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PDF - 182 KB, Table S1. Kinase selectivity profile for LY3023414 in Cerep and Life Technologies assays based on a 3-point concentration response curve; Table S2. In vitro inhibitory activity of LY3023414 against kinases in a PC3 cell lysate measured in the KiNativ assay based on a 3-point concentration response curve; Table S3. In vitro cell-based target inhibition in SKOV3, A2780, and AsPC-1; Table S4. Summary of pathway-relevant genetic alterations in cancer cell line panel and in selected patient-derived xenograft tumor models tested; Table S5. Activated caspase-3/7 levels in cell lines treated with 2.5 μM LY3023414 for 48 hours; Table S6. In vitro combination studies with LY3023414 and standard of care agents in various cancer cell lines; Table S7. Time course of in vivo target inhibition in U87 MG xenograft tumors after oral administration of 15 and 30 mg/kg LY3023414; Table S8. Summary of LY3023414 monotherapy efficacy in patient-derived xenograft tumor models; Table S9. Summary of LY3023414 combination efficacy in patient-derived xenograft squamous non-small cell lung cancer (NSCLC) tumor models.

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LY573636-sodium (tasisulam) is a small molecule antitumor agent with a novel mechanism of action currently being investigated in a variety of human cancers. In vitro, tasisulam induced apoptosis via the intrinsic pathway, resulting in cytochrome c release and caspase-dependent cell death. Using high content cellular imaging and subpopulation analysis of a wide range of in vitro and in vivo cancer models, tasisulam increased the proportion of cells with 4N DNA content and phospho-histone H3 expression, leading to G2–M accumulation and subsequent apoptosis. Tasisulam also blocked VEGF, epidermal growth factor, and fibroblast growth factor–induced endothelial cell cord formation but did not block acute growth factor receptor signaling (unlike sunitinib, which blocks VEGF-driven angiogenesis at the receptor kinase level) or induce apoptosis in primary endothelial cells. Importantly, in vivo phenocopying of in vitro effects were observed in multiple human tumor xenografts. Tasisulam was as effective as sunitinib at inhibiting neovascularization in a Matrigel plug angiogenesis assay in vivo and also caused reversible, non G2–M–dependent growth arrest in primary endothelial cells. Tasisulam also induced vascular normalization in vivo. Interestingly, the combination of tasisulam and sunitinib significantly delayed growth of the Caki-1 renal cell carcinoma model, whereas neither agent was active alone. These data show that tasisulam has a unique, dual-faceted mechanism of action involving mitotic catastrophe and antiangiogenesis, a phenotype distinct from conventional chemotherapies and published anticancer agents. Mol Cancer Ther; 10(11); 2168–78. ©2011 AACR.

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PDF - 564 KB, Figure S1. Duodenal compound concentration profile of multiple compounds from a gastroduodenal passage simulation model; Figure S2. Kinetics of LY3023414 inhibition of PI3Kα; Figure S3. LY3023414 inhibition of mTORC1 in a concentration-dependent manner after ex vivo treatment of PMBC or whole blood; Figure S4. Activity of LY3023414 in a tumor clonogenic assay with a panel of patient-derived tumor xenografts passaged in nude mice and then grown in soft agar; Figure S5. Mean (±SD) concentrations of LY3023414 in male mice plasma, male rat plasma, and male beagle dog plasma following single dose; Figure S6. In vivo efficacy of LY3023414 and rapamycin alone and in combination using (A) H1975 and (B) 786-O xenograft tumor models.

