Hannah W. Mbatia

Photochemical Tools for Studying Metal Ion Signaling and Homeostasis

Metal ions have well-established catalytic and structural roles in proteins. Much of the knowledge acquired about metalloenzymes has been derived using spectroscopic techniques and X-ray crystallography, but these methodologies are less effective for studying metal ions that are not tightly bound to biomacromolecules. In order to prevent deleterious chemistry, cells tightly regulate the uptake, distribution, and intracellular concentrations of metal ions. Investigation into these homeostasis mechanisms has necessitated the development of alternative ways to study metal ions. Photochemical tools such as small molecule and protein-based fluorescent sensors as well as photocaged complexes have provided insight into the homeostasis and signaling mechanisms of Ca(2+), Zn(2+), and Cu(+), but a comprehensive picture of metal ions in biology will require additional development of these techniques, which are reviewed in this Current Topics article.

Novel C-4 Heteroaryl 13-cis-Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.

Understanding the Relationship Between Photolysis Efficiency and Metal Binding Using ArgenCast Photocages

ArgenCast‐1 (1), a photocage for silver utilizing acyclic polythioether 3,6,12,15‐tetrathia‐9‐azaheptadecane receptor and 4,5‐dimethoxy‐2‐nitrobenzyl (DMNB) chromophore has been prepared using trimethylsilyl trifluoromethanesulfonate‐assisted electrophilic aromatic substitution. Metal binding studies with ArgenCast‐1 reveal interactions with Ag+, Hg2+ and Cu+, but only Ag+ coordinates in both aqueous and organic solvents. The uncaging mechanism of ArgenCast‐1 metal complex involves a photoreaction that converts the nitrobenzydrol into the electron withdrawing nitrosobenzophenone that participates in a resonance interaction with a metal‐bound aniline nitrogen atom. The structural change following photolysis decreases availability of the nitrogen lone pair for Ag+ coordination, but strong interactions between Ag+ and the thioether ligands mitigates metal ion release. A resonance interaction with a key aci‐nitro intermediate reduces the photolysis quantum yield of ArgenCast‐1, so several naphthyl‐based nitrobenzyl groups were screened as alternatives to DMNB. The naphthyl Ag+ photocages, ArgenCast‐2 and ‐3, exhibit nearly identical quantum yield to ArgenCast‐1 owing to the dominance of resonance between aci‐nitro intermediate and the aniline nitrogen atom.

VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

Abstract 756: First MNK degrading agents block phosphorylation of eIF4E, induce apoptosis, and inhibit cell growth, migration and invasion in triple-negative and HER2-overexpressing breast cancer cell lines

Introduction: Retinoic acid metabolism blocking agents are known to exhibit a range of anticarcinogenic properties. This study investigated the anticancer efficacy of novel, potent retinamide retinoic acid metabolism blocking agents (RRs) in triple negative and Her 2 overexpressing breast cancer cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine 209 phosphorylation by upstream MAPK-interacting kinases (Mnk). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating breast cancers particularly for HER-2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. Methods: Triple negative and Her 2 overexprssing breast cancer cells were treated with retinamide RAMBAs and Mnk inhibitors and evaluated the effects of RRs on growth inhibition, MAPK/Mnk mediated eIF4E protein translational machinery and downstream biological effects in triple negative and Her 2 overexpressing breast cancer cells Results: We report that the degradation of Mnk by RRs in breast cancer cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, invasion and metastasis in addition to inducing apoptosis. Most importantly the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (Cercosporamide and CGP 57380). RRs effect on p-eIF4E downregulation and growth inhibition were also far more potent than the clinically relevant retinoids and Mnk inhibitors, cercosporamide and CGP 57380. Conclusion: Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk based therapeutic treatment of breast cancers. Citation Format: Senthilmurugan Ramalingam, Lalji Gediya, Puranik Purushottamachar, Andrew Kwegyir-Afful, Vidya priyadarsini Ramamurthy, Hannah Mbatia, Vincent Njar. First MNK degrading agents block phosphorylation of eIF4E, induce apoptosis, and inhibit cell growth, migration and invasion in triple-negative and HER2-overexpressing breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2014-756