ABSTRACT
Aldehyde dehydrogenase 1A (ALDH1A) isoforms may be a useful target for overcoming chemotherapy resistance in high-grade serous ovarian cancer (HGSOC) and other solid tumor cancers. However, as different cancers express different ALDH1A isoforms, isoform selective inhibitors may have a limited therapeutic scope. Furthermore, resistance to an ALDH1A isoform selective inhibitor could arise via induction of expression of other ALDH1A isoforms. As such, we have focused on the development of pan-ALDH1A inhibitors, rather than on ALDH1A isoform selective compounds. Herein, we report the development of a new group of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in HGSOC. Optimization of the CM10 scaffold, aided by ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular efficacy as demonstrated by reduction in ALDEFLUOR signal in HGSOC cells, and substantial improvements in liver microsomal stability. Based on this work we identified two compounds 17 and 25 suitable for future in vivo proof of concept experiments.
Subject(s)
Isoenzymes , Neoplasms , Humans , Aldehyde Dehydrogenase/metabolism , Retinal Dehydrogenase/metabolism , Aldehyde Oxidoreductases/metabolismABSTRACT
Survival in high-risk pediatric neuroblastoma has remained around 50% for the last 20 years, with immunotherapies and targeted therapies having had minimal impact. Here, we identify the small molecule CX-5461 as selectively cytotoxic to high-risk neuroblastoma and synergistic with low picomolar concentrations of topoisomerase I inhibitors in improving survival in vivo in orthotopic patient-derived xenograft neuroblastoma mouse models. CX-5461 recently progressed through phase I clinical trial as a first-in-human inhibitor of RNA-POL I. However, we also use a comprehensive panel of in vitro and in vivo assays to demonstrate that CX-5461 has been mischaracterized and that its primary target at pharmacologically relevant concentrations, is in fact topoisomerase II beta (TOP2B), not RNA-POL I. This is important because existing clinically approved chemotherapeutics have well-documented off-target interactions with TOP2B, which have previously been shown to cause both therapy-induced leukemia and cardiotoxicity-often-fatal adverse events, which can emerge several years after treatment. Thus, while we show that combination therapies involving CX-5461 have promising anti-tumor activity in vivo in neuroblastoma, our identification of TOP2B as the primary target of CX-5461 indicates unexpected safety concerns that should be examined in ongoing phase II clinical trials in adult patients before pursuing clinical studies in children.
Subject(s)
DNA Topoisomerases, Type II/metabolism , Indoles/therapeutic use , Morpholines/therapeutic use , Neuroblastoma/drug therapy , Neuroblastoma/metabolism , Pyrimidines/therapeutic use , Sulfonamides/therapeutic use , Animals , Benzothiazoles , Blotting, Western , Cell Line, Tumor , Drug Synergism , Enzyme Activation/drug effects , Flow Cytometry , Fluorescent Antibody Technique , Mice , Mice, Nude , Molecular Dynamics Simulation , Naphthyridines , Real-Time Polymerase Chain ReactionABSTRACT
There is strong evidence that inhibition of one or more Aldehyde Dehydrogenase 1A (ALDH1A) isoforms may be beneficial in chemotherapy-resistant ovarian cancer and other tumor types. While many previous efforts have focused on development of ALDH1A1 selective inhibitors, the most deadly ovarian cancer subtype, high-grade serous (HGSOC), exhibits elevated expression of ALDH1A3. Herein, we report continued development of pan-ALDH1A inhibitors to assess whether broad spectrum ALDH1A inhibition is an effective adjunct to chemotherapy in this critical tumor subtype. Optimization of the CM39 scaffold, aided by metabolite ID and several new ALDH1A1 crystal structures, led to improved biochemical potencies, improved cellular ALDH inhibition in HGSOC cell lines, and substantial improvements in microsomal stability culminating in orally bioavailable compounds. We demonstrate that two compounds 68 and 69 are able to synergize with chemotherapy in a resistant cell line and patient-derived HGSOC tumor spheroids, indicating their suitability for future in vivo proof of concept experiments.
Subject(s)
Aldehyde Dehydrogenase/antagonists & inhibitors , Aldehyde Dehydrogenase/therapeutic use , Ovarian Neoplasms/drug therapy , Aldehyde Dehydrogenase/pharmacology , Female , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.
