Stuart T. Hazeldine

Low Molecular Weight Amidoximes that Act as Potent Inhibitors of Lysine-Specific Demethylase 1

The recently discovered enzyme lysine-specific demethylase 1 (LSD1) plays an important role in the epigenetic control of gene expression, and aberrant gene silencing secondary to LSD1 dysregulation is thought to contribute to the development of cancer. We reported that (bis)guanidines, (bis)biguanides, and their urea- and thiourea isosteres are potent inhibitors of LSD1 and induce the re-expression of aberrantly silenced tumor suppressor genes in tumor cells in vitro. We now report a series of small molecule amidoximes that are moderate inhibitors of recombinant LSD1 but that produce dramatic changes in methylation at the histone 3 lysine 4 (H3K4) chromatin mark, a specific target of LSD1, in Calu-6 lung carcinoma cells. In addition, these analogues increase cellular levels of secreted frizzle-related protein (SFRP) 2, H-cadherin (HCAD), and the transcription factor GATA4. These compounds represent leads for an important new series of drug-like epigenetic modulators with the potential for use as antitumor agents.

Dual‐Function CXCR4 Antagonist Polyplexes To Deliver Gene Therapy and Inhibit Cancer Cell Invasion

A bicyclam-based biodegradable polycation with CXCR4 antagonistic activity was developed with potential for combined drug/gene cancer therapies. The dual-function polycation prevents cancer cell invasion by inhibiting CXCL12 stimulated CXCR4 activation, while at the same time efficiently and safely delivers plasmid DNA into cancer cells.

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

Background: Many bacteria synthesize spermidine but lack orthologues of polyamine biosynthetic enzymes S-adenosylmethionine decarboxylase and spermidine synthase. Results: An alternative spermidine biosynthetic pathway is essential in Campylobacter jejuni. Conclusion: The alternative route via carboxyspermidine is the dominant pathway in the human gut microbiota and deep sea hydrothermal vents. Significance: A multiplicity of polyamine biosynthetic pathways exist, providing novel targets for development of antimicrobial drugs. 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.

Bioisosteric Heterocyclic Versions of 7-{[2-(4-Phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol: Identification of Highly Potent and Selective Agonists for Dopamine D3 Receptor with Potent in Vivo Activity

In the current report, we extend the SAR study on our hybrid structure 7-{[2-(4-phenyl-piperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol further to include heterocyclic bioisosteric analogues. Binding assays were carried out with HEK-293 cells expressing either D2 or D3 receptors with tritiated spiperone to evaluate inhibition constants (Ki). Functional activity of selected compounds in stimulating GTPgammaS binding was assessed with CHO cells expressing human D2 receptors and AtT-20 cells expressing human D3 receptors. The highest binding affinity and selectivity for D3 receptors were exhibited by (-)-34 (Ki=0.92 nM and D2/D3=253). In the functional GTPgammaS binding assay, (-)-34 exhibited full agonist activity with picomolar affinity for D3 receptor with high selectivity (EC50=0.08 nM and D2/D3=248). In the in vivo rotational study, (-)-34 exhibited potent rotational activity in 6-OH-DA unilaterally lesioned rats with long duration of action, which indicates its potential application in neuroprotective treatment of Parkinsons disease.

The role of autophagy in the death of L1210 leukemia cells initiated by the new antitumor agents, XK469 and SH80

The phenoxypropionic acid derivative 2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid (XK469) and an analogue termed 2-{4-[(7-bromo-2-quinalinyl)oxy]phenoxy}propionic acid (SH80) can eradicate malignant cell types resistant to many common antitumor agents. Colony formation assays indicated that a 24 h exposure of L1210 cells to XK469 or SH80 inhibited clonogenic growth with CI90 values of 10 and 13 μmol/L, respectively. This effect was associated with G2-M arrest and the absence of any detectable markers of apoptosis (i.e., plasma membrane blebbing, procaspase 3 activation, loss of mitochondrial membrane potential, and formation of condensed chromatin). Drug-treated cells increased in size and eventually exhibited the characteristics of autophagy (i.e., appearance of autophagosomes and conversion of microtubule-associated protein light chain 3-I to 3-II). The absence of apoptosis was not related to an inhibition of the apoptotic program. Cultures treated with XK469 or SH80 readily underwent apoptosis upon exposure to the Bcl-2/Bcl-xL antagonist ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate. Continued incubation of drug-treated cells led to a reciprocal loss of large autophagic cells and the appearance of smaller cells that could not be stained with Höechst dye HO33342, had a chaotic morphology, were trypan blue–permeable, and lacked mitochondrial membrane potential. L1210 cells cotreated with the phosphatidylinositol-3-kinase inhibitor wortmannin, or having reduced Atg7 protein content, underwent G2-M arrest, but not autophagy, following XK469 treatment. Hence, the therapeutic actions of XK469/SH80 with L1210 cultures reflect both the initiation of a cell cycle arrest as well as the initiation of autophagy. [Mol Cancer Ther 2007;6(1):370–9]