The PI3K/AKT/mTOR pathway is among the most frequently altered pathways in cancer cell growth and survival. LY3023414 is a complex fused imidazoquinolinone with high solubility across a wide pH range designed to inhibit class I PI3K isoforms and mTOR kinase. Here, we describe the in vitro and in vivo activity of LY3023414. LY3023414 was highly soluble at pH 2–7. In biochemical testing against approximately 266 kinases, LY3023414 potently and selectively inhibited class I PI3K isoforms, mTORC1/2, and DNA-PK at low nanomolar concentrations. In vitro, inhibition of PI3K/AKT/mTOR signaling by LY3023414 caused G1 cell-cycle arrest and resulted in broad antiproliferative activity in cancer cell panel screens. In vivo, LY3023414 demonstrated high bioavailability and dose-dependent dephosphorylation of PI3K/AKT/mTOR pathway downstream substrates such as AKT, S6K, S6RP, and 4E-BP1 for 4 to 6 hours, reflecting the drug's half-life of 2 hours. Of note, equivalent total daily doses of LY3023414 given either once daily or twice daily inhibited tumor growth to similar extents in multiple xenograft models, indicating that intermittent target inhibition is sufficient for antitumor activity. In combination with standard-of-care drugs, LY3023414 demonstrated additive antitumor activity. The novel, orally bioavailable PI3K/mTOR inhibitor LY3023414 is highly soluble and exhibits potent in vivo efficacy via intermittent target inhibition. It is currently being evaluated in phase I and II trials for the treatment of human malignancies. Mol Cancer Ther; 15(10); 2344–56. ©2016 AACR.

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD+, essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD+ for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic. Background: NAMPT catalyzes the rate-limiting reaction in converting nicotinamide to NAD+ in cancers. Results: NAMPT inhibition attenuates glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step, resulting in perturbing metabolic pathways related to glycolysis. Conclusion: The metabolic basis of NAMPT inhibition is the attenuation of glycolysis by reducing NAD+ available to glyceraldehyde 3-phosphate dehydrogenase. Significance: This study sheds new light on how NAMPT regulates cancer metabolism.

Fluorescent analogs were synthesized of the potent PI 3-kinase inhibitors, wortmannin and demethoxyviridin. The esterification of 11-deacetylwortmannin, 17-hydroxywortmannin, and demethoxyviridin with the fluorescent carboxylic acids NBD-sarcosine and 7-dimethylaminocoumarin-4-acetic acid generated six novel fluorescent esters. Potent inhibition of PI 3-kinase-alpha was observed for the derivatives of 11-desacetylwortmannin and demethoxyviridin.

Gene expression from the human immunodeficiency virus 1 (HIV-1) is greatly enhanced by binding of the virally encoded Tat protein to a 59-base RNA stem-loop structure, the Transactivation Responsive Element (TAR), located at the 5'-termini of all viral transcripts. This interaction was investigated in vitro using 32P-labelled TAR and highly purified Tat in which cysteine residues were blocked by sulpitolysis (S-Tat). It is shown that specific complex formation between S-Tat and TAR can occur in the presence of urea, with urea concentrations between 5 and 6 M causing an approximately two-fold increase in the level of binding. Two conditions favoring RNA secondary structure, low temperature (0 degree C) and the presence of divalent cations (Mg2+), diminished the level of specific binding. These observations suggest that the presence of mild denaturants promoted macromolecular refolding or rearrangement in a manner that increased the number of molecules available for binding, and present a general method for studying protein/RNA interactions where analysis has been obstructed by improper protein or RNA conformation.

Owing to the prevalence of the JAK2V617F mutation in myeloproliferative neoplasms (MPNs), its constitutive activity, and ability to recapitulate the MPN phenotype in mouse models, JAK2V617F kinase is an attractive therapeutic target. We report the discovery and initial characterization of the orally bioavailable imidazopyridazine, LY2784544, a potent, selective and ATP-competitive inhibitor of janus kinase 2 (JAK2) tyrosine kinase. LY2784544 was discovered and characterized using a JAK2-inhibition screening assay in tandem with biochemical and cell-based assays. LY2784544 in vitro selectivity for JAK2 was found to be equal or superior to known JAK2 inhibitors. Further studies showed that LY2784544 effectively inhibited JAK2V617F-driven signaling and cell proliferation in Ba/F3 cells (IC50=20 and 55 nM, respectively). In comparison, LY2784544 was much less potent at inhibiting interleukin-3-stimulated wild-type JAK2-mediated signaling and cell proliferation (IC50=1183 and 1309 nM, respectively). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2V617F-GFP (green fluorescent protein) ascitic tumor cells (TED50=12.7 mg/kg) and significantly reduced (P

