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Supplementary Figure 1: Standard curves of recombinant modified histone proteins for quantification of western blots

Supplementary Figure 5: H3K27me3 enrichment across genes modulated by EZH2 inhibition

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Supplementary Figure 6: Gene Set Enrichment Analysis (GSEA) for SUP-B8 cells treated with GSK126

Supplementary Table 2: Biochemical activity of WT and A687V EZH2 complexes with modified histone peptides

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Supplementary Figure 2: Alignment of all human SET domain methyltransferases

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Supplementary Table 1: Primers utilized for A687V EZH2 mutagenesis

Supplementary Table 3: Functional analysis of genes significantly up- and down-regulated in SUP-B8 and NALM-6 cells.

Supplementary Figure 4: Sanger sequencing chromatograms of EZH2 mutations in acute lymphoblastic leukemia cell lines

The EZH2 methyltransferase silences gene expression through methylation of histone H3 on lysine 27 (H3K27). Recently, EZH2 mutations have been reported at Y641, A677, and A687 in non-Hodgkin lymphoma. Although the Y641F/N/S/H/C and A677G mutations exhibit clearly increased activity with substrates dimethylated at lysine 27 (H3K27me2), the A687V mutant has been shown to prefer a monomethylated lysine 27 (H3K27me1) with little gain of activity toward H3K27me2. Herein, we demonstrate that despite this unique substrate preference, A687V EZH2 still drives increased H3K27me3 when transiently expressed in cells. However, unlike the previously described mutants that dramatically deplete global H3K27me2 levels, A687V EZH2 retains normal levels of H3K27me2. Sequencing of B-cell–derived cancer cell lines identified an acute lymphoblastic leukemia cell line harboring this mutation. Similar to exogenous expression of A687V EZH2, this cell line exhibited elevated H3K27me3 while possessing H3K27me2 levels higher than Y641- or A677-mutant lines. Treatment of A687V EZH2-mutant cells with GSK126, a selective EZH2 inhibitor, was associated with a global decrease in H3K27me3, robust gene activation, caspase activation, and decreased proliferation. Structural modeling of the A687V EZH2 active site suggests that the increased catalytic activity with H3K27me1 may be due to a weakened interaction with an active site water molecule that must be displaced for dimethylation to occur. These findings suggest that A687V EZH2 likely increases global H3K27me3 indirectly through increased catalytic activity with H3K27me1 and cells harboring this mutation are highly dependent on EZH2 activity for their survival. Mol Cancer Ther; 13(12); 3062–73. ©2014 AACR.

Advances in understanding the role and molecular mechanisms underlying immune surveillance and control of (pre)malignancies is revolutionizing clinical practice in the treatment of cancer. Presently, multiple biologic drugs targeting the immune checkpoint proteins PD(L)1 or CTLA4 have been approved and/or are in advanced stages of clinical development for many cancers. In addition, combination therapy with these agents and other immunomodulators is being intensively explored with the aim of improving primary response rates or prolonging overall survival. The effectiveness of cancer immunotherapy with biologics is spurring research in alternate approaches including small-molecule-mediated targeting of intracellular pathways modulating the innate and adaptive immune response. This focus of this review is on some of the key intracellular pathways where the development of a small-molecule therapeutic is attractive, tractable, and potentially synergistic with extracellular biologic-mediated immune checkpoint blockade.

The transcription factor NF-kappaB (NF-kB) is a key regulator of cytokine and chemokine production in melanoma and is responsible for symptoms such as anorexia, fatigue, and weight loss. In addition, NF-kB is believed to contribute to progression of the disease by upregulation of cell cycle and anti-apoptotic genes and to contribute to resistance against targeted therapies and immunotherapy. In this study, we have examined the ability of the bromodomain and extra-terminal (BET) protein inhibitor I-BET151 to inhibit NF-kB in melanoma cells. We show that I-BET151 is a potent, selective inhibitor of a number of NF-kB target genes involved in induction of inflammation and cell cycle regulation and downregulates production of cytokines such as IL-6 and IL-8. SiRNA studies indicate that BRD2 is the main BET protein involved in regulation of NF-kB and that I-BET151 caused transcriptional downregulation of the NF-kB subunit p105/p50. These results suggest that BET inhibitors may have an important role in treatment of melanoma where activation of NF-kB may have a key pathogenic role.

