Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2′-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Inducible T-cell costimulator (ICOS), a homodimeric protein expressed on the surface of activated T-cells, is being investigated as a potential therapeutic target to treat various cancers. Recent studies have reported aberrant increases in the soluble form of ICOS (sICOS) in human serum in disease-state patients, primarily using commercial ELISA kits. However, results from our in-house immunoassay did not show these aberrant increases, leading us to speculate that commercial sICOS ELISAs may be prone to interference. We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody interference. We then analyzed a panel of healthy, cancer, chronic hepatitis C virus, systemic lupus erythematosus, and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations. Since even well characterized immunoassay methods are prone to non-specific interference, we also developed a novel sICOS LC-MS/MS method to confirm the results. Using these orthogonal approaches, we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.

IQGAP1 is a key scaffold protein that regulates numerous cellular processes and signaling pathways. Analogous to many other cellular proteins, IQGAP1 undergoes post-translational modifications, including phosphorylation. Nevertheless, very little is known about the specific sites of phosphorylation or the effects on IQGAP1 function. Here, using several approaches, including MS, site-directed mutagenesis, siRNA-mediated gene silencing, and chemical inhibitors, we identified the specific tyrosine residues that are phosphorylated on IQGAP1 and evaluated the effect on function. Tyr-172, Tyr-654, Tyr-855, and Tyr-1510 were phosphorylated on IQGAP1 when phosphotyrosine phosphatase activity was inhibited in cells. IQGAP1 was phosphorylated exclusively on Tyr-1510 under conditions with enhanced MET or c-Src signaling, including in human lung cancer cell lines. This phosphorylation was significantly reduced by chemical inhibitors of MET or c-Src or by siRNA-mediated knockdown of MET. To investigate the biological sequelae of phosphorylation, we generated a nonphosphorylatable IQGAP1 construct by replacing Tyr-1510 with alanine. The ability of hepatocyte growth factor, the ligand for MET, to promote AKT activation and cell migration was significantly greater when IQGAP1-null cells were reconstituted with IQGAP1 Y1510A than when cells were reconstituted with WT IQGAP1. Collectively, our data suggest that phosphorylation of Tyr-1510 of IQGAP1 alters cell function. Because increased MET signaling is implicated in the development and progression of several types of carcinoma, IQGAP1 may be a potential therapeutic target in selected malignancies.

A persistent problem in early small-molecule drug discovery is the frequent lack of rank-order correlation between biochemical potencies derived from initial screens using purified proteins and the diminished potency and efficacy observed in subsequent disease-relevant cellular phenotypic assays. The introduction of the cellular thermal shift assay (CETSA) has bridged this gap by enabling assessment of drug target engagement directly in live cells based on ligand-induced changes in protein thermal stability. Initial success in applying CETSA across multiple drug target classes motivated our investigation into replacing the low-throughput, manually intensive Western blot readout with a quantitative, automated higher-throughput assay that would provide sufficient capacity to use CETSA as a primary hit qualification strategy. We introduce a high-throughput dose-response cellular thermal shift assay (HTDR-CETSA), a single-pot homogenous assay adapted for high-density microtiter plate format. The assay features titratable BacMam expression of full-length target proteins fused to the DiscoverX 42 amino acid ePL tag in HeLa suspension cells, facilitating enzyme fragment complementation–based chemiluminescent quantification of ligand-stabilized soluble protein. This simplified format can accommodate determination of full-dose CETSA curves for hundreds of individual compounds/analyst/day in replicates. HTDR-CETSA data generated for substrate site and alternate binding mode inhibitors of the histone-lysine N-methyltransferase SMYD3 in HeLa suspension cells demonstrate excellent correlation with rank-order potencies observed in cellular mechanistic assays and direct translation to target engagement of endogenous Smyd3 in cancer-relevant cell lines. We envision this workflow to be generically applicable to HTDR-CETSA screening spanning a wide variety of soluble intracellular protein target classes.