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for cellular metabolism, energy production, and DNA repair. NAMPT has been extensively studied because of its critical role in these cellular processes and the prospect of developing therapeutics against the target, yet how it regulates cellular metabolism is not fully understood. In this study we utilized liquid chromatography-mass spectrometry to examine the effects of FK866, a small molecule inhibitor of NAMPT currently in clinical trials, on glycolysis, the pentose phosphate pathway, the tricarboxylic acid (TCA) cycle, and serine biosynthesis in cancer cells and tumor xenografts. We show for the first time that NAMPT inhibition leads to the attenuation of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step due to the reduced availability of NAD(+) for the enzyme. The attenuation of glycolysis results in the accumulation of glycolytic intermediates before and at the glyceraldehyde 3-phosphate dehydrogenase step, promoting carbon overflow into the pentose phosphate pathway as evidenced by the increased intermediate levels. The attenuation of glycolysis also causes decreased glycolytic intermediates after the glyceraldehyde 3-phosphate dehydrogenase step, thereby reducing carbon flow into serine biosynthesis and the TCA cycle. Labeling studies establish that the carbon overflow into the pentose phosphate pathway is mainly through its non-oxidative branch. Together, these studies establish the blockade of glycolysis at the glyceraldehyde 3-phosphate dehydrogenase step as the central metabolic basis of NAMPT inhibition responsible for ATP depletion, metabolic perturbation, and subsequent tumor growth inhibition. These studies also suggest that altered metabolite levels in tumors can be used as robust pharmacodynamic markers for evaluating NAMPT inhibitors in the clinic.

LY573636-sodium (tasisulam) is a small molecule antitumor agent with a novel mechanism of action currently being investigated in a variety of human cancers. In vitro, tasisulam induced apoptosis via the intrinsic pathway, resulting in cytochrome c release and caspase-dependent cell death. Using high content cellular imaging and subpopulation analysis of a wide range of in vitro and in vivo cancer models, tasisulam increased the proportion of cells with 4N DNA content and phospho-histone H3 expression, leading to G2–M accumulation and subsequent apoptosis. Tasisulam also blocked VEGF, epidermal growth factor, and fibroblast growth factor–induced endothelial cell cord formation but did not block acute growth factor receptor signaling (unlike sunitinib, which blocks VEGF-driven angiogenesis at the receptor kinase level) or induce apoptosis in primary endothelial cells. Importantly, in vivo phenocopying of in vitro effects were observed in multiple human tumor xenografts. Tasisulam was as effective as sunitinib at inhibiting neovascularization in a Matrigel plug angiogenesis assay in vivo and also caused reversible, non G2–M–dependent growth arrest in primary endothelial cells. Tasisulam also induced vascular normalization in vivo. Interestingly, the combination of tasisulam and sunitinib significantly delayed growth of the Caki-1 renal cell carcinoma model, whereas neither agent was active alone. These data show that tasisulam has a unique, dual-faceted mechanism of action involving mitotic catastrophe and antiangiogenesis, a phenotype distinct from conventional chemotherapies and published anticancer agents. Mol Cancer Ther; 10(11); 2168–78. ©2011 AACR.

A method has been developed to prepare, purify, and fully characterize poly-iodinated insulin-like growth factor II (IGF-II) which can then be catalytically deiodinated to produce IGF-II with its native disulfide bonded structure. This method can potentially be adapted to prepare tritiated IGF-II with the use of tritium gas in the hydrogenolysis step. IGF-II was iodinated at all three tyrosines using lactoperoxidase with a three-fold excess of sodium iodide. The iodinated products were purified using reversed-phase HPLC and characterized by peptide mapping. The tyrosine-containing peptides generated by pepsin digestion were characterized by amino acid sequence analysis. Monoand di-iodinated phenylthiohydantoin tyrosine derivatives were synthesized and used to identify the idodination state of the modified tyrosine residues in the sequence analysis. Purified poly-iodinated IGF-II was deiodinated by hydrogenolysis, over a prereduced palladium (II) oxide catalyst to form IGF-II with its native disulfide bonds intact, as shown by peptide mapping.

