John E. Kerrigan

Topoisomerase IIβ–Mediated DNA Double-Strand Breaks: Implications in Doxorubicin Cardiotoxicity and Prevention by Dexrazoxane

Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.

Discovery of a small-molecule inhibitor and cellular probe of Keap1-Nrf2 protein-protein interaction.

A high-throughput screen (HTS) of the MLPCN library using a homogenous fluorescence polarization assay identified a small molecule as a first-in-class direct inhibitor of Keap1-Nrf2 protein-protein interaction. The HTS hit has three chiral centers; a combination of flash and chiral chromatographic separation demonstrated that Keap1-binding activity resides predominantly in one stereoisomer (SRS)-5 designated as ML334 (LH601A), which is at least 100× more potent than the other stereoisomers. The stereochemistry of the four cis isomers was assigned using X-ray crystallography and confirmed using stereospecific synthesis. (SRS)-5 is functionally active in both an ARE gene reporter assay and an Nrf2 nuclear translocation assay. The stereospecific nature of binding between (SRS)-5 and Keap1 as well as the preliminary but tractable structure-activity relationships support its use as a lead for our ongoing optimization.

A G-quadruplex stabilizer induces M-phase cell cycle arrest.

G-quadruplex stabilizers such as telomestatin and HXDV bind with exquisite specificity to G-quadruplexes, but not to triplex, duplex, or single-stranded DNAs. Studies have suggested that the antiproliferative and possibly anti-tumor activities of these compounds are linked to their inhibitory effect on telomerase and/or telomere function. In the current studies, we show that HXDV, a synthetic analog of telomestatin, exhibits antiproliferative activity against both telomerase-positive and -negative cells and induces robust apoptosis within 16 h of treatment, suggesting a mode of action independent of telomerase. HXDV was also shown to inhibit cell cycle progression causing M-phase cell cycle arrest, as evidenced by accumulation of cells with 4 n DNA content, increased mitotic index, separated centrosomes, elevated histone H3 phosphorylation at Ser-10 (an M-phase marker), and defective chromosome alignment and spindle fiber assembly (revealed by time-lapse microscopy). The M-phase arrest caused by HXDV paralleled with reduction in the expression level of the major M-phase checkpoint regulator Aurora A. All these cellular effects appear to depend on the G-quadruplex binding activity of HXDV as its non-G-quadruplex binding analog, TXTLeu, is completely devoid of all these effects. In the aggregate, our results suggest that HXDV, which exhibits anti-proliferative and apoptotic activities, is also a novel M-phase blocker, with a mode of action dependent on its G-quadruplex binding activity.

Models of glycoprotein hormone receptor interaction

The glycoprotein hormones regulate reproduction and development through their interactions with receptors in ovarian, testicular, and thyroid tissues. Efforts to design hormone agonists and antagonists useful for treating infertility and hyperthyroidism would benefit from a molecular understanding of hormone-receptor interaction. The structure of a complex containing FSH bound to a fragment of its receptor has been determined at 2.9 Å resolution, but this does not explain several observations made with cell-surface G protein receptors and may reflect the manner in which FSH binds a short alternate spliced receptor form. We discuss observations that must be explained by any model of the cell-surface G protein-coupled glycoprotein hormone receptors and suggest structures for these receptors that satisfy these requirements. Glycoprotein hormones appear to contact two distinct sites in the extracellular domains of their receptors, not just the leucine-rich repeat domain. These dual contacts contribute to ligand binding specificity and appear to be essential for signal transduction. As outlined in this minireview, differences in the manners in which these ligands contact their receptors explain why some ligands and ligand analogs interact with more than one class of receptor and why some receptors and receptor analogs bind more than one ligand. The unique manner in which these ligands appear to interact with their receptors may have facilitated hormone and receptor co-evolution during early vertebrate speciation.

Dietary Isothiocyanate-induced Apoptosis via Thiol Modification of DNA Topoisomerase IIα

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.

Molecular dynamics simulations in drug design.

This minireview focuses on recent developments in the application of molecular dynamics to drug design. Recent applications of endpoint free-energy computational methods such as molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and generalized Born surface area (MM-GBSA) and linear response methods are described. Recent progress in steered molecular dynamics applied to drug design is reviewed.

