New Transmission-Selective Antimalarial Agents through Hit-to-Lead Optimization of 2-([1,1'-Biphenyl]-4-carboxamido)benzoic Acid Derivatives.

Malaria elimination requires multipronged approaches, including the application of antimalarial drugs able to block human-to-mosquito transmission of malaria parasites. The transmissible gametocytes of Plasmodium falciparum seem to be highly sensitive towards epidrugs, particularly those targeting demethylation of histone post-translational marks. Here, we report exploration of compounds from a chemical library generated during hit-to-lead optimization of inhibitors of the human histone lysine demethylase, KDM4B. Derivatives of 2-([1,1'-biphenyl]-4-carboxamido) benzoic acid, around either the amide or a sulfonamide linker backbone (2-(arylcarboxamido)benzoic acid, 2-carboxamide (arylsulfonamido)benzoic acid and N-(2-(1H-tetrazol-5-yl)phenyl)-arylcarboxamide), showed potent activity towards late-stage gametocytes (stage IV/V) of P. falciparum, with the most potent compound reaching single digit nanomolar activity. Structure-activity relationship trends were evident and frontrunner compounds also displayed microsomal stability and favourable solubility profiles. Simplified synthetic routes support further derivatization of these compounds for further development of these series as malaria transmission-blocking agents.

Polyamine catabolism and oxidative damage.

Polyamines (PAs) are indispensable polycations ubiquitous to all living cells. Among their many critical functions, PAs contribute to the oxidative balance of the cell. Beginning with studies by the Tabor laboratory in bacteria and yeast, the requirement for PAs as protectors against oxygen radical-mediated damage has been well established in many organisms, including mammals. However, PAs also serve as substrates for oxidation reactions that produce hydrogen peroxide (H2O2) both intra- and extracellularly. As intracellular concentrations of PAs can reach millimolar concentrations, the H2O2 amounts produced through their catabolism, coupled with a reduction in protective PAs, are sufficient to cause the oxidative damage associated with many pathologies, including cancer. Thus, the maintenance of intracellular polyamine homeostasis may ultimately contribute to the maintenance of oxidative homeostasis. Again, pioneering studies by Tabor and colleagues led the way in first identifying spermine oxidase in Saccharomyces cerevisiae. They also first purified the extracellular bovine serum amine oxidase and elucidated the products of its oxidation of primary amine groups of PAs when included in culture medium. These investigations formed the foundation for many polyamine-related studies and experimental procedures still performed today. This Minireview will summarize key innovative studies regarding PAs and oxidative damage, starting with those from the Tabor laboratory and including the most recent advances, with a focus on mammalian systems.

Targeting polyamine metabolism for cancer therapy and prevention.

The chemically simple, biologically complex eukaryotic polyamines, spermidine and spermine, are positively charged alkylamines involved in many crucial cellular processes. Along with their diamine precursor putrescine, their normally high intracellular concentrations require fine attenuation by multiple regulatory mechanisms to keep these essential molecules within strict physiologic ranges. Since the metabolism of and requirement for polyamines are frequently dysregulated in neoplastic disease, the metabolic pathway and functions of polyamines provide rational drug targets; however, these targets have been difficult to exploit for chemotherapy. It is the goal of this article to review the latest findings in the field that demonstrate the potential utility of targeting the metabolism and function of polyamines as strategies for both chemotherapy and, possibly more importantly, chemoprevention.

Transcriptional activity of the islet ß cell factor Pdx1 is augmented by lysine methylation catalyzed by the methyltransferase Set7/9.

The transcription factor Pdx1 is crucial to islet ß cell function and regulates target genes in part through interaction with coregulatory factors. Set7/9 is a Lys methyltransferase that interacts with Pdx1. Here we tested the hypothesis that Lys methylation of Pdx1 by Set7/9 augments Pdx1 transcriptional activity. Using mass spectrometry and mutational analysis of purified proteins, we found that Set7/9 methylates the N-terminal residues Lys-123 and Lys-131 of Pdx1. Methylation of these residues occurred only in the context of intact, full-length Pdx1, suggesting a specific requirement of secondary and/or tertiary structural elements for catalysis by Set7/9. Immunoprecipitation assays and mass spectrometric analysis using ß cells verified Lys methylation of endogenous Pdx1. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed a requirement of Pdx1 residue Lys-131, but not Lys-123, for transcriptional augmentation by Set7/9. Lys-131 was not required for high-affinity interactions with DNA in vitro, suggesting that its methylation likely enhances post-DNA binding events. To define the role of Set7/9 in ß cell function, we generated mutant mice in which the gene encoding Set7/9 was conditionally deleted in ß cells (Set(Δ)ß). Set(Δ)ß mice exhibited glucose intolerance similar to Pdx1-deficient mice, and their isolated islets showed impaired glucose-stimulated insulin secretion with reductions in expression of Pdx1 target genes. Our results suggest a previously unappreciated role for Set7/9-mediated methylation in the maintenance of Pdx1 activity and ß cell function.