Expression plasmids encoding random sequence mutant proteins of insulin-like growth factor II (IGFII) were constructed by cassette mutagenesis, to improve the efficiency of IGFII synthesis in Escherichia coli . A pool of oligodeoxyribonucleotide linkers containing random trinucleotide sequences were used to introduce second-codon substitutions into the gene encoding Met-Xaa-Trp-IGFII in expression vectors. E. coli RV308 cells transformed with these vectors synthesized IGFII at levels varying from 0–22% of total cell protein. This variable synthesis is a function of the random second-codon sequence and its corresponding amino acid, Xaa. Our data showed that mRNA stability, protein stability and translational efficiency all contributed to variable expression levels of Met-Xaa-Trp- IGFII in E. coli . Furthermore, an efficiently synthesized IGFII mutant protein, Met-His-Trp-IGFII, was converted to natural sequence IGFII by a simple oxidative cleavage reaction.

The Class I phosphoinositide 3-kinases (PI3Ks) are lipid kinases that phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositides. Although closely related, experimental evidence suggests that the four Class I PI3Ks may be functionally distinct. To further study their unique biochemical properties, the three human Class Ia PI3K (alpha, beta, and delta) p110 catalytic domains were cloned and co-expressed with the p85alpha regulatory domain in Sf9 cells. None of the p110 subunits were successfully expressed in the absence of p85alpha. Successful expression and purification of each p85alpha/p110 protein required using an excess of the p110 vector over the p85 vector during co-infection of Sf9 cells. Proteins were purified as the p85alpha/p110 complex by nickel affinity chromatography through an N-terminal His-tag on the p110 subunit using an imidazole gradient. The purification yields were high using the optimized ratio of p85/p110 vector and small culture volumes, with 24mg/L cell culture media for p85alpha/p110alpha, 17.5mg/L for p85alpha/p110delta, and 3.5mg/L for p85alpha/p110beta. The identity of each purified isoform was confirmed by mass spectral analysis and immunoblotting. The activities of the three p85alpha/p110 proteins and the Class Ib p110gamma catalytic domain were investigated using phosphatidylinositol 4,5-bisphosphate (PIP2) as the substrate in a PIP2/phosphatidylserine (PS) liposome. All four enzymes exhibited reaction velocities that were dependent on the surface concentration of PIP2. The surface concentrations that gave maximal activity for each human isoform with 0.5mM PIP2 were 2.5mol% PIP2 for p110gamma, 7.5mol% for p85alpha/p110beta, and 10mol% PIP2 for p85alpha/p110alpha and p85alpha/p110delta. The specific activity of p85alpha/p110alpha was three to five times higher than that of the other human isoforms. These kinetic differences may contribute to the unique roles of these isoforms in cells.

This review summarizes the types of evidence that can be invoked in order to demonstrate that a virally encoded protein possesses ion channel activity that is intrinsic to the life cycle of the virus. Ion channel activity has been proposed to be a key step in the life cycle of influenza virus, and the protein responsible for this activity has been proposed to be the M2 protein encoded by the virus. This review contrasts the evidence supporting the conclusion that the A/M2 protein of influenza A virus has intrinsic ion channel activity with the evidence that the 3AB protein encoded by the human rhinovirus possesses intrinsic ion channel activity.

The first potent inhibitors of glutamate racemase (MurI) enzyme that show whole cell antibacterial activity are described. Optically pure 4-substituted D-glutamic acid analogues with (2R,4S) stereochemistry and bearing aryl-, heteroaryl-, cinnamyl-, or biaryl-methyl substituents represent a novel class of glutamate racemase inhibitors. Exploration of the D-Glu core led to the identification of lead compounds (-)-8 and 10. 2-Naphthylmethyl derivative 10 was found to be a potent competitive inhibitor of glutamate racemase activity (K(i) = 16 nM, circular dichroism assay; IC(50) = 0.1 microg/mL high-performance liquid chromatography (HPLC) assay). Thorough structure-activity relationship (SAR) studies led to benzothienyl derivatives such as 69 and 74 with increased potency (IC(50) = 0.036 and 0.01 microg/mL, respectively, HPLC assay). These compounds showed potent whole cell antibacterial activity against S. pneumoniae PN-R6, and good correlation with the enzyme assay. Compounds 69, 74 and biaryl derivative 52 showed efficacy in an in vivo murine thigh infection model against Streptococcus pneumoniae. Data described herein suggest that glutamate racemase may be a viable target for developing new antibacterial agents.

