Evgeny Kiselev

7-azaindenoisoquinolines as topoisomerase I inhibitors and potential anticancer agents.

A series of 7-azaindenoisoquinoline topoisomerase I (Top1) inhibitors have been prepared to investigate the effect of increased electron affinity of the aromatic system on the ability to stabilize the Top1-DNA cleavage complex. Ab initio calculations suggest that introduction of nitrogen into the aromatic system of the indenoisoquinolines would facilitate charge transfer complex formation with DNA, thus improving the π-π stacking interactions. The present study shows that 7-azaindenoisoquinolines demonstrate improved water solubility without any decrease in Top1 inhibitory activity or cytotoxicity. Analysis of the biological results reveals that smaller lactam ring substituents enable intercalation into both free DNA and Top1-DNA cleavage complex, whereas larger substituents only allow binding to the cleavage complex but not free DNA. Free DNA binding suppresses Top1-catalyzed DNA cleavage at high drug concentrations, whereas DNA cleavage and inhibition of religation occurs at low drug concentration.

Design, Synthesis and Evaluation of Dibenzo[c,h][1,6]naphthyridines as Topoisomerase I Inhibitors and Potential Anticancer Agents

Indenoisoquinoline topoisomerase I (Top1) inhibitors are a novel class of anticancer agents. Modifications of the indenoisoquinoline A, B, and D rings have been extensively studied in order to optimize Top1 inhibitory activity and cytotoxicity. To improve understanding of the forces that stabilize drug-Top1-DNA ternary complexes, the five-membered cyclopentadienone C-ring of the indenoisoquinoline system was replaced by six-membered nitrogen heterocyclic rings, resulting in dibenzo[c,h][1,6]naphthyridines that were synthesized by a novel route and tested for Top1 inhibition. This resulted in several compounds that have unique DNA cleavage site selectivities and potent antitumor activities in a number of cancer cell lines.

Induction of Retinoid X Receptor Activity and Consequent Upregulation of p21WAF1/CIP1 by Indenoisoquinolines in MCF7 Cells

Retinoid X receptor (RXR) has been targeted for the chemoprevention and treatment of cancer. To discover potential agents acting through RXRs, we utilized an RXR response element (RXRE)-luciferase reporter gene assay. Following extensive screening, 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline dihydrochloride (AM6-36) was found to induce RXRE-luciferase activities. AM6-36 inhibited COX-2 expression and anchorage-independent growth with 12-O-tetradecanoylphorbol 13-acetate-stimulated JB6 Cl41 cells, induced the expression of CD38 in HL-60 cells, and attenuated the growth of N-methyl-N-nitrosourea–induced mammary tumors in rats. Consistent with other reports describing the antiproliferative effects of RXR agonists in breast cancers, AM6-36 showed growth inhibition with cultured MCF7 breast cancer cells, accompanied by G2/M-phase arrest at lower concentrations and enhanced S-phase arrest at higher concentrations. On the basis of DNA microarray analysis, AM6-36 upregulated the expression of CDKN1A, a target gene of RXR, by 35-fold. In accord with this response, the expression of the corresponding protein, p21WAF1/CIP1, was increased in the presence of AM6-36. Induction of p21 by AM6-36 was abrogated following transient knockdown of RXRα, demonstrating that the effect of AM6-36 on the expression of p21 is closely related to modulation of RXRα transcriptional activity. Intestinal permeability was suggested with Caco-2 cells and limited metabolism resulted when AM6-36 was incubated with human liver microsomes. Oral administration with rats resulted in 0.8 μg/mL, 4.3 μg/g, and 0.3 μg/g in serum, liver, and mammary gland, respectively. In sum, these data suggest that AM6-36 is a promising lead for the treatment or prevention of breast cancer and provide a strong rationale for testing in more advanced antitumor systems. Cancer Prev Res; 4(4); 592–607. ©2011 AACR.

Optimization of the lactam side chain of 7-azaindenoisoquinoline topoisomerase I inhibitors and mechanism of action studies in cancer cells.