Transplantable Syngeneic Rodent Tumors: Solid Tumors in Mice

As preclinical chemotherapists, we are often asked to identify experimental tumor models that can accurately predict for the drug response characteristics of all tumors of a given cellular subtype or molecular target. Unfortunately, it is impossible to give satisfactory answers to these inquiries. Because of the unique character of each independently arising tumor (whether spontaneous or induced), it does not take very long to realize that each tumor is a unique biologic entity with its own tumor growth behavior, histological appearance, drug response and molecular expression profiles. This is true whether the tumor is an experimental animal model or one originally derived from a patient. Further, many factors can influence the tumor growth and therapy response of experimental tumor models. Still, in vivo models are needed to adequately assess pharmacodynamics, toxicity and efficacy of any potential novel therapy. Presented herein is what we hope will be useful information regarding the transplant characteristics of tumor models, with some of the “pitfalls” to look out for when using any given tumor model for chemotherapy evaluations. Although most of the examples given use syngeneic models, the methodologies for assessing the predictive worth and maintaining model usefulness can be applied to almost any given transplantable tumor system (whether syngeneic or xenograft).

Development of functional poly(amido amine) CXCR4 antagonists with the ability to mobilize leukocytes and deliver nucleic acids.

CXCR4 chemokine receptor plays a crucial role in metastatic spread of multiple types of cancer. The present study reports on synthesis of functional polymers based on newly synthesized CXCR4-inhibiting monomers. The resultant linear polymeric CXCR4 antagonists (PCX) show improved ability to inhibit CXCR4 when compared with the monomers. The CXCR4 antagonism provides PCX with the ability to mobilize leukocytes from bone marrow to peripheral blood and to inhibit cancer cell invasion. Due to their cationic nature, PCX can form polyplexes with DNA and mediate efficient transfection. The reported findings validate PCX as promising dual-function polymeric drugs that can deliver therapeutic nucleic acids and improve cancer therapy by simultaneously inhibiting CXCR4 chemokine receptor.

Polyamine-based small molecule epigenetic modulators

Chromatin remodelling enzymes such as the histone deacetylases (HDACs) and histone demethylases such as lysine-specific demethylase 1 (LSD1) have been validated as targets for cancer drug discovery. Although a number of HDAC inhibitors have been marketed or are in human clinical trials, the search for isoform-specific HDAC inhibitors is an ongoing effort. In addition, the discovery and development of compounds targeting histone demethylases are in their early stages. Epigenetic modulators used in combination with traditional antitumor agents such as 5-azacytidine represent an exciting new approach to cancer chemotherapy. We have developed multiple series of HDAC inhibitors and LSD1 inhibitors that promote the re-expression of aberrantly silenced genes that are important in human cancer. The design, synthesis and biological activity of these analogues is described herein.

Cyclam-based polymeric copper chelators for gene delivery and potential PET imaging.

A series of reducible polycationic copper chelators (RPCs) based on 1,4,8,11-tetraazacyclotetradecane (cyclam) were synthesized by Michael addition. Molecular weight of the polycations was controlled by reaction stoichiometry and reaction conditions, resulting in polymers with molecular weights ranging from 4400 to 13 800. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II). The presence of disulfide bonds in the polycations resulted in substantially lower cytotoxicity than control 25 kDa poly(ethyleneimine). RPC as well as their complexes with Cu(II) exhibited high transfection activity in vitro. The reported polycationic Cu(II) chelates represent promising nucleic acid delivery vectors with potential for future theranostic applications.

Transplantable Syngeneic Rodent Tumors

For many decades, the results from transplantable tumor models have been viewed with considerable skepticism. The perception has long been that these models are excessively sensitive, and not predictive of the human disease. Although we do not intend to debate the many issues involved, it is our view that a better understanding of the transplant properties (e.g., take-rate) of the models—as well as a better understanding of the potential shortcomings in data presentation—will greatly aid the reader in the interpretation of these data. This chapter is an effort to summarize some of the basic operating characteristics of a wide range of solid-tumor models. Since this is a chemotherapy group, we can best explain some of the behavioral characteristics of these models within therapeutic experiments. Most of the data is drawn from the use of transplantable, syngeneic mouse tumors, but a few human tumors have been used for contrast.