The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly transactivates gene expression from the viral long terminal repeat (LTR) and is required for virus efficient replication. Previous studies have shown that cells scrape-loaded in the presence of purified recombinant Tat can absorb the protein in a receptor-independent fashion. Using recombinant Tat in which cysteine residues were blocked by sulfitolysis to prevent disulfide aggregation (S-Tat) we were unable to observe this phenomenon, possibly because of improper protein folding. In this study we report that the block to cellular uptake could be overcome by mixing S-Tat with a cationic liposome, Lipofectin. When mixed with Lipofectin, S-Tat effected a specific, concentration-dependent transactivation of HIV-1 LTR-directed reporter gene activity in Hela Cells. Cellular uptake was confirmed by Western blot analysis with an anti-Tat antibody. The method described utilizes cells plated in a 96-well format, requires only nanogram quantities of S-Tat protein and is much less labor-intensive than assays involving scrape-loading, making it suitable for use as a high-throughput screen for detecting Tat inhibitors. The method may have applications for the analysis of other recombinant proteins that require uptake into intact cells for determination of functionality and presents a general technique for introducing exogenous proteins into cells.

Publisher Summary This chapter describes expression and preparation of pure human cytomegalovirus (HCMV) assemblin with the help of a chelating peptide (CP) purification handle. Chelating peptide-immobilized metal ion affinity chromatography (CP-IMAC) uses an engineered metal-binding site or CP at either the N terminus or C terminus of a recombinant protein for a one-step affinity purification using IMAC. The CP sequences are easily incorporated into the protein with recombinant DNA cloning techniques. Cells are lysed with lysozyme and sonicated. Inclusion bodies are collected by centrifugation, lyophilized, and 10 mg/ml of solids dissolved in 0.5 M Tris, 7 M urea, pH 8.2. All purification steps are carried out in buffers containing 7 M urea, which maintains the enzyme in an unfolded inactive conformation. Blocking the cysteine residues as S-sulfonates facilitates the purification by solubilizing more protein from the inclusion bodies and preventing the formation of intermolecular disulfide bonds and aggregates. The activity of HCMV CP-assemblin is measured by following the hydrolysis of FITC, fluorescein isothiocyanate, a labeled peptide mimic of the maturational site of the assembly protein precursor.