Optimization of the lactam ω-aminoalkyl substituents in a series of 7-azaindenoisoquinolines resulted in new anticancer agents with improved Top1 inhibitory potencies and cancer cell cytotoxicities. The new compounds 14–17 and 19 exhibited mean graph midpoint cytotoxicity (GI50) values of 21–71 nM in the NCI panel of 60 human cancer cell cultures. Ternary 7-azaindenoisoquinoline–DNA–Top1 cleavage complexes that persist for up to 6 h were detected in HCT116 colon cancer cells. Ternary complexes containing 7-azaindenoisoquinolines were significantly more stable than those in which camptothecin was incorporated. DNA content distribution histograms showed S-phase block 3 h after drug removal. Drug-induced DNA damage in HCT116 cells was revealed by induction of the histone γ-H2AX marker. The 7-azaindenoisoquinolines were able to partially overcome resistance in several drug-resistant cell lines, and they were not substrates for the ABCB1 drug efflux transporter. Molecular modeling studies indicate that the 7-azaindenoisoquinolines intercalate at the DNA cleavage site in DNA–Top1 covalent complexes with the lactam side chain projecting into the major groove. Overall, the results indicate that the 7-azaindenoisoquinolines are promising anticancer agents that merit further development.

Dibenzo[c,h][1,5]naphthyridinediones as Topoisomerase I Inhibitors: Design, Synthesis, and Biological Evaluation

Dibenzo[c,h][1,5]naphthyridinediones were prepared via a novel synthetic pathway. The compounds were designed as topoisomerase I (Top1) inhibitors based on the indenoisoquinoline series of drugs. The results of biological evaluation demonstrate that, unlike very closely related dibenzo[c,h][1,6]naphthyridinediones, dibenzo[c,h][1,5]naphthyridinediones retain the Top1 inhibitory activity of similarly substituted indenoisoquinolines.

Synthesis and Biological Evaluation of the First Triple Inhibitors of Human Topoisomerase 1, Tyrosyl–DNA Phosphodiesterase 1 (Tdp1), and Tyrosyl–DNA Phosphodiesterase 2 (Tdp2)

Tdp1 and Tdp2 are two tyrosyl-DNA phosphodiesterases that can repair damaged DNA resulting from topoisomerase inhibitors and a variety of other DNA-damaging agents. Both Tdp1 and Tdp2 inhibition could hypothetically potentiate the cytotoxicities of topoisomerase inhibitors. This study reports the successful structure-based design and synthesis of new 7-azaindenoisoquinolines that act as triple inhibitors of Top1, Tdp1, and Tdp2. Enzyme inhibitory data and cytotoxicity data from human cancer cell cultures establish that modification of the lactam side chain of the 7-azaindenoisoquinolines can modulate their inhibitory potencies and selectivities vs Top1, Tdp1, and Tdp2. Molecular modeling of selected target compounds bound to Top1, Tdp1, and Tdp2 was used to design the inhibitors and facilitate the structure-activity relationship analysis. The monitoring of DNA damage by γ-H2AX foci formation in human PBMCs (lymphocytes) and acute lymphoblastic leukemia CCRF-CEM cells documented significantly more DNA damage in the cancer cells vs normal cells.

Induction of apoptosis by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via modulation of MAPKs (p38 and c-Jun N-terminal kinase) and c-Myc in HL-60 human leukemia cells.