Structure-activity study for (bis)ureidopropyl- and (bis)thioureidopropyldiamine LSD1 inhibitors with 3-5-3 and 3-6-3 carbon backbone architectures.

Methylation at specific histone lysine residues is a critical post-translational modification that alters chromatin architecture, and dysregulated lysine methylation/demethylation is associated with the silencing of tumor suppressor genes. The enzyme lysine-specific demethylase 1 (LSD1) complexed to specific transcription factors catalyzes the oxidative demethylation of mono- and dimethyllysine 4 of histone H3 (H3K4me and H3K4me2, respectively). We have previously reported potent (bis)urea and (bis)thiourea LSD1 inhibitors that increase cellular levels of H3K4me and H3K4me2, promote the re-expression of silenced tumor suppressor genes and suppress tumor growth in vitro. Here we report the design additional (bis)urea and (bis)thiourea LSD1 inhibitors that feature 3-5-3 or 3-6-3 carbon backbone architectures. Three of these compounds displayed single-digit IC50 values in a recombinant LSD1 assay. In addition, compound 6d exhibited an IC50 of 4.2µM against the Calu-6 human lung adenocarcinoma line, and 4.8µM against the MCF7 breast tumor cell line, in an MTS cell viability assay. Following treatment with 6b-6d, Calu-6 cells exhibited a significant increase in the mRNA expression for the silenced tumor suppressor genes SFRP2, HCAD and p16, and modest increases in GATA4 message. The compounds described in this paper represent the most potent epigenetic modulators in this series, and have potential for use as antitumor agents.

Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites.

A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure-activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea-polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites.

The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines.

Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl-H3K4 histone marks that are associated with active gene expression. As most post-translational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N (5)-[3,3-(diphenyl)propyl]-N(1)-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the re-expression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the e-cadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated promising clinical responses to agents targeting epigenetic silencing, this polyamine analogue LSD1 inhibitor presents an exciting new avenue for the development of novel therapeutic agents for the treatment of AML.

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites.

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5 weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.

Design of small molecule epigenetic modulators.

The field of epigenetics has expanded rapidly to reveal multiple new targets for drug discovery. The functional elements of the epigenomic machinery can be categorized as writers, erasers and readers, and together these elements control cellular gene expression and homeostasis. It is increasingly clear that aberrations in the epigenome can underly a variety of diseases, and thus discovery of small molecules that modulate the epigenome in a specific manner is a viable approach to the discovery of new therapeutic agents. In this Digest, the components of epigenetic control of gene expression will be briefly summarized, and efforts to identify small molecules that modulate epigenetic processes will be described.

Loss of LSD1 (lysine-specific demethylase 1) suppresses growth and alters gene expression of human colon cancer cells in a p53- and DNMT1(DNA methyltransferase 1)-independent manner.

Epigenetic silencing of gene expression is important in cancer. Aberrant DNA CpG island hypermethylation and histone modifications are involved in the aberrant silencing of tumour-suppressor genes. LSD1 (lysine-specific demethylase 1) is a H3K4 (histone H3 Lys4) demethylase associated with gene repression and is overexpressed in multiple cancer types. LSD1 has also been implicated in targeting p53 and DNMT1 (DNA methyltransferase 1), with data suggesting that the demethylating activity of LSD1 on these proteins is necessary for their stabilization. To examine the role of LSD1 we generated LSD1 heterozygous (LSD1+/-) and homozygous (LSD1-/-) knockouts in the human colorectal cancer cell line HCT116. The deletion of LSD1 led to a reduced cell proliferation both in vitro and in vivo. Surprisingly, the knockout of LSD1 in HCT116 cells did not result in global increases in its histone substrate H3K4me2 (dimethyl-H3K4) or changes in the stability or function of p53 or DNMT1. However, there was a significant difference in gene expression between cells containing LSD1 and those null for LSD1. The results of the present study suggested that LSD1 is critical in the regulation of cell proliferation, but also indicated that LSD1 is not an absolute requirement for the stabilization of either p53 or DNMT1.