Abstract 4087 Introduction: The JAK2-V617F mutation is the most common molecular abnormality in the BCR/ABL-negative myeloid proliferative neoplasms (MPNs) and is present in approximately 95% of patients with polycythemia vera (PV) and roughly 60% of patients with either essential thrombocythemia (ET) or primary myelofibrosis (PMF). The JAK2 V617F kinase is constitutively active, oncogenic and recapitulates MPN in murine models. Therefore, mutant JAK2 is a potential therapeutic target for MPNs. Here, we describe the efficacy of the clinical candidate LY2784544; a novel small molecule inhibitor selective for JAK2 V617F mutant, in JAK2 V617F-induced in vitro and in vivo models. Methods: Assessment of the ability of LY2784544 to inhibit the signaling of the V617F mutant or wild type JAK2 was evaluated in Ba/F3 cells expressing V617F or wild type JAK2, whereby the cellular level of phospho-STAT5 (pSTAT5) was measured by a high content imaging (Cellomics) assay. The antiproliferative activity of LY2784544 and its ability to induce apoptosis were examined by Celltiter-Glo Cell Viability and Caspase-Glo 3/7 Apoptosis Assays, respectively. The effect of LY2784544 on JAK3 was investigated in IL-2 stimulated NK-92 (nature killer) cells by measuring the inhibition of JAK3-STAT5 signaling with the Cellomics assay. To examine the in vivo target inhibition, a JAK2 V617F-induced ascites tumor model was established by implanting Ba/F3-JAK2 V617F-GFP cells into the intraperitoneal cavity of severe combined immunodeficiency (SCID) mice. After orally administering LY2784544, the inhibition of pSTAT5 was measured in Ba/F3-JAK2 V617F-GFP ascites tumor cells by fluorescence activated cell sorting (FACS) assay. The anti-tumor efficacy of LY2784544 was investigated in a SCID mouse model of MPN developed by intravenously implanting Ba/F3-JAK2 V617F-GFP cells. After oral treatment for 7 or 14 days, twice daily (BID), tumor burden reduction and the effect on erythroid progenitor cells were determined by measuring the GFP positive tumor cells and CD71/Ter119 positive cells in the spleens with FACS assays, respectively. Results: In the in vitro tests using Ba/F3 cells expressing either wild type or V617F mutant JAK2, LY2784544 potently inhibited the JAK2 V617F-STAT5 signaling at a concentration that was 41-fold lower than that required to inhibit IL-3-activated wild type JAK2-STAT5 signaling (IC50=0.055 μ M for JAK2 V617F vs. 2.26 μ M for WT JAK2). Similarly in the proliferation assay, LY2784544 selectively inhibited the JAK2 V617F-driven cell proliferation (IC50= 0.068 μ M). Inhibition of JAK2 V617F signaling correlated well with the induction of apoptosis (EC50= 0.113 μ M) in Ba/F3 cell model. Consistent with the observed apparent selectivity for the V617F mutant and reduced sensitivity against wild type JAK2, a lower potency was observed with LY2784544 in the IL-3-dependent cell proliferation assay (IC50=1.356 μ M) as well as less effective inhibition of IL-2 dependent JAK3-STAT5 signaling in NK-92 cells (IC50=0.94 μ M). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2 V617F-GFP ascites tumor cells with a Threshold Effective Dose 50 (TED50) of 12.7 mg/kg. Consistent with this effective inhibition of JAK2V617F, LY2784544 also significantly reduced Ba/F3-JAK2 V617F-GFP tumor burden in the JAK2 V617F-induced MPN model with a TED50 of 13.7 mg/kg after oral treatment (BID) for 14 days, indicating that the efficacy of LY2784544 is mediated by pharmacological inhibition of JAK2 V16F-STAT5 pathway. Furthermore, LY2784544 selectively reduced Ba/F3-JAK2 V617F-GFP tumor cells with no effect on CD71/Ter119 positive erythroid progenitors in spleens of SCID mice after oral treatment (BID) for seven days. Conclusions: The in vitro data suggest that constitutively active JAK2 V617F is more sensitive to LY2784544 than cytokine-activated wild type JAK2. Consistent with this observation, in MPN disease model testing, LY2784544 selectively reduces JAK2 V617F tumor cell burden with no effect on erythroid progenitor cells. These findings suggest that as a JAK2 V617F selective targeted therapy, LY2784544 has the potential to induce apoptosis in JAK2 V617F malignant clones while potentially minimizing unintended effects on the normal hematopoietic progenitor cells. These results helped to support the advancement of LY2784544 into an ongoing phase I trial in the MPNs (I3X-MC-JHTA, NCT01134120). Disclosures: Ma: Eli Lilly and Company: Employment. Zhao:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment. Clayton:Eli Lilly and Company: Employment. Blosser:Eli Lilly and Company: Employment. Burkholder:Eli Lilly and Company: Employment. Smith:Eli Lilly and Company: Employment.

The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.