Over the past several years, there has been rapidly expanding evidence of epigenetic dysregulation in cancer, in which histone and DNA modification play a critical role in tumor growth and survival. These findings have gained the attention of the drug discovery and development community, and offer the potential for a second generation of cancer epigenetic agents for patients following the approved “first generation” of DNA methylation (e.g., Dacogen, Vidaza) and broad-spectrum HDAC inhibitors (e.g., Vorinostat, Romidepsin). This Review provides an analysis of prospects for discovery and development of novel cancer agents that target epigenetic proteins. We will examine key examples of epigenetic dysregulation in tumors as well as challenges to epigenetic drug discovery with emerging biology and novel classes of drug targets. We will also highlight recent successes in cancer epigenetics drug discovery and consider important factors for clinical success in this burgeoning area. Epigenetic dysregulation in cancer Epigenetic information is contained in the cell in multiple forms that include DNA methylation, histone modification (methylation, acetylation, phosphorylation, etc.), nucleosome positioning, and microRNA expression, among others. This combined information constitutes the epigenome. A comprehensive understanding of epigenomic dysregulation in specific cancer types has not been elucidated yet. Currently, there is an understanding of tumor-specific types of epigenetic modifications without a full appreciation of the context of the entire cancer epigenome in the specific tumor. Cancer epigenetic dysregulation can be categorized into three types: (a) altered DNA or histone modification, (b) somatic alteration in an epigenetic protein, and (c) altered expression of an epigenetic protein. Those types of cancer epigenome dysregulation have been reviewed comprehensively elsewhere (1–3), and only will be referred to here.

EZH2/PRC2 catalyzes transcriptionally repressive methylation at lysine 27 of histone H3 and has been associated with numerous cancer types. Point mutations in EZH2 at Tyr641 and Ala677 identified in non-Hodgkin lymphomas alter substrate specificity and result in increased trimethylation at histone H3K27. Interestingly, EZH2/PRC2 is activated by binding H3K27me3 marks on histones, and this activation is proposed as a mechanism for self-propagation of gene silencing. Recent work has identified GSK126 as a potent, selective, SAM-competitive inhibitor of EZH2 capable of globally decreasing H3K27 trimethylation in cells. Here we show that activation of PRC2 by an H3 peptide trimethylated at K27 is primarily an effect on the rate-limiting step (kcat) with no effect on substrate binding (Km). Additionally, GSK126 is shown to have a significantly longer residence time of inhibition on the activated form of EZH2/PRC2 as compared to unactivated EZH2/PRC2. Overall inhibition constant (Ki*) values for GSK126 were determined to be as low as 93 pM and appear to be driven by slow dissociation of inhibitor from the activated enzyme. The data suggest that activation of EZH2 allows the enzyme to adopt a conformation that possesses greater affinity for GSK126. The long residence time of GSK126 may be beneficial in vivo and may result in durable target inhibition after drug systemic clearance.

BET (bromodomain and extra-terminal) proteins regulate gene expression through their ability to bind to acetylated chromatin and subsequently activate RNA PolII-driven transcriptional elongation. Small molecule BET inhibitors prevent binding of BET proteins to acetylated histones and inhibit transcriptional activation of BET target genes. BET inhibitors attenuate cell growth and survival in several hematologic cancer models, partially through the down-regulation of the critical oncogene, MYC. We hypothesized that BET inhibitors will regulate MYC expression in solid tumors that frequently over-express MYC. Here we describe the effects of the highly specific BET inhibitor, I-BET762, on MYC expression in prostate cancer models. I-BET762 potently reduced MYC expression in prostate cancer cell lines and a patient-derived tumor model with subsequent inhibition of cell growth and reduction of tumor burden in vivo. Our data suggests that I-BET762 effects are partially driven by MYC down-regulation and underlines the critical importance of additional mechanisms of I-BET762 induced phenotypes.

The histone H3-lysine 27 (H3K27) methyltransferase EZH2 plays a critical role in regulating gene expression, and its aberrant activity is linked to the onset and progression of cancer. As part of a drug discovery program targeting EZH2, we have identified highly potent, selective, SAM-competitive, and cell-active EZH2 inhibitors, including GSK926 (3) and GSK343 (6). These compounds are small molecule chemical tools that would be useful to further explore the biology of EZH2.

