared with traditional MRM analysis of the same samples analyzed using the high-sensitivity QTRAP 5500 system.. Data mining was then performed on the full scan analysis to identify known metabolites such as glucuronide, de-methylated, and hydroxylated metabolites from metabolic assays, as well as to determine possible non-hepatic compound instability and isomerization from in vitro plasma stability and permeability samples. Results indicate that the use of the Xevo-Q-TOF, combined with narrow window mass extraction and full scan analysis, gives comparable quantitative data (e.g. percent loss of parent compound) to conventional MRM techniques without the need for time-consuming compound optimization of new chemical entities (NCEs) or known CYP-dependent metabolites. In addition, valuable qualitative information could be obtained only from Xevo-Q-TOF full scans showing the susceptibility of reference compounds and NCEs for Phase I oxidative metabolism, Phase II conjugation, and non-hepatic, compound instability.
    17th North American Regional International society for the study of xenobiotics Meeting;

Publication Stats

139 Citations
133.76 Total Impact Points

Institutions

  • 2014
    • SickKids
      Toronto, Ontario, Canada
    • The University of Calgary
      • Hotchkiss Brain Institute
      Calgary, Alberta, Canada
    • University of Toronto
      Toronto, Ontario, Canada
  • 2013
    • Ontario Institute for Cancer Research
      Toronto, Ontario, Canada