Anthony S. Fischl

Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) catalyzes the formation of holo‐ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3′5′‐ADP, a product of AcpS, at 2.0 and 1.9 Å resolution, respectively. The crystal structure reveals an α/β fold and shows that AcpS assembles as a tightly packed functional trimer, with a non‐crystallographic pseudo‐symmetric 3‐fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure‐based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.

Biochemical and Molecular Analyses of the Streptococcus pneumoniae Acyl Carrier Protein Synthase, an Enzyme Essential for Fatty Acid Biosynthesis

Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4′-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To further understand the physiological role of AcpS, we identified, cloned, and expressed the acpS and acpP genes ofStreptococcus pneumoniae and purified both products to homogeneity. Both acpS and acpP form operons with the genes whose functions are required for other cellular metabolism. The acpS gene complements an Escherichia coli mutant defective in the production of AcpS and appears to be essential for the growth of S. pneumoniae. Gel filtration and cross-linking analyses establish that purified AcpS exists as a homotrimer. AcpS activity was significantly stimulated by apo-ACP at concentrations over 10 μm and slightly inhibited at concentrations of 5–10 μm. Double reciprocal analysis of initial velocities of AcpS at various concentrations of CoA or apo-ACP indicated a random or compulsory ordered bi bi type of reaction mechanism. Further analysis of the inhibition kinetics of the product (3′,5′-ADP) suggested that it is competitive with respect to CoA but mixed (competitive and noncompetitive) with respect to apo-ACP. Finally, apo-ACP bound tightly to AcpS in the absence of CoA, but CoA failed to do so in the absence of apo-ACP. Together, these results suggest that AcpS may be allosterically regulated by apo-ACP and probably proceeds by an ordered reaction mechanism with the first formation of the AcpS-apo-ACP complex and the subsequent transfer of 4′-phosphopantetheine to the apo-ACP of the complex.

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A(2) on the Caliper 250 HTS system: throughput of 80min per 384-well plate can be achieved with unattended running time of 5.4h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

INOSITOLPHOSPHORYL CERAMIDE SYNTHASE FROM YEAST

Publisher Summary Inositolphosphoryl ceramide (IPC) synthase catalyzes the transfer of inositol phosphate from phosphatidylinositol (PI) to ceramide forming diacylglycerol and IPC, a major yeast sphingolipid and precursor of the yeast sphingolipids, mannosylinositolphosphoryl ceramide and mannosyldiinositolphosphoryl ceramide. The enzyme, encoded by the yeast AUR1 gene, and its product, IPC, are essential to the growth and viability of Saccharornyces cerevisiae. IPC synthase activity is associated with the microsomal membrane fraction of yeast, and the expression of IPC synthase activity is regulated by inositol and the growth phase of S. cerevisiae . Methods to measure IPC synthase activity from yeast microsomal membranes and Triton X-100-solubilized yeast microsomes have been reported. This chapter describes the purification and properties of the enzyme. The overall purification of IPC synthase over the cell extract is 3425-fold with an activity yield of 23%. Examination of the purified enzyme by sodium dodecyl sulfate gel electrophoresis reveals four major protein bands, suggesting that the enzyme is highly purified but not to homogeneity. The four major protein bands have minimum subunit molecular weights of 97,000, 66,000, 50,000, and 46,000.

Stromal-Based Signatures for the Classification of Gastric Cancer

Treatment of metastatic gastric cancer typically involves chemotherapy and monoclonal antibodies targeting HER2 (ERBB2) and VEGFR2 (KDR). However, reliable methods to identify patients who would benefit most from a combination of treatment modalities targeting the tumor stroma, including new immunotherapy approaches, are still lacking. Therefore, we integrated a mouse model of stromal activation and gastric cancer genomic information to identify gene expression signatures that may inform treatment strategies. We generated a mouse model in which VEGF-A is expressed via adenovirus, enabling a stromal response marked by immune infiltration and angiogenesis at the injection site, and identified distinct stromal gene expression signatures. With these data, we designed multiplexed IHC assays that were applied to human primary gastric tumors and classified each tumor to a dominant stromal phenotype representative of the vascular and immune diversity found in gastric cancer. We also refined the stromal gene signatures and explored their relation to the dominant patient phenotypes identified by recent large-scale studies of gastric cancer genomics (The Cancer Genome Atlas and Asian Cancer Research Group), revealing four distinct stromal phenotypes. Collectively, these findings suggest that a genomics-based systems approach focused on the tumor stroma can be used to discover putative predictive biomarkers of treatment response, especially to antiangiogenesis agents and immunotherapy, thus offering an opportunity to improve patient stratification. Cancer Res; 76(9); 2573-86. ©2016 AACR.