EZH2, the catalytic subunit of the polycomb repressive complex 2 (PRC2), is involved in repressing gene expression through methylation of histone H3 on lysine 27 (H3K27). Overexpression of EZH2 is implicated in tumorigenesis, and mutations within its catalytic domain occur in lymphoma. Here, Caretha Creasy and colleagues describe a potent small-molecule inhibitor of EZH2 methyltransferase activity that decreases levels of methylated H3K27 and reactivates silenced PRC2 target genes. It also inhibits the proliferation of EZH2 mutant cell lines and the growth of EZH2 mutant xenografts in mice. Pharmacological inhibition of EZH2 activity may therefore be a viable strategy for treating EZH2 mutant lymphoma.

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.

Summaryc-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

The Aurora kinases play critical roles in the regulation of mitosis and are frequently overexpressed or amplified in human tumors. Selective inhibitors may provide a new therapy for the treatment of tumors with Aurora kinase amplification. Herein we describe our lead optimization efforts within a 7-azaindole-based series culminating in the identification of GSK1070916 (17k). Key to the advancement of the series was the introduction of a 2-aryl group containing a basic amine onto the azaindole leading to significantly improved cellular activity. Compound 17k is a potent and selective ATP-competitive inhibitor of Aurora B and C with K(i)* values of 0.38 +/- 0.29 and 1.5 +/- 0.4 nM, respectively, and is >250-fold selective over Aurora A. Biochemical characterization revealed that compound 17k has an extremely slow dissociation half-life from Aurora B (>480 min), distinguishing it from clinical compounds 1 and 2. In vitro treatment of A549 human lung cancer cells with compound 17k results in a potent antiproliferative effect (EC(50) = 7 nM). Intraperitoneal administration of 17k in mice bearing human tumor xenografts leads to inhibition of histone H3 phosphorylation at serine 10 in human colon cancer (Colo205) and tumor regression in human leukemia (HL-60). Compound 17k is being progressed to human clinical trials.

Myelosuppression has been observed with several multikinase angiogenesis inhibitors in clinical studies, although the frequency and severity varies among the different agents. Inhibitors targeting vascular endothelial growth factor receptor (VEGFR) often inhibit other kinases, which may contribute to their adverse-event profiles. Kinase selectivity of pazopanib, sorafenib, and sunitinib was evaluated in a panel of 242 kinases. Cellular potency was measured using autophosphorylation assays. Effect on human bone marrow progenitor growth in the presence of multiple growth factors was evaluated and correlated with the kinase selectivity. Sunitinib inhibited more kinases than pazopanib and sorafenib, at potencies within 10-fold of VEGFR-2. All three compounds potently inhibited VEGFR-2, platelet-derived growth factor receptor-β and c-Kit, However, pazopanib was less active against Flt-3 in both kinase and cellular assays. The inhibitory properties of pazopanib, sorafenib, and sunitinib were dependent on the growth factor used to initiate bone marrow colony formation. Addition of stem cell factor and/or Flt-3 ligand with granulocyte-macrophage colony stimulating factor resulted in significant shifts in potency for sorafenib and sunitinib but less so for pazopanib. Activity against c-kit and Flt-3 by multikinase angiogenesis inhibitors provide a potential explanation for the differences in myelosuppression observed with these agents in patients.

Aurora kinases are a family of serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. AurB (Aurora B kinase) has shown a clear link to cancer and is being pursued as an attractive cancer target. Multiple small molecules targeting AurB have entered the clinic for the treatment of cancer. A protein cofactor, INCENP (inner centromere protein), regulates the cellular localization and activation of AurB. In the present study, we examined the effect of INCENP on the activation kinetics of AurB and also elucidated the kinetic mechanism of AurB-catalysed substrate phosphorylation. We have concluded that: (i) substoichoimetric concentrations of INCENP are sufficient for AurB autophosphorylation at the activation loop residue Thr232, and hence INCENP plays a catalytic role in AurB autophosphorylation; (ii) AurB/INCENP-catalysed phosphorylation of a peptide substrate proceeds through a rapid equilibrium random Bi Bi kinetic mechanism; and (iii) INCENP has relatively minor effects on the specific activity of AurB using a peptide substrate when compared with its role in AurB autoactivation. These results indicate that the effects of INCENP, and probably accessory proteins in general, may differ when enzymes are acting on different downstream targets.