We report our experimental results supporting the hypothesis that a specific metal-chelating peptide (CP) on the NH2 terminus of a protein can be used to purify that protein using immobilized metal ion affinity chromatography (IMAC). The potential utility of this approach resides with recombinant proteins since the nucleotide sequence that codes for the protein can be extended to include codons for the chelating peptide and thereby generate the gene for a chimeric CP-protein that can be cloned, expressed, and affinity-purified with immobilized metal ions. The chelating peptide purification handle could then be removed chemically or enzymatically after purification has been achieved to generate a protein with the natural amino acid sequence. The feasibility of using a chelating peptide as a purification handle has been demonstrated using a leuteinizing hormone-releasing hormone (LHRH) analog, 2-10 LHRH, which contains the previously identified chelating peptide, His-Trp, on the NH2 terminus. 2-10 LHRH had a high affinity for a Ni(II) IMAC column due to the NH2-terminal dipeptide sequence His-Trp, forming a coordination complex with Ni(II), whereas the controls, 3-10 LHRH and 4-10 LHRH, lacking the CP sequence, did not bind. Furthermore, 2-10 LHRH could be purified from a mixture of histidine-containing peptides on a Ni(II) IMAC column in one step. His-Trp proinsulin was used as a model of a recombinant CP-protein. The S-sulfonates of His-Trp-proinsulin and proinsulin were isolated from Escherichia coli engineered to overproduce these proteins as trpLE' fusion proteins. His-Trp-proinsulin(SSO3-)6 had a higher affinity for immobilized Ni(II) than proinsulin (SSO3-)6. Both proteins were eluted by decreasing the pH or by introducing a displacing ligand into the buffer. Ni(II) eluted from the column with much higher concentrations of displacing ligand than the proteins.

The complete peptide map of purified folded recombinant human insulin-like growth factor II (rhIGF-II) was determined to verify its sequence and disulfide bonding scheme. Each peptide generated by digestion with pepsin was purified and characterized by amino acid analysis, amino acid sequence analysis, and fast atom bombardment/mass spectrometry. Some peptides were also sequenced using tandem mass spectrometry. The rhIGF-II peptide map was compared to that of rat insulin-like growth factor II and to that of a disulfide-bonded isomer of rhIGF-II. The data obtained in these studies are consistent with the conclusion that the rhIGF-II obtained from Escherichia coli has the correct amino acid composition, sequence, and the native disulfide-bonded structure. The binding affinities of these forms of recombinant IGF-II for IGF carrier proteins were measured in an IGF binding protein assay. The disulfide isomer of rhIGF-II was 160-fold less potent than native rhIGF-II in the competitive protein binding assay. These studies illustrate the need to characterize recombinant polypeptides containing disulfide bonds to allow the native structure to be verified before characterizing the biological properties of such molecules in hopes of elucidating their physiologic functions.

Recently, the sequence of the human receptor for insulin-like growth factor II (IGF-II) was found to be 80% identical [Morgan et al., (1987) Nature 329 , 301–307] to the sequence of a partial clone of the bovine cation-independent mannose-6-phosphate receptor [Lobel et al., (1987) Proc. Natl. Acad. Sci. USA 84 , 2233–2237]. In the present study, the purified receptor for insulin-like growth factor II (IGF-II) was found to react with two different polyclonal antibodies to the purified mannose-6-phosphate receptor. Moreover, mannose-6-phosphate was found to stimulate the binding of labeled IGF-II to the IGF-II receptor by two-fold. This effect had the same specificity and affinity as the reported binding of mannose-6-phosphate to its receptor; mannose-1-phosphate and mannose had no effect on the binding of labeled IGF-II to its receptor, and the half-maximally effective concentration of mannose-6-phosphate was 0.3 mM. Also, mannose-6-phosphate did not affect labeled IGF-II binding to the insulin receptor. These results support the hypothesis that a single protein of Mr∼250,000 binds both IGF-II and mannose-6-phosphate. Furthermore, they indicate that mannose-6-phosphate can modulate the interaction of IGF-II to its receptor.

The primary structure of human insulin-like growth factor II receptor, predicted from the complementary DNA sequence, reveals a transmembrane receptor molecule with a large extracellular domain made up of fifteen repeat sequences and a small region homologous to the collagen-binding domain of fibronectin. The structural and biochemical features of the IGF-II receptor appear identical to those of the cation-independent mannose-6-phosphate receptor.