5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-ones and related compounds as TOP1-targeting agents: influence of structure on the ternary cleavable complex formation.

In this paper, we present our results from a docking study of the title compounds with the DNA/topoisomerase I complex based on the recently published X-ray crystal structure of the topotecan/DNA/topoisomerase I ternary cleavable complex (Staker, B.L., et al. PNAS 2002, 99, 15387) using the Autodock program. Simple intermolecular docking energies (E(dock)) correlate well with in vitro DNA cleavage data suggesting that the binding mode from the crystal structure is a reasonable binding mode for these compounds.

Functional annotation of rare gene aberration drivers of pancreatic cancer

As we enter the era of precision medicine, characterization of cancer genomes will directly influence therapeutic decisions in the clinic. Here we describe a platform enabling functionalization of rare gene mutations through their high-throughput construction, molecular barcoding and delivery to cancer models for in vivo tumour driver screens. We apply these technologies to identify oncogenic drivers of pancreatic ductal adenocarcinoma (PDAC). This approach reveals oncogenic activity for rare gene aberrations in genes including NAD Kinase (NADK), which regulates NADP(H) homeostasis and cellular redox state. We further validate mutant NADK, whose expression provides gain-of-function enzymatic activity leading to a reduction in cellular reactive oxygen species and tumorigenesis, and show that depletion of wild-type NADK in PDAC cell lines attenuates cancer cell growth in vitro and in vivo. These data indicate that annotating rare aberrations can reveal important cancer signalling pathways representing additional therapeutic targets.

Enhanced Degradation of Dihydrofolate Reductase through Inhibition of NAD Kinase by Nicotinamide Analogs

Dihydrofolate reductase (DHFR), because of its essential role in DNA synthesis, has been targeted for the treatment of a wide variety of human diseases, including cancer, autoimmune diseases, and infectious diseases. Methotrexate (MTX), a tight binding inhibitor of DHFR, is one of the most widely used drugs in cancer treatment and is especially effective in the treatment of acute lymphocytic leukemia, non-Hodgkin’s lymphoma, and osteosarcoma. Limitations to its use in cancer include natural resistance and acquired resistance due to decreased cellular uptake and decreased retention due to impaired polyglutamylate formation and toxicity at higher doses. Here, we describe a novel mechanism to induce DHFR degradation through cofactor depletion in neoplastic cells by inhibition of NAD kinase, the only enzyme responsible for generating NADP, which is rapidly converted to NADPH by dehydrogenases/reductases. We identified an inhibitor of NAD kinase, thionicotinamide adenine dinucleotide phosphate (NADPS), which led to accelerated degradation of DHFR and to inhibition of cancer cell growth. Of importance, combination treatment of NADPS with MTX displayed significant synergy in a metastatic colon cancer cell line and was effective in a MTX-transport resistant leukemic cell line. We suggest that NAD kinase is a valid target for further inhibitor development for cancer treatment.

NAD+ Kinase as a Therapeutic Target in Cancer

NAD+ kinase (NADK) catalyzes the phosphorylation of nicotinamide adenine dinucleotide (NAD+) to nicotinamide adenine dinucleotide phosphate (NADP+) using ATP as the phosphate donor. NADP+ is then reduced to NADPH by dehydrogenases, in particular glucose-6-phosphate dehydrogenase and the malic enzymes. NADPH functions as an important cofactor in a variety of metabolic and biosynthetic pathways. The demand for NADPH is particularly high in proliferating cancer cells, where it acts as a cofactor for the synthesis of nucleotides, proteins, and fatty acids. Moreover, NADPH is essential for the neutralization of the dangerously high levels of reactive oxygen species (ROS) generated by increased metabolic activity. Given its key role in metabolism and regulation of ROS, it is not surprising that several recent studies, including in vitro and in vivo assays of tumor growth and querying of patient samples, have identified NADK as a potential therapeutic target for the treatment of cancer. In this review, we will discuss the experimental evidence justifying further exploration of NADK as a clinically relevant drug target and describe our studies with a lead compound, thionicotinamide, an NADK inhibitor prodrug. Clin Cancer Res; 22(21); 5189–95. ©2016 AACR.