Subject(s)
Fluorescent Dyes/chemistry , Cell Line , Fluorescence , Humans , Microscopy, FluorescenceABSTRACT
It is estimated that Trichomonas vaginalis affects an astonishing 3.9% of the world's population, and while many of those infected are asymptomatic, progression of the disease can lead to serious health problems. Currently, the nitroimidazoles constitute the only drug class approved to treat trichomoniasis in the United States, which makes the spread of drug resistance a realistic concern. We developed a new image-based, high-throughput, and high-content assay for testing natural products (purified compounds and extracts) for antitrichomonal activity. Applying this assay system to a library of fungal natural product extracts led to the identification of three general classes of natural product inhibitors that exhibited moderate to strong activities against T. vaginalis: anthraquinones, xanthone-anthraquinone heterodimers, and decalin-linked tetramic-acid-containing metabolites. The tetramate natural products emerged as the most promising candidate molecules with pyrrolocin A (51) exhibiting potent activity against the parasite (EC50 = 60 nM), yet this metabolite showed limited toxicity to mammalian cell lines (selectivity index values of 100 and 167 versus 3T3 fibroblast and Ect1 normal cervical cells, respectively). The imaging-based assay system is a powerful tool for the bioassay-guided purification of single-component antitrichomonal biomolecules from complex natural product mixtures.
Subject(s)
Antiprotozoal Agents/pharmacology , Biological Products/pharmacology , Drug Discovery/methods , High-Throughput Screening Assays/methods , Image Processing, Computer-Assisted/methods , Trichomonas vaginalis/drug effects , Antiprotozoal Agents/isolation & purification , Biological Products/isolation & purification , Cell Line , Female , Fibroblasts/drug effects , Fungi/chemistry , Humans , Pyrrolidinones/isolation & purification , Pyrrolidinones/pharmacology , Quinones/isolation & purification , Quinones/pharmacology , Sensitivity and Specificity , Trichomonas Vaginitis/drug therapyABSTRACT
Ovarian cancer is typified by the development of chemotherapy resistance. Chemotherapy resistance is associated with high aldehyde dehydrogenase (ALDH) enzymatic activity, increased cancer "stemness," and expression of the stem cell marker CD133. As such, ALDH activity has been proposed as a therapeutic target. Although it remains controversial which of the 19 ALDH family members drive chemotherapy resistance, ALDH1A family members have been primarily linked with chemotherapy resistant and stemness. We identified two ALDH1A family selective inhibitors (ALDH1Ai). ALDH1Ai preferentially kills CD133+ ovarian cancer stem-like cells (CSCs). ALDH1Ai induce necroptotic CSC death, mediated, in part, by the induction of mitochondrial uncoupling proteins and reduction in oxidative phosphorylation. ALDH1Ai is highly synergistic with chemotherapy, reducing tumor initiation capacity and increasing tumor eradication in vivo. These studies link ALDH1A with necroptosis and confirm the family as a critical therapeutic target to overcome chemotherapy resistance and improve patient outcomes.
Subject(s)
Aldehyde Dehydrogenase 1 Family/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Necroptosis , Neoplastic Stem Cells/metabolism , Ovarian Neoplasms/metabolism , Retinal Dehydrogenase/antagonists & inhibitors , AC133 Antigen/genetics , AC133 Antigen/metabolism , Aldehyde Dehydrogenase 1 Family/metabolism , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm , Female , Humans , Mice , Neoplastic Stem Cells/drug effects , Oxidative Phosphorylation , Retinal Dehydrogenase/metabolismABSTRACT
Aldehyde dehydrogenase (ALDH) activity is commonly used as a marker to identify cancer stem-like cells. The three ALDH1A isoforms have all been individually implicated in cancer stem-like cells and in chemoresistance; however, which isoform is preferentially expressed varies between cell lines. We sought to explore the structural determinants of ALDH1A isoform selectivity in a series of small-molecule inhibitors in support of research into the role of ALDH1A in cancer stem cells. An SAR campaign guided by a cocrystal structure of the HTS hit CM39 (7) with ALDH1A1 afforded first-in-class inhibitors of the ALDH1A subfamily with excellent selectivity over the homologous ALDH2 isoform. We also discovered the first reported modestly selective single isoform 1A2 and 1A3 inhibitors. Two compounds, 13g and 13h, depleted the CD133+ putative cancer stem cell pool, synergized with cisplatin, and achieved efficacious concentrations in vivo following IP administration. Compound 13h additionally synergized with cisplatin in a patient-derived ovarian cancer spheroid model.