Phosphorylation is perhaps the most common and physiologically versatile reversible protein modification. More than 150,000 sites have been identified on over 18,000 human proteins. Catalyzing the addition of phosphate to these proteins is one of the largest families of genes in the genome. More than 500 protein kinases have been identified in humans, and many of these are shared across all eukaryotes. The removal of phosphate from proteins is carried out by a much smaller family of protein phosphatases. Together these enzymes and their regulatory partners modulate an exquisitely complex and integrated network of pathways that controls many aspects of cell growth, metabolism, division, motility, and differentiation. Dysregulation of these signals is one of the major mechanistic drivers of many human diseases. Dissecting the pathways that lead to these effects often involves understanding how phosphorylation regulates the behavior of individual proteins in the pathway.Since the development of soft ionization techniques beginning in the mid-1980s, mass spectrometry (MS) has emerged as a key technology in the study of protein phosphorylation and how it drives cellular events. Combined with newly developed methods in cell biology and low flow separation strategies mass spectrometry has proven to be very effective in addressing which proteins are phosphorylated; where they are phosphorylated; if the sites are regulated; and to what extent they are regulated. In this chapter, we describe the current state-of-the-art MS-based approaches to answer these key questions. We explore the reasons why localizing specific sites of phosphorylation and determining absolute stoichiometry is difficult and describe successful approaches to circumvent some of these difficulties. Moving from the unbiased analysis of an isolated protein to exploring the phosphorylation content of an entire proteome has been made possible by the development of high-resolution, high mass accuracy MS systems with greater than 10 Hz MS/MS data acquisition rates, but poses new challenges. Here, we describe current and emerging techniques for separating complex mixtures of phosphopeptides and efficient strategies for enriching the phosphopeptide pool to better take advantage of the power of modern MS systems. Quantitative strategies, which can assign functional significance to an individual phosphorylation sites or highlight the activation of an entire biological pathway, are discussed with their relative strengths and weaknesses reviewed. Finally, data analysis and bioinformatic tools critical to facilitate an understanding of the consequences of these events are discussed.

The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

Although therapeutic interventions of signal-transduction cascades with targeted kinase inhibitors are a well-established strategy, drug-discovery efforts to identify targeted phosphatase inhibitors have proven challenging. Herein we report a series of allosteric, small-molecule inhibitors of wild-type p53-induced phosphatase (Wip1), an oncogenic phosphatase common to multiple cancers. Compound binding to Wip1 is dependent on a 'flap' subdomain located near the Wip1 catalytic site that renders Wip1 structurally divergent from other members of the protein phosphatase 2C (PP2C) family and that thereby confers selectivity for Wip1 over other phosphatases. Treatment of tumor cells with the inhibitor GSK2830371 increases phosphorylation of Wip1 substrates and causes growth inhibition in both hematopoietic tumor cell lines and Wip1-amplified breast tumor cells harboring wild-type TP53. Oral administration of Wip1 inhibitors in mice results in expected pharmacodynamic effects and causes inhibition of lymphoma xenograft growth. To our knowledge, GSK2830371 is the first orally active, allosteric inhibitor of Wip1 phosphatase.

Abstract Many cancer cells generate energy by rapidly converting glucose to lactate in the cytosol, a process termed aerobic glycolysis. This metabolic phenotype is recognized as one of the hallmarks of cancer and is enabled by lactate dehydrogenase (LDH), which catalyzes pyruvate to lactate inter-conversion. We find that hepatocellular carcinoma cells express micromolar quantities of LDH5 and that LDH5 protein down-regulation takes about 5 days allowing time for the cells to adapt their metabolism. Since metabolic processes happen in minutes, addressing consequences of LDH5 inhibition by protein down-regulation is inadequate. We screened the GSK compound library and identified a series of quinoline acids as NADH-competitive LDH5 inhibitors. Subsequent lead optimization yielded molecules with LDH5 inhibitory potencies as low as 2-3 nM and selectivity over LDH1 on the order of 10-100-fold. These molecules were cell-permeable and did not have any appreciable activity against a panel of approximately fifty common enzymes, receptors and ion channels, making them the most potent and selective LDH5 inhibitors identified to date. Using these tool inhibitors, we find that rapid chemical inhibition of LDH5 in Snu398 hepatocellular carcinoma cells results in profound inhibition of lactate production and increase in pyruvate as measured by mass spectrometric analysis. Real-time analysis by NMR spectroscopy of live Snu398 cells fed with 13C-labeled glucose demonstrated that chemical LDH5 inhibition led to a rapid decrease in glucose uptake and concomitant slow-down of lactate production. Comprehensive analysis of more than 500 metabolites upon LDH5 inhibition in Snu398 cells revealed that the cytosolic glycolysis pathway was significantly impeded with some up-stream intermediates increasing as much as 40-fold. As the cell lost its ability for cytosolic glucose processing, the TCA cycle activity increased indicating that pyruvate entered the mitochondria and restored their activity resulting in increased oxygen consumption upon LDH5 inhibition. Several pathways that rely on glycolytic and TCA intermediates were also upregulated, including fatty acid metabolism and pentose phosphate pathway. LDH5 inhibition also strongly potentiated PKM2 activity. These profound metabolic alterations greatly impaired cell survival and induced cell death in Snu398 cells. In summary, we have shown that rapid chemical inhibition of LDH5 leads to profound metabolic alterations and impairs cell survival in hepatocellular carcinoma cells making it a compelling strategy for treating solid tumors relying on aerobic glycolysis. Citation Format: Julia Billiard, Roland Annan, Jennifer Ariazi, Jacques Briand, Kristin Brown, Nino Campobasso, Subhas Chakravorty, Deping Chai, Mariela Colón, Elizabeth Davenport, Christopher Dodson, Nathan Gaul, Seth Gilbert, Anthony Jurewicz, Hong Lu, Dean McNulty, Jeanelle McSurdy-Freed, Lisa Miller, Kelvin Nurse, Paru Rao Nuthulaganti, Chad Quinn, Jessica Schneck, Gilbert Scott, Tony Shaw, Christian Sherk, Angela Smallwood, Sharon Sweitzer, James Villa, Gregory Waitt, Richard Wooster, Kevin Duffy. Rapid LDH5 inhibition reverses malignant metabolic phenotype and impairs survival of hepatocellular carcinoma cells . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5418. doi:10.1158/1538-7445.AM2013-5418