Matrix metalloproteinase 13 (MMP13) is a key enzyme implicated in the degradation of the extracellular matrix in osteoarthritis. Clinical administration of broad spectrum MMP inhibitors such as marimastat has been implicated in severe musculo-skeletal side effects. Consequently, research has been focused on designing inhibitors that selectively inhibit MMP13, thereby circumventing musculo-skeletal toxicities. A series of pyrimidine dicarboxamides were recently shown to be highly selective inhibitors of MMP13 with a novel binding mode. We have applied a molecular ruler to this exosite by dual inhibition studies involving a potent dicarboxamide in the presence of two metal chelators of different sizes. A larger hydroxamate mimic overlaps and antagonizes binding of the dicarboxamide to the exosite whereas the much smaller acetohydroxamate synergizes with the dicarboxamide. These studies elucidate the steric requirement for compounds that fit exclusively into the active site, a mandate for generating highly selective MMP13 inhibitors.

The Aurora kinases are a family of serine/threonine kinases involved in mitosis. The expression of AurA is ubiquitous and cell cycle regulated. It is overexpressed in many tumor types, including breast, colon, and ovarian. TPX2 is a binding partner and activator of AurA. A fragment of TPX2 (residues 1-43) has been shown to be sufficient for binding, kinase activation, and protection from dephosphorylation. We have shown that the addition of TPX2(1-43) increases the catalytic efficiency of AurA. While TPX2 binding has no effect on the turnover number of AurA and does not change the reaction mechanism (characterized here to be a rapid equilibrium random mechanism), it increases the binding affinity of both ATP and a peptide substrate. We have also demonstrated differences in the inhibitor structure-activity relationship (SAR) in the presence or absence of TPX2(1-43). To better understand the differential SAR, we carried out computer modeling studies to gain insight into the effect of TPX2 on the binding interactions between AurA and inhibitors. Our working hypothesis is that TPX2 binding decreases the size and accessibility of a hydrophobic pocket, adjacent to the ATP site, to inhibitors.

SummaryEpigenetic dysregulation has emerged as an important mechanism in cancer. Alterations in epigenetic machinery have become a major focus for targeted therapies. The current report describes the discovery and biological activity of a cyclopropylamine containing inhibitor of Lysine Demethylase 1 (LSD1), GSK2879552. This small molecule is a potent, selective, orally bioavailable, mechanism-based irreversible inactivator of LSD1. A proliferation screen of cell lines representing a number of tumor types indicated that small cell lung carcinoma (SCLC) is sensitive to LSD1 inhibition. The subset of SCLC lines and primary samples that undergo growth inhibition in response to GSK2879552 exhibit DNA hypomethylation of a signature set of probes, suggesting this may be used as a predictive biomarker of activity.

Several potent, cell permeable 4-aryl-dihydropyrimidinones have been identified as inhibitors of FATP4. Lipophilic ester substituents at the 5-position and substitution at the para-position (optimal groups being -NO(2) and CF(3)) of the 4-aryl group led to active compounds. In two cases racemates were resolved and the S enantiomers shown to have higher potencies.

Mdm2 negatively regulates p53 by inhibiting its transcriptional activity and promoting its degradation by functioning as an E3 ubiquitin ligase. The primary p53 binding site on mdm2 is located in its N-terminal domain. Through binding to p53 at its N-terminal transactivation domain, mdm2 directly blocks the transcriptional activation function of p53. We discovered that truncated mdm2 protein constructs without the N-terminal p53 binding domain are at least as active as full-length mdm2 in catalyzing p53 ubiquitination. Furthermore, the deletion of the central acidic domain significantly reduces the E3 ligase activity of mdm2 toward p53. We have also performed GST pull-down experiments to probe the direct binding of various mdm2 domain constructs toward full length p53 and found that mdm2 constructs without the N-terminal p53 binding domain retain the ability to bind to p53. Our kinetic and binding data localize the second p53 binding site between amino acids 211 and 361, including the acidic domain and the zinc finger region. Our work, consistent with other reports, suggests that the p53 tetramer interacts with at least two sites on mdm2. Although the interaction between the N-termini of mdm2 and p53 blocks the transactivation activity of p53, the interaction between the central domain of mdm2 and the core domain of p53 is critical for the ubiquitination and degradation of p53. This second mdm2-p53 interaction site represents an alternative target for small molecule modulators of the mdm2-p53 pathway.