Reproducibility of total choline/water ratios in mouse U87MG xenograft tumors by 1H‐MRS

To evaluate the reproducibility of the measurement of the total choline‐to‐water ratio, and the effect of repositioning the subject between scans, using 1H‐magnetic resonance spectroscopy in a mouse U87MG xenograft model.

Direct analysis of PI(3,4,5)P3 using liquid chromatography electrospray ionization tandem mass spectrometry

Phosphatidylinositol (3,4,5) trisphosphate (PIP3) is a biologically active membrane phospholipid that is essential for the growth and survival of all eukaryotic cells. We describe a new method that directly measures PIP3 and describe the HPLC separation and measurement of the positional isomers of phosphatidylinositol bisphosphate, PI(3,5)P2, PI(3,4)P2 and PI(4,5)P2. Mass spectrometric analyses were performed online using ultra-high performance liquid chromatography (UHPLC)-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the negative multiple-reaction monitoring (MRM) modes. Rapid separation of PIP3 from PI, phosphatidylinositol phosphate (PIP) and PIP2 was accomplished by C18 reverse phase chromatography with the addition of the ion pairing reagents diisopropylethanolamine (DiiPEA) and ethylenediamine tetraacetic acid tetrasodium salt dihydrate (EDTA) to the samples and mobile phase with a total run time, including equilibration, of 12 min. Importantly, these chromatography conditions result in no carryover of PIP, PIP2, and PIP3 between samples. To validate the new method, U87MG cancer cells were serum starved and treated with PDGF to stimulate PIP3 biosynthesis in the presence or absence of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. Results generated with the LC/MS method were in excellent agreement with results generated using [33P] phosphate radiolabeled U87MG cells and anion exchange chromatography analysis, a well validated method for measuring PIP3. To demonstrate the usefulness of the new method, we generated reproducible IC50 data for several well-characterized PI3K small molecule inhibitors using a U87MG cell-based assay as well as showing PIP3 can be measured from additional cancer cell lines. Together, our results demonstrate this novel method is sensitive, reproducible and can be used to directly measure PIP3 without radiolabeling or complex lipid derivatization.

Abstract 1823: Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor

Merestinib (LY2801653) is an orally bioavailable small molecule inhibitor of several oncokinases, including MET, AXL, DDR1/2, MERTK, ROS1, Tie2 (aka TEK), and MKNK1/2. Merestinib has been extensively characterized in a wide range of preclinical tumor xenograft models and shown to potently inhibit MET driven and non-MET driven tumor growth. In addition to its direct antitumor activity, merestinib inhibits angiogenesis and induces a tumor vessel normalization phenotype in xenograft tumors 1 . While MET signaling is important for angiogenesis, the effect of merestinib on angiogenesis is likely not exclusively driven by MET inhibition. In co-culture angiogenesis assays, merestinib inhibited VEGF-dependent and VEGF-independent endothelial cell cord formation 2,3 and sprouting 4 with potencies in the low nM range (3-30 nM). In contrast, the MET-specific kinase inhibitor, PF04217903, only weakly inhibited cord formation and endothelial sprouting. In an established in vivo matrigel co-implant vasculogenesis model where VEGFR2 or MET selective inhibition had minimal effect, merestinib decreased vascular density by 69%. In addition, while MET antibody emibetuzumab (human anti-MET antibody) plus ramucirumab (human anti-VEGFR2 antibody) decreased vascular density by 64%, merestinib plus ramucirumab decreased it by 92%. In a mouse adenovirus-driven VEGF-A ear angiogenesis model 5 , treatment with DC101, a mouse anti-VEGFR2 antibody, or merestinib inhibited angiogenesis; however the combination of DC101 and merestinib appeared to inhibit it even more. Finally, in the MKN45 gastric tumor xenograft model, merestinib (T/C = 4.8%) and DC101 (T/C = 15.3%) each significantly inhibited tumor growth alone and the combination resulted in 27.6% tumor regression and was significantly better than either single agent alone. Together, these studies indicate that merestinib has greater effects on angiogenesis than selective MET inhibition and its actions are not dependent on VEGFR2. In addition, while in vitro studies show reductions in VEGFR2 phosphorylation with high concentration of merestinib, treatment with merestinib did not inhibit VEGF dependent phosphorylation of VEGFR2 in mouse lung tissue at clinically relevant exposures. These data suggest that the anti-angiogenic activity of merestinib includes activities of other kinases targeted by merestinib. These data provide rationale and support for the clinical evaluation of combination of merestinib with ramucirumab (NCT02745769). 1 -Yan et. al. Invest New Drugs. 2013;31:833-844, 2 - Falcon et. al. J Hematol Oncol. 2013;6:31, 3 - Falcon et. al. PLoS ONE. 2014;9:e106901, 4 - Nakatsu et. al. Methods Enzymol. 2008;433:65-82, 5 - Nagy et. al. Methods Enzymol. 2008;444:43-64. Citation Format: Diane M. Bodenmiller, Julie A. Stewart, Glenn F. Evans, Victoria L. Peek, Jennifer R. Stephens, Xi Lin, Seema Iyer, Beverly L. Falcon, Sudhakar Chintharlapalli, Sau-Chi Betty Yan, Anthony S. Fischl. Characterization of the anti-angiogenic properties of merestinib (LY2801653), an oncokinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1823. doi:10.1158/1538-7445.AM2017-1823