Cancer is a leading cause of death of men and women worldwide. Tumor cell motility contributes to metastatic invasion that causes the vast majority of cancer deaths. Extracellular receptors modified by α2,3-sialic acids that promote this motility can serve as ideal chemotherapeutic targets. For example, the extracellular domain of the mucin receptor podoplanin (PDPN) is highly O-glycosylated with α2,3-sialic acid linked to galactose. PDPN is activated by endogenous ligands to induce tumor cell motility and metastasis. Dietary lectins that target proteins containing α2,3-sialic acid inhibit tumor cell growth. However, anti-cancer lectins that have been examined thus far target receptors that have not been identified. We report here that a lectin from the seeds of Maackia amurensis (MASL) with affinity for O-linked carbohydrate chains containing sialic acid targets PDPN to inhibit transformed cell growth and motility at nanomolar concentrations. Interestingly, the biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular system to inhibit melanoma cell growth, migration, and tumorigenesis. These studies demonstrate how lectins may be used to help develop dietary agents that target specific receptors to combat malignant cell growth.

Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formidable challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. Reduction of sample complexity and enrichment of the phosphopeptide pool is a necessary prerequisite for global phosphoproteomics. Metal affinity chromatography and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic and enrichment strategies. However, each is not without shortcomings. The former suffers from selectivity over highly acidic peptides, while the latter is a low resolution technique that provides little in the way of enrichment for phosphorylated peptides. Here we describe a phosphopeptide fractionation scheme using hydrophilic interaction chromatography (HILIC) which both enriches the phosphopeptide pool and efficiently fractionates the remaining peptides. When HILIC is used in front of metal affinity chromatography, the selectivity of the metal affinity resin is improved to greater than 90%. The lack of significant numbers of nonphosphorylated peptides also allows for more efficient use of the mass spectrometer duty cycle in that the instrument spends nearly all of its time sequencing phosphopeptides.

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca2+ attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser1443, a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1-3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography-tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

Centromere-associated protein-E (CENP-E) is a kinetochore-associated mitotic kinesin that is thought to function as the key receptor responsible for mitotic checkpoint signal transduction after interaction with spindle microtubules. We have identified GSK923295, an allosteric inhibitor of CENP-E kinesin motor ATPase activity, and mapped the inhibitor binding site to a region similar to that bound by loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, which block release of ADP and destabilize motor-microtubule interaction, GSK923295 inhibited release of inorganic phosphate and stabilized CENP-E motor domain interaction with microtubules. Inhibition of CENP-E motor activity in cultured cells and tumor xenografts caused failure of metaphase chromosome alignment and induced mitotic arrest, indicating that tight binding of CENP-E to microtubules is insufficient to satisfy the mitotic checkpoint. Consistent with genetic studies in mice suggesting that decreased CENP-E function can have a tumor-suppressive effect, inhibition of CENP-E induced tumor cell apoptosis and tumor regression.