Heat shock protein (Hsp)90 is emerging as an important therapeutic target for the treatment of cancer. Two analogues of the Hsp90 inhibitor geldanamycin are currently in clinical trials. Geldanamycin (GA) and its analogues have been reported to bind purified Hsp90 with low micromolar potency, in stark contrast to their low nanomolar antiproliferative activity in cell culture and their potent antitumor activity in animal models. Several models have been proposed to account for the ≈100-fold-greater potency in cell culture, including that GA analogues bind with greater affinity to a five-protein Hsp90 complex than to Hsp90 alone. We have determined that GA and the fluorescent analogue BODIPY-GA (BDGA) both demonstrate slow, tight binding to purified Hsp90. BDGA, used to characterize the kinetics of ligand–Hsp90 interactions, was found to bind Hsp90α with k off = 2.5 × 10 −3 min −1 , t 1/2 = 4.6 h, and K i * = 10 nM. It was found that BDGA binds to a functional multiprotein Hsp90 complex with kinetics and affinity identical to that of Hsp90 alone. Also, BDGA binds to Hsp90 from multiple cell lysates in a time-dependent manner with similar kinetics. Therefore, our results indicate that the high potency of GA in cell culture and in vivo can be accounted for by its time-dependent, tight binding to Hsp90 alone. In the broader context, these studies highlight the essentiality of detailed biochemical characterization of drug–target interactions for the effective translation of in vitro pharmacology to cellular and in vivo efficacy.

Human angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a zinc metalloprotease whose closest homolog is angiotensin I-converting enzyme. To begin to elucidate the physiological role of ACE2, ACE2 was purified, and its catalytic activity was characterized. ACE2 proteolytic activity has a pH optimum of 6.5 and is enhanced by monovalent anions, which is consistent with the activity of ACE. ACE2 activity is increased approximately 10-fold by Cl(-) and F(-) but is unaffected by Br(-). ACE2 was screened for hydrolytic activity against a panel of 126 biological peptides, using liquid chromatography-mass spectrometry detection. Eleven of the peptides were hydrolyzed by ACE2, and in each case, the proteolytic activity resulted in removal of the C-terminal residue only. ACE2 hydrolyzes three of the peptides with high catalytic efficiency: angiotensin II () (k(cat)/K(m) = 1.9 x 10(6) m(-1) s(-1)), apelin-13 (k(cat)/K(m) = 2.1 x 10(6) m(-1) s(-1)), and dynorphin A 1-13 (k(cat)/K(m) = 3.1 x 10(6) m(-1) s(-1)). The ACE2 catalytic efficiency is 400-fold higher with angiotensin II () as a substrate than with angiotensin I (). ACE2 also efficiently hydrolyzes des-Arg(9)-bradykinin (k(cat)/K(m) = 1.3 x 10(5) m(-1) s(-1)), but it does not hydrolyze bradykinin. An alignment of the ACE2 peptide substrates reveals a consensus sequence of: Pro-X((1-3 residues))-Pro-Hydrophobic, where hydrolysis occurs between proline and the hydrophobic amino acid.