Polyamine catabolism contributes to enterotoxigenic Bacteroides fragilis-induced colon tumorigenesis.

It is estimated that the etiology of 20-30% of epithelial cancers is directly associated with inflammation, although the direct molecular events linking inflammation and carcinogenesis are poorly defined. In the context of gastrointestinal disease, the bacterium enterotoxigenic Bacteroides fragilis (ETBF) is a significant source of chronic inflammation and has been implicated as a risk factor for colorectal cancer. Spermine oxidase (SMO) is a polyamine catabolic enzyme that is highly inducible by inflammatory stimuli resulting in increased reactive oxygen species (ROS) and DNA damage. We now demonstrate that purified B. fragilis toxin (BFT) up-regulates SMO in HT29/c1 and T84 colonic epithelial cells, resulting in SMO-dependent generation of ROS and induction of γ-H2A.x, a marker of DNA damage. Further, ETBF-induced colitis in C57BL/6 mice is associated with increased SMO expression and treatment of mice with an inhibitor of polyamine catabolism, N(1),N(4)-bis(2,3-butandienyl)-1,4-butanediamine (MDL 72527), significantly reduces ETBF-induced chronic inflammation and proliferation. Most importantly, in the multiple intestinal neoplasia (Min) mouse model, treatment with MDL 72527 reduces ETBF-induced colon tumorigenesis by 69% (P < 0.001). The results of these studies indicate that SMO is a source of bacteria-induced ROS directly associated with tumorigenesis and could serve as a unique target for chemoprevention.

Establishing New Isosexual Pairs in Adult Male Guinea Pigs (Cavia porcellus) to Facilitate Social Housing.

Guinea pigs (Cavia porcellus) are a commonly used species in biomedical research. As social creatures, compatible guinea pigs should be housed together unless scientific objectives or veterinary care require otherwise. Extensive literature suggests that adult male guinea pigs are highly aggressive in the presence of females, but data are lacking regarding the compatibility of cohoused adult males in the absence of females. Most studies that use adult males do not report housing densities. We used serial wound scoring and observations of behavior to determine whether unfamiliar adult male guinea pigs will develop stable, prosocial isosexual pairs. Wound scoring was performed before and 24 h after pairing. Serial behavioral observations assessed affiliative and agonistic behaviors at 0.5, 2, 24, and 48 h after pairing. Wound scoring and behavioral observations continued weekly for 1 mo and monthly thereafter. Wound scores were significantly higher at 24 h after pairing as compared with baseline and all other time points. Wounding was rare after week 2, indicating reduced aggression. Furthermore, affiliative behaviors significantly increased over time while agonistic behaviors were rare. Together, these data suggest that unfamiliar adult male guinea pigs establish stable prosocial pairs after an acclimation period. As was done in the present study, providing ample space, separate shelters for each animal, and the absence of female guinea pigs will likely facilitate successful pairing. We recommend consideration of a social housing program for adult male guinea pigs to provide companionship and enrich their housing environment.

Polyamines and cancer: implications for chemotherapy and chemoprevention.

Polyamines are small organic cations that are essential for normal cell growth and development in eukaryotes. Under normal physiological conditions, intracellular polyamine concentrations are tightly regulated through a dynamic network of biosynthetic and catabolic enzymes, and a poorly characterised transport system. This precise regulation ensures that the intracellular concentration of polyamines is maintained within strictly controlled limits. It has frequently been observed that the metabolism of, and the requirement for, polyamines in tumours is frequently dysregulated. Elevated levels of polyamines have been associated with breast, colon, lung, prostate and skin cancers, and altered levels of rate-limiting enzymes in both biosynthesis and catabolism have been observed. Based on these observations and the absolute requirement for polyamines in tumour growth, the polyamine pathway is a rational target for chemoprevention and chemotherapeutics. Here we describe the recent advances made in the polyamine field and focus on the roles of polyamines and polyamine metabolism in neoplasia through a discussion of the current animal models for the polyamine pathway, chemotherapeutic strategies that target the polyamine pathway, chemotherapeutic clinical trials for polyamine pathway-specific drugs and ongoing clinical trials targeting polyamine biosynthesis.