Mass spectrometry-based protein phosphorylation analysis on a proteome-wide scale remains a formidable challenge, hampered by the complexity and dynamic range of protein expression on the global level and multi-site phosphorylation at substoichiometric ratios at the individual protein level. It is recognized that reduction of sample complexity or enrichment of the phosphopeptide pool is a necessary prerequisite for global phospho-proteomics. Immobilized metal affinity chromatography (IMAC) and strong cation exchange chromatography, either alone or in tandem, have emerged as the most widely used chromatographic-based enrichment strategies. However, each is not without shortcomings. Both techniques provide little fractionation of phosphorylated species and are compromised by competition and co-elution of highly acidic peptides. Here, we describe a phosphopeptide prefractionation scheme using hydrophilic interaction chromatography, which both enriches the phosphopeptide pool and efficiently fractionates the remaining peptides. When used in front of IMAC, the selectivity of the metal affinity resin is improved to greater than 95%. The lack of significant numbers of nonphosphorylated peptides also allows for more efficient use of the mass spectrometer duty cycle in that the instrument spends nearly all of its time in sequencing the phosphopeptides.

Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.

The diversity and complexity of proteins and peptides in biological systems requires powerful liquid chromatography-based separations to optimize resolution and detection of components. Proteomics strategies often combine two orthogonal separation modes to meet this challenge. In nearly all cases, the second dimension is a reverse phase separation interfaced directly to a mass spectrometer. Here we report on the use of hydrophilic interaction chromatography (HILIC) as part of a multidimensional chromatography strategy for proteomics. Tryptic peptides are separated on TSKgel Amide-80 columns using a shallow inverse organic gradient. Under these conditions, peptide retention is based on overall hydrophilicity, and a separation truly orthogonal to reverse phase is produced. Analysis of tryptic digests from HeLa cells yielded numbers of protein identifications comparable to that obtained using strong cation exchange. We also demonstrate that HILIC represents a significant advance in phosphoproteomics analysis. We exploited the strong hydrophilicity of the phosphate group to selectively enrich and fractionate phosphopeptides based on their increased retention under HILIC conditions. Subsequent IMAC enrichment of phosphopeptides from HILIC fractions showed better than 99% selectivity. This was achieved without the use of derivatization or chemical modifiers. In a 300-mu g equivalent of HeLa cell lysate we identified over 1000 unique phosphorylation sites. More than 700 novel sites were added to the HeLa phosphoproteome.

Human Relaxin 2 is an insulin-related peptide hormone with a mass of 19,084 Da. The mRNA contains a number of arginine codons that are rarely used by Escherichia coli to produce highly expressed proteins. As a result, expressing this recombinant protein in E. coli is problematic. When human Relaxin 2 was expressed in E. coli BL21 (DE3), several forms of the protein were made. One species had the expected molecular weight (19,084 Da). A second species observed had a molecular weight of 21,244 Da. A third minor species had a molecular weight of 17,118 Da. These aberrant molecular weights can be explained as follows. First, a sequence CGA-AAA-AAG-AGA, containing the rare arginine codons CGA and AGA was the site of the +1 frameshift that generated the 21,244 Da species. Since there was a limited supply of this arginyl-tRNA, the peptidyl-tRNA moved +1 nucleotide to occupy the codon and resumed protein synthesis. Second, a -1 frameshift associated with 'slippery A' sequence XXA-AAA-AAG accounted for 10% of the product with a mass of 17,118 Da. Presumably, the shift to -1 also occurred because there was a paucity of the arginyl-tRNAArgucu. Introduction of a plasmid coding for the cognate tRNA for AGA and site directed mutagenesis prevented the formation of both frameshift species.

In addition to selecting molecules of pharmacological interest, high-throughput screening campaigns often generate hits of undesirable mechanism, which cannot be exploited for drug discovery as they lead to obvious problems of specificity and developability. Examples of undesirable mechanisms are target alkylation/acylation and compound aggregation. Both types of "promiscuous" mechanisms have been described in the literature, as have methods for their detection. In addition to these mechanisms, compounds can also inhibit by oxidizing susceptible enzyme targets, such as metalloenzymes and cysteine-using enzymes. However, this redox phenomenon has been documented infrequently, and an easy method for detecting this behavior is missing. In this article, the authors describe direct proof of small-molecule oxidation of a cysteine protease by liquid chromatography/tandem mass spectrometry, develop a simple assay to predict this oxidizing behavior by compounds, and show the utility of this assay by demonstrating its ability to distinguish nuisance redox compounds from well-behaved inhibitors in 3 historical GlaxoSmithKline drug discovery efforts.