3-Deoxy- D - manno -2-octulosonate-8-phosphate (KDO-8-P) synthase catalyzes the aldol-type condensation of phosphoenolpyruvate and D -arabinose-5-phosphate (A-5-P) to produce KDO-8-P and inorganic phosphate. All KDO-8-P synthases, as exemplified by the enzyme from Escherichia coli , were believed not to require a metal cofactor for catalytic activity. However, recent studies have demonstrated that the KDO-8-P synthase from Aquifex aeolicus is a metalloenzyme. Moreover, sequence alignments and phylogenetic analysis of KDO-8-P synthase protein sequences strongly suggested that there is a whole subfamily of KDO-8-P synthases that are also metalloenzymes. One of these putative metalloenzymes is the ortholog from the human pathogen Helicobacter pylori . In order to test this model, we have cloned the kdsa gene encoding H. pylori KDO-8-P synthase, and overexpressed and purified the protein. This enzyme was found to bind one mol Zn/mol monomer, and the removal of this metal by treatment with 2,6-pyridine dicarboxylic acid abolished enzymatic activity. The Zn 2+ in the enzyme could be quantitatively replaced by Cd 2+ , which increased the observed k cat by ∼2-fold, and decreased the apparent K m (A-5-P) by ∼6.5-fold. Furthermore, removal of the Zn 2+ from the enzyme did not greatly perturb its circular dichroism spectra. Thus, the divalent metal most likely serves as cofactor directly involved in catalysis.

Smyd3 is a lysine methyltransferase implicated in chromatin and cancer regulation. Here we show that Smyd3 catalyzes histone H4 methylation at lysine 5 (H4K5me). This novel histone methylation mark is detected in diverse cell types and its formation is attenuated by depletion of Smyd3 protein. Further, Smyd3-driven cancer cell phenotypes require its enzymatic activity. Thus, Smyd3, via H4K5 methylation, provides a potential new link between chromatin dynamics and neoplastic disease.

Deregulation of lysine methylation signalling has emerged as a common aetiological factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumours. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP kinase signalling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma and lung adenocarcinoma, we found that abrogating SMYD3 catalytic activity inhibits tumour development in response to oncogenic Ras. We used protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signalling module and SMYD3 depletion synergizes with a MEK inhibitor to block Ras-driven tumorigenesis. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signalling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signalling.

The bromodomain and extraterminal (BET) protein BRD2-4 inhibitors hold therapeutic promise in preclinical models of hematologic malignancies. However, translation of these data to molecules suitable for clinical development has yet to be accomplished. Herein we expand the mechanistic understanding of BET inhibitors in multiple myeloma by using the chemical probe molecule I-BET151. I-BET151 induces apoptosis and exerts strong antiproliferative effect in vitro and in vivo. This is associated with contrasting effects on oncogenic MYC and HEXIM1, an inhibitor of the transcriptional activator P-TEFb. I-BET151 causes transcriptional repression of MYC and MYC-dependent programs by abrogating recruitment to the chromatin of the P-TEFb component CDK9 in a BRD2-4-dependent manner. In contrast, transcriptional upregulation of HEXIM1 is BRD2-4 independent. Finally, preclinical studies show that I-BET762 has a favorable pharmacologic profile as an oral agent and that it inhibits myeloma cell proliferation, resulting in survival advantage in a systemic myeloma xenograft model. These data provide a strong rationale for extending the clinical testing of the novel antimyeloma agent I-BET762 and reveal insights into biologic pathways required for myeloma cell proliferation.

SUMMARY Helicobacter pylori is a gram-negative bacteria which colonizes the gastric mucosa of humans and is implicated in a wide range of gastroduodenal diseases. This paper reviews the physiology of this bacterium as predicted from the sequenced genomes of two unrelated strains and reconciles these predictions with the literature. In general, the predicted capabilities are in good agreement with reported experimental observations. H. pylori is limited in carbohydrate utilization and will use amino acids, for which it has transporter systems, as sources of carbon. Energy can be generated by fermentation, and the bacterium possesses components necessary for both aerobic and anaerobic respiration. Sulfur metabolism is limited, whereas nitrogen metabolism is extensive. There is active uptake of DNA via transformation and ample restriction-modification activities. The cell contains numerous outer membrane proteins, some of which are porins or involved in iron uptake. Some of these outer membrane proteins and the lipopolysaccharide may be regulated by a slipped-strand repair mechanism which probably results in phase variation and plays a role in colonization. In contrast to a commonly held belief that H. pylori is a very diverse species, few differences were predicted in the physiology of these two unrelated strains, indicating that host and environmental factors probably play a significant role in the outocme of H. pylori-related disease.