Ligand modifications to reduce the relative resistance of multi-drug resistant HIV-1 protease.

Proper proteolytic processing of the HIV-1 Gag/Pol polyprotein is required for HIV infection and viral replication. This feature has made HIV-1 protease an attractive target for antiretroviral drug design for the treatment of HIV-1 infected patients. To examine the role of the P1 and P1'positions of the substrate in inhibitory efficacy of multi-drug resistant HIV-1 protease 769 (MDR 769), we performed a series of structure-function studies. Using the original CA/p2 cleavage site sequence, we generated heptapeptides containing one reduced peptide bond with an L to F and A to F double mutation at P1 and P1' (F-r-F), and an A to F at P1' (L-r-F) resulting in P1/P1' modified ligands. Here, we present an analysis of co-crystal structures of CA/p2 F-r-F, and CA/p2 L-r-F in complex with MDR 769. To examine conformational changes in the complex structure, molecular dynamic (MD) simulations were performed with MDR769-ligand complexes. MD trajectories show the isobutyl group of both the lopinavir analog and the CA/p2 L-r-F substrate cause a conformational change of in the active site of MDR 769. IC50 measurements suggest the non identical P1/P1' ligands (CA/p2 L-r-F and lopinavir analog) are more effective against MDR proteases as opposed to identical P1/P1'ligands. Our results suggest that a non identical P1/P1'composition may be more favorable for the inhibition of MDR 769 as they induce conformational changes in the active site of the enzyme resulting in disruption of the two-fold symmetry of the protease, thus, stabilizing the inhibitor in the active site.

Stimulation of natural killer cells with small molecule inhibitors of CD38 for the treatment of neuroblastoma.

High-risk neuroblastoma (NB) accounts for 15% of all pediatric cancer deaths. Refractory disease for high-risk NB patients is attributed to chemotherapy resistance and immunotherapy failure. The poor prognosis for high-risk NB patients demonstrates an unmet medical need for the development of new, more efficacious therapeutics. CD38 is an immunomodulating protein that is expressed constitutively on natural killer (NK) cells and other immune cells in the tumor microenvironment (TME). Furthermore, CD38 over expression is implicated in propagating an immunosuppressive milieu within the TME. Through virtual and physical screening, we have identified drug-like small molecule inhibitors of CD38 with low micromolar IC50 values. We have begun to explore structure activity relationships for CD38 inhibition through derivatization of our most effective hit molecule to develop a new compound with lead-like physicochemical properties and improved potency. We have demonstrated that our derivatized inhibitor, compound 2, elicits immunomodulatory effects in NK cells by increasing cell viability by 190 ± 36% in multiple donors and by significantly increasing interferon gamma. Additionally, we have illustrated that NK cells exhibited enhanced cytotoxicity toward NB cells (14% reduction of NB cells over 90 minutes) when given a combination treatment of our inhibitor and the immunocytokine ch14.18-IL2. Herein we describe the synthesis and biological evaluation of small molecule CD38 inhibitors and demonstrate their potential utility as a novel approach to NB immunotherapy. These compounds represent the first examples of small molecules that stimulate immune function for the treatment of cancer.

Alternative spermidine biosynthetic route is critical for growth of Campylobacter jejuni and is the dominant polyamine pathway in human gut microbiota.