Abstract 930: Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth

Lung cancer is the leading cause of cancer death worldwide. MAPK activation via KRAS mutation is present in up to 30% of lung cancer patients. NSCLC patients with KRAS mutation is associated with poor prognosis and represents an unmet medical need. LY3009120, a pan-RAF and RAF dimer inhibitor which is in phase I clinical trial, was previously demonstrated to have anti-tumor activities in BRAF or RAS mutant tumor cells in vitro and in vivo. Ramucirumab, a fully-human antagonist monoclonal antibody to human VEGFR-2 was recently approved as an anti-angiogenic treatment for several cancer indications including second-line NSCLC. Combination strategies in cancer including targeting both tumor cells and the surrounding stroma cells have been shown to be effective in various disease subtypes. In this study, the combination effect of LSN3074753 (a surrogate and an analogue of LY3009120) with VEGFR-2 inhibitor DC101 (a monoclonal antibody specific for murine VEGFR-2 and a surrogate for ramucirumab) were evaluated in KRAS mutant NSCLC models, including NCI-H2122 (G-12C), A549 (G-12S) and NCI-H441 (G-12V). LSN3074753 treatment alone resulted in 66.9% and 82.4% tumor growth inhibition in H2122 and A549 xenograft tumors, respectively; and 41.4% tumor regression in H441 xenograft tumors. DC101 treatment alone resulted in 64.5%, 75% and 102.2% tumor growth inhibition in H2122, A549 and H441, respectively. The combination of LSN3074753 and DC101 led to more significant tumor growth inhibition (87.2% of tumor growth inhibition for H2122, p Citation Format: Wenjuan Wu, Julie Stewart, Constance King, Bonita Jones, Robert Flack, Susan Pratt, Randi Berryman, Michelle Swearingen, Diane Bodenmiller, Xi Lin, Mark Uhlik, Beverly Falcon, Anthony Fischl, Jason Manro, Ramon Tiu, Sudhakar Chintharlapalli, Bronislaw Pytowski, Shripad V. Bhagwat, Sean Buchanan, Sheng-Bin Peng. Combined inhibition of pan-RAF and VEGFR-2 mediates antitumor activity in KRAS mutant non-small cell lung cancer (NSCLC) through enhanced inhibition of tumor angiogenesis and growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 930.

Abstract B117: Early detection of a metabolic tumor response to p70S6 kinase/AKT inhibition by LYS6KAKT1 using in vivo choline proton magnetic resonance spectroscopy.

As part of a comprehensive drug discovery platform aimed at targeting the PI3K pathway, we have developed a potent small molecule dual inhibitor of p70 S6 kinase and AKT targeting two key nodes of the pathway. LYS6KAKT1 is a potent, highly selective ATP competitive inhibitor against p70S6 kinase and AKT with an IC50 of 6 nM and 14 nM respectively. LYS6KAKT1 inhibits phosphorylation of S6 ribosomal protein and phosphorylation of GSK3b in U87MG glioblastoma cells as well as phosphorylation of other downstream AKT substrates such as PRAS40 and FOXO (Geeganage et al, Abstract 62, 22nd Molecular Targets and Cancer Therapeutics, 16–19 November 2010). In vivo, LYS6KAKT1 demonstrate potent phospho-S6 inhibition in nude mice bearing glioblastoma cells, with an ED50 value of 3 mg/kg and an ED90 value of 8 mg/kg 4 hours after a single oral dose. Based on pre-clinical observations, LYS6KAKT1 is currently being evaluated in Phase I clinical studies for cancer. The purpose of this study was to investigate the effect of LYS6KAKT1 on tumor choline metabolites, a potential tumor response biomarker for PI3K pathway inhibition. Proton Magnetic Resonance Spectroscopy ([1H] MRS) was used to monitor the changes in tumor choline metabolites following treatment of U87MG xenografts in athymic nude mice. LYS6KAKT1 (12.5 or 25 mg/kg, PO, BID) significantly reduced total choline tumor metabolites after 7-days of dosing (p = 0.0006, n = 36). The total tumor choline metabolites, after 7 days of dosing, correlated significantly with relative changes in tumor volumes (r = 0.68, p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B117.