Dihydropyran-2-one possessing a sulfamate moiety at the 4-position of the thiophenyl ring were designed to reach S 3 ′ pocket of the HIV protease. Synthetic routes for the preparation of thiotosylates possessing 3-(2- t -butyl-5-methyl-4-sulfamate) phenylthio moiety were established. SAR of various sulfamate analogs including HIV protease binding affinities, antiviral activities and therapeutic indices will be described.

5,6-Dihydro-2H-pyran-2-ones are potent inhibitors of HIV-1 protease, which bind to the S1, S2, S1', and S2' pockets and have a unique binding mode with the catalytic aspartyl groups and the flap region of the enzyme. Efforts to explore 3-position heterocyclic scaffolds that bind to the S1' and S2' pockets have provided a number of selected analogs that display high HIV-1 protease inhibitory activity. reserved.

Dihydropyran-2-ones possessing amino and carboxamide functionalities on 3-SPh (2-tert-butyl, 5-methyl) ring were synthesized and evaluated for their antiviral activities. Both the enantiomers of inhibitor 15 were synthesized. The in vitro resistance profile, inhibitory activities against cytochrome P450 isozymes and pharmacokinetic properties of inhibitor 15S will be discussed.

Helicobacter pylori, one of the most common bacterial pathogens of humans, colonizes the gastric mucosa, where it appears to persist throughout the host's life unless the patient is treated. Colonization induces chronic gastric inflammation which can progress to a variety of diseases, ranging in severity from superficial gastritis and peptic ulcer to gastric cancer and mucosal-associated lymphoma. Strain-specific genetic diversity has been proposed to be involved in the organism's ability to cause different diseases or even be beneficial to the infected host and to participate in the lifelong chronicity of infection. Here we compare the complete genomic sequences of two unrelated H. pylori isolates. This is, to our knowledge, the first such genomic comparison. H. pylori was believed to exhibit a large degree of genomic and allelic diversity, but we find that the overall genomic organization, gene order and predicted proteomes (sets of proteins encoded by the genomes) of the two strains are quite similar. Between 6 to 7% of the genes are specific to each strain, with almost half of these genes being clustered in a single hypervariable region.

The 4-hydroxy-5,6-dihydropyrone template was utilized as a flexible scaffolding from which to build potent active site inhibitors of HIV protease. Dihydropyrone 1c (5,6-dihydro-4-hydroxy-6-phenyl-3-[(2-phenylethyl)thio]-2H-pyran-2-one) was modeled in the active site of HIV protease utilizing a similar binding mode found for the previously reported 4-hydroxybenzopyran-2-ones. Our model led us to pursue the synthesis of 6,6-disubstituted dihydropyrones with the aim of filling S1 and S2 and thereby increasing the potency of the parent dihydropyrone 1c which did not fill S2. Toward this end we attached various hydrophobic and hydrophilic side chains at the 6-position of the dihydropyrone to mimic the natural and unnatural amino acids known to be effective substrates at P2 and P2'. Parent dihydropyrone 1c (IC50 = 2100 nM) was elaborated into compounds with greater than a 100-fold increase in potency [18c, IC50 = 5 nM, 5-(3,6-dihydro-4-hydroxy-6-oxo-2-phenyl-5-[2-phenylethyl)thio] -2H-pyran-2-yl)pentanoic acid and 12c, IC50 = 51 nM, 5,6-dihydro-4-hydroxy-6-phenyl-6-(2-phenylethyl)-3- [(2-phenyl-ethyl)thio]-2H-pyran-2-one]. Optimization of the 3-position fragment to fill S1' and S2' afforded potent HIV protease inhibitor 49 [IC50 = 10 nM, 3-[(2-tert-butyl-5-methylphenyl)sulfanyl]-5,6-dihydro-4 -hydroxy-6-phenyl-6-(2-phenylethyl)-2H-pyran-2-one]. The resulting low molecular weight compounds (475) have one or no chiral centers and are readily synthesized.