The availability of fully sequenced bacterial genomes has revealed that many species known to synthesize the polyamine spermidine lack the spermidine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. We found that such species possess orthologues of the sym-norspermidine biosynthetic enzymes carboxynorspermidine dehydrogenase and carboxynorspermidine decarboxylase. By deleting these genes in the food-borne pathogen Campylobacter jejuni, we found that the carboxynorspermidine decarboxylase orthologue is responsible for synthesizing spermidine and not sym-norspermidine in vivo. In polyamine auxotrophic gene deletion strains of C. jejuni, growth is highly compromised but can be restored by exogenous sym-homospermidine and to a lesser extent by sym-norspermidine. The alternative spermidine biosynthetic pathway is present in many bacterial phyla and is the dominant spermidine route in the human gut, stomach, and oral microbiomes, and it appears to have supplanted the S-adenosylmethionine decarboxylase/spermidine synthase pathway in the gut microbiota. Approximately half of the gut Firmicutes species appear to be polyamine auxotrophs, but all encode the potABCD spermidine/putrescine transporter. Orthologues encoding carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase are found clustered with an array of diverse putrescine biosynthetic genes in different bacterial genomes, consistent with a role in spermidine, rather than sym-norspermidine biosynthesis. Due to the pervasiveness of ε-proteobacteria in deep sea hydrothermal vents and to the ubiquity of the alternative spermidine biosynthetic pathway in that phylum, the carboxyspermidine route is also dominant in deep sea hydrothermal vents. The carboxyspermidine pathway for polyamine biosynthesis is found in diverse human pathogens, and this alternative spermidine biosynthetic route presents an attractive target for developing novel antimicrobial compounds.

Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds.

Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.

Polyamine analogs modulate gene expression by inhibiting lysine-specific demethylase 1 (LSD1) and altering chromatin structure in human breast cancer cells.

Aberrant epigenetic repression of gene expression has been implicated in most cancers, including breast cancer. The nuclear amine oxidase, lysine-specific demethylase 1 (LSD1) has the ability to broadly repress gene expression by removing the activating mono- and di-methylation marks at the lysine 4 residue of histone 3 (H3K4me1 and me2). Additionally, LSD1 is highly expressed in estrogen receptor α negative (ER-) breast cancer cells. Since epigenetic marks are reversible, they make attractive therapeutic targets. Here we examine the effects of polyamine analog inhibitors of LSD1 on gene expression, with the goal of targeting LSD1 as a therapeutic modality in the treatment of breast cancer. Exposure of the ER-negative human breast cancer cells, MDA-MB-231 to the LSD1 inhibitors, 2d or PG11144, significantly increases global H3K4me1 and H3K4me2, and alters gene expression. Array analysis indicated that 98 (75 up and 23 down) and 477 (237 up and 240 down) genes changed expression by at least 1.5-fold or greater after treatment with 2d and PG11144, respectively. The expression of 12 up-regulated genes by 2d and 14 up-regulated genes by PG11144 was validated by quantitative RT-PCR. Quantitative chromatin immunoprecipitation (ChIP) analysis demonstrated that up-regulated gene expression by polyamine analogs is associated with increase of the active histone marks H3K4me1, H3K4me2 and H3K9act, and decrease of the repressive histone marks H3K9me2 and H3K27me3, in the promoter regions of the relevant target genes. These data indicate that the pharmacologic inhibition of LSD1 can effectively alter gene expression and that this therapeutic strategy has potential.

Knockdown of ornithine decarboxylase antizyme 1 causes loss of uptake regulation leading to increased N1, N11-bis(ethyl)norspermine (BENSpm) accumulation and toxicity in NCI H157 lung cancer cells.

Ornithine decarboxylase antizyme 1 (AZ1) is a major regulatory protein responsible for the regulation and degradation of ornithine decarboxylase (ODC). To better understand the role of AZ1 in polyamine metabolism and in modulating the response to anticancer polyamine analogues, a small interfering RNA strategy was used to create a series of stable clones in human H157 non-small cell lung cancer cells that expressed less than 5-10% of basal AZ1 levels. Antizyme 1 knockdown clones accumulated greater amounts of the polyamine analogue N (1),N (11)-bis(ethyl)norspermine (BENSpm) and were more sensitive to analogue treatment. The possibility of a loss of polyamine uptake regulation in the knockdown clones was confirmed by polyamine uptake analysis. These results are consistent with the hypothesis that AZ1 knockdown leads to dysregulation of polyamine uptake, resulting in increased analogue accumulation and toxicity. Importantly, there appears to be little difference between AZ1 knockdown cells and cells with normal levels of AZ1 with respect to ODC regulation, suggesting that another regulatory protein, potentially AZ2, compensates for the loss of AZ1. The results of these studies are important for the understanding of both the regulation of polyamine homeostasis and in understanding the factors that regulate tumor cell sensitivity to the anti-tumor polyamine analogues.