Recently, we reported that 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (AM6-36), sharing structural similarity with naturally occurring isoquinolines, induced activities mediated by retinoid X receptor (RXR) response element accompanied by antiproliferative effects on breast cancer cells. To further characterize the biologic potential of AM6-36, we currently report studies conducted with HL-60 human leukemia cells. AM6-36 significantly inhibited cellular proliferation in a dose- and time-dependent manner with an IC(50) value of 86 nM. When evaluated at low test concentrations (≤0.25 μM), AM6-36 induced arrest in the G2/M phase of the cell cycle. At higher concentrations (1 and 2 μM), the response shifted to apoptosis, which was consistent with the effect of AM6-36 on other apoptotic signatures including an increase of apoptotic annexin V(+) 7-AAD(-) cells, loss of mitochondrial membrane potential, induction of poly(ADP-ribose) polymerase cleavage, and activation of several caspases. These apoptotic effects are potentially due to up-regulation of p38 MAPK and JNK phosphorylation and down-regulation of c-Myc oncogene expression. Taken together, AM6-36 might serve as an effective anticancer agent by inducing G2/M cell cycle arrest and apoptosis through the activation of MAPKs and inhibition of c-Myc.

Structure-Based Design of 3-(4-Aryl-1H-1,2,3-triazol-1-yl)-Biphenyl Derivatives as P2Y14 Receptor Antagonists

UDP and UDP-glucose activate the P2Y14 receptor (P2Y14R) to modulate processes related to inflammation, diabetes, and asthma. A computational pipeline suggested alternatives to naphthalene of a previously reported P2Y14R antagonist (3, PPTN) using docking and molecular dynamics simulations on a hP2Y14R homology model based on P2Y12R structures. By reevaluating the binding of 3 to P2Y14R computationally, two alternatives, i.e., alkynyl and triazolyl derivatives, were identified. Improved synthesis of fluorescent antagonist 4 enabled affinity quantification (IC50s, nM) using flow cytometry of P2Y14R-expressing CHO cells. p-F3C-phenyl-triazole 65 (32) was more potent than a corresponding alkyne 11. Thus, additional triazolyl derivatives were prepared, as guided by docking simulations, with nonpolar aryl substituents favored. Although triazoles were less potent than 3 (6), simpler synthesis facilitated further structural optimization. Additionally, relative P2Y14R affinities agreed with predicted binding of alkynyl and triazole analogues. These triazoles, designed through a structure-based approach, can be assessed in disease models.

Design and Synthesis of Chlorinated and Fluorinated 7-Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I

The 7-azaindenoisoquinolines are cytotoxic topoisomerase I (Top1) inhibitors. Previously reported representatives bear a 3-nitro group. The present report documents the replacement of the potentially genotoxic 3-nitro group by 3-chloro and 3-fluoro substituents, resulting in compounds with high Top1 inhibitory activities and potent cytotoxicities in human cancer cell cultures and reduced lethality in an animal model. Some of the new Top1 inhibitors also possess moderate inhibitory activities against tyrosyl-DNA phosphodiesterase 1 (TDP1) and tyrosyl-DNA phosphodiesterase 2 (TDP2), two enzymes that are involved in DNA damage repair resulting from Top1 inhibitors, and they produce significantly more DNA damage in cancer cells than in normal cells. Eighteen of the new compounds had cytotoxicity mean-graph midpoint (MGM) GI50 values in the submicromolar (0.033-0.630 μM) range. Compounds 16b and 17b are the most potent in human cancer cell cultures with MGM GI50 values of 0.063 and 0.033 μM, respectively. Possible binding modes to Top1 and TDP1were investigated by molecular modeling.

Chapter 2:Interfacial Inhibitors

Interfacial inhibition is a widespread molecular mechanism by which natural products, such as toxins, inactivate macromolecular complexes by trapping normally transient conformational intermediates. This mechanism is effectively harnessed for therapeutic purposes, primarily in the case of antibacterial and chemotherapeutic anticancer drugs. This chapter provides case studies for drugs targeting protein–DNA interfaces, such as topoisomerase inhibitors and HIV integrase inhibitors. Examples of interfacial inhibitors for protein–protein interfaces are also provided for gyrase, STING and Arp2–3 inhibitors. Interfacial inhibition offers prospects for targeting previously ‘undruggable’ targets, including transcription, replication and chromatin-remodelling complexes. From a discovery viewpoint, the interfacial inhibition principle establishes the value of natural-product screening, the importance of non-competitive inhibitors and the value of high-throughput assays based on enhancement of macromolecular complex formation rather than disruption.