As part of the National Cancer Institute's Drug Screening Program, a new class of antiretrovirals active against the human immunodeficiency virus HIV-1 has been identified, and the HIV-1 nucleocapsid protein NCp7 was proposed as the target of antiviral action. The 2,2′-dithiobis-[4′-(sulfamoyl)benzanilide] ( 3x ) and the 2,2′-dithiobis(5-acetylamino)benzamide ( 10 ) represented the prototypic lead structures. A wide variety of 2,2′-dithiobisbenzamides were prepared and tested for anti-HIV-1 activity, cytotoxicity, and their ability to extrude zinc from the zinc fingers for NCp7. The structure-activity relationships demonstrated that the ability to extrude zinc from NCp7 resided in the 2,2′-dithiobisbenzamide core structure. The 3,3′ and the 4,4′ isomers were inactive. While many analogs based upon the core structure retained the zinc extrusion activity, the best overall anti-HIV-1 activity was only found in a narrow set of derivatives possessing carboxylic acid, carboxamide, or phenylsulfonamide functional groups. These functional groups were more important for reducing cytotoxicity than improving antiviral potency or activity vs NCp7. All of the compounds with antiviral activity also extruded zinc from NCp7. From this study several classes of low μM anti-HIV agents with simple chemical structures were identified as possible chemotherapeutic agents for the treatment of AIDS.

BET family proteins are epigenetic regulators known to control expression of genes involved in cell growth and oncogenesis. Selective inhibitors of BET proteins exhibit potent anti-proliferative activity in a number of hematologic cancer models, in part through suppression of the MYC oncogene and downstream Myc-driven pathways. However, little is currently known about the activity of BET inhibitors in solid tumor models, and whether down-regulation of MYC family genes contributes to sensitivity. Here we provide evidence for potent BET inhibitor activity in neuroblastoma, a pediatric solid tumor associated with a high frequency of MYCN amplifications. We treated a panel of neuroblastoma cell lines with a novel small molecule inhibitor of BET proteins, GSK1324726A (I-BET726), and observed potent growth inhibition and cytotoxicity in most cell lines irrespective of MYCN copy number or expression level. Gene expression analyses in neuroblastoma cell lines suggest a role of BET inhibition in apoptosis, signaling, and N-Myc-driven pathways, including the direct suppression of BCL2 and MYCN. Reversal of MYCN or BCL2 suppression reduces the potency of I-BET726-induced cytotoxicity in a cell line-specific manner; however, neither factor fully accounts for I-BET726 sensitivity. Oral administration of I-BET726 to mouse xenograft models of human neuroblastoma results in tumor growth inhibition and down-regulation MYCN and BCL2 expression, suggesting a potential role for these genes in tumor growth. Taken together, our data highlight the potential of BET inhibitors as novel therapeutics for neuroblastoma, and suggest that sensitivity is driven by pleiotropic effects on cell growth and apoptotic pathways in a context-specific manner.

Systematic substitutions on 6-phenyl and 3-SCH 2 Ph rings of inhibitor 1 , were carried out to optimize the inhibitory activity against HIV PR. These studies lead to 3-Sbenzyl esters with enhanced potency. The X-ray crystal structure of 32 bound to HIV PR revealed that the 3-SCH 2 phenyl group is occupying the P 2 ′ pocket, which is contrary to the binding mode of 1 (derivative lacking ortho isopropyl ester group). In the latter case, benzyl group occupies the P 1 ′ pocket.

In a study of 4-hydroxy-pyran-2-ones as possible inhibitors of HIV protease, a series of compounds were synthesized following the Topliss operational scheme for substitution on a phenyl group at the 6 position of the pyrone. In addition, a number of compounds with polar substituents were made.

Publications from our laboratories have recently described a series of 3-thioaryl substituted-4-hydroxy-pyrones1 as HIV protease inhibitors. The current work examines the analogous 5,6-dihydro-2H-pyran-2-ones with 6,6-substitutions focusing on the use of 1°, 2°, and 3° alkyl amides of various chain lengths to fill the S1 through S3 enzyme pockets.

New templates were designed and prepared which straddle the active site of HIV-1 protease. These templates were designed to be "flexible scaffolds' upon which substituents could be appended to fill the pockets of HIV protease. The new templates prepared and analysed were 4-hydroxy-5H-furan-2-ones, 4-hydroxy-5,6-dihydropyrones, 3-hydroxy-cyclohex-2-enones, and 4-hydroxy-2(1H)-pyridinones, of which the 4-hydroxy-5,6-dihydropyrones were found to be the most potent inhibitors of HIV-1 protease.

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.