Human Insulin-like growth factor II (IGF-II) was produced in Escherichia coli as a 113 amino acid chimeric protein, LE1-Met-IGF-II consisting of 45 N-terminal amino acids from the E. coli trp leader peptide and the trpE polypeptide, and 67 C-terminal amino acids for IGF-II. High-level expression of the fusion protein, LE1-Met-IGF-II, was obtained with pCZ21, a plasmid containing a thermoinducible runaway replicon and the kanamycin resistance marker from Tn5. In E. coli, LE1-Met-IGF-II formed granules that were isolated and cleaved with cyanogen bromide to liberate IGF-II. IGF-II was sulfitolyzed, purified by anion exchange chromatography, refolded through a disulfide interchange reaction, and further purified by reverse-phase HPLC and gel filtration. The biological activity of this recombinant human IGF-II was measured in a competitive protein binding assay for IGF-II and by its ability to stimulate amino isobutyric acid uptake and protein synthesis in NCTC 2414 fibroblast cultures.

Autoradiography was used to visualize insulin-like growth factor II (IGF-II) receptors in the cerebellar cortex of weaver and Purkinje cell degeneration (pcd) mice. These mutants were selected for their respective absence of granule or Purkinje cells. Histological preparations confirmed a severe loss of granule cells in the cerebella of weaver mutants and an absence of Purkinje cells in those of pcd mutants. Autoradiographs showed specific IGF-II binding to the granule cell layer of the cerebellar cortex in control mice, and in pcd mutants. No specific [125I]human IGF-II binding was observed in the cerebellar cortex of weaver mutants. These studies suggest that specific IGF-II receptor sites are located on the granule cells of the cerebellum.

Several properties of the FeMo-cofactor (co) of nitrogenase in N-methylformamide solution at ambient temperature have been investigated by means of 19F NMR spectroscopy. With C6H5CF3 reference signals the magnetic moment per Mo atom was found to be approximately equal to 3.9 BM, consistent with S = 3/2 ground state identified by other spectroscopic methods at low temperature. Reaction of FeMo-co with 1.0 eq of RFS- (RF = p-C6H4CF3, p-C6H4F) afforded isotropically shifted signals indicative of binding to a paramagnetic cluster. By comparison with the spectra of Fe-S and Fe-Mo-S species derivatized with RFS-, including the cubane-type MoFe3S4 clusters with S = 3/2 ground states, it was concluded that the essential FeMo-co cluster structure remains intact and a Fe atom is the probable thiolate binding site. An interaction of FeMo-co with C6H5S- had been detected earlier by low temperature EPR spectroscopy. The binding site assignment is based on large observed isotropic shifts (ca. -12ppm) compared to the much smaller values found for Mo-SRF ligands in MoFe3S4 clusters and anticipated in FeMo-co on the basis of recent spectroscopic results. Isotropic 19F shifts have proven extremely sensitive to electronic and structural features of Fe-S and Fe-Mo-S clusters. The inclusion of a 19F NMR label in FeMo-co should prove of utility in further investigation of cofactor properties and reactions.

The presence of insulin-like growth factor-II (IGF-II) in brain and cerebral spinal fluid prompted us to investigate the distribution of receptors for this peptide in rat brain slices. Human 125I-IGF-II (10 pM) was incubated for 16 h at 4 degrees C with thaw-mounted slices of rat brain from 11 different brain regions. Incubations in the absence or presence of excess unlabeled human IGF-II or insulin were performed and the labeled tissues were exposed to X-ray film for 4-7 days. Autoradiographs showed dense labeling in the granule layers of the olfactory bulbs, deep layers of the cerebral cortex, pineal gland, anterior pituitary, hippocampus (CA1-CA4, and dentate gyrus), and the granule cell layers of the cerebellum. Unlabeled IGF-II eliminated most of the binding in these brain regions while insulin produced only a minimal reduction in the amount of 125I-IGF-II bound. These results indicate that a neural receptor for IGF-II is uniquely distributed in rat brain tissue supporting the notion that this peptide might play an important role in neuronal functioning.