Browsing by Subject "Benzimidazoles"

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    Design, Synthesis and Study of DNA-Targeted Benzimidazole-Amino Acid Conjugates
    (2013-07-12) Garner, Matthew L.; Long, Eric C. (Eric Charles); Minto, Robert; O'Donnell, Martin J.
    The DNA minor groove continues to be an important biological target in the development of anticancer, antiviral, and antimicrobial compounds. Among agents that target the minor groove, studies of well-established benzimidazole-based DNA binders such as Hoechst 33258 have made it clear that the benzimidazole-amidine portion of these molecules promotes an efficient, site-selective DNA association. Building on the beneficial attributes of existing benzimidazole-based DNA binding agents, a series of benzimidazole-amino acid conjugates was synthesized to investigate their DNA recognition and binding properties. In this series of compounds, the benzimidazole-amidine moiety was utilized as a core DNA “anchoring” element accompanied by different amino acids to provide structural diversity that may influence DNA binding affinity and site-selectivity. Single amino acid conjugates of benzimidazole-amidines were synthesized, as well as a series of conjugates containing 20 dipeptides with the general structure Xaa-Gly. These conjugates were synthesized through a solid-phase synthetic route building from a resin-bound amino acid (or dipeptide). The synthetic steps involved: (1) the coupling of 4-formylbenzoic acid to the resin-bound amino acid (via diisopropylcarbodiimide and hydroxybenzotriazole); followed by (2) introduction of a 3,4-diaminobenzamidoxime in the presence of 1,4-benzoquinone to construct the benzimidazole ring; and, finally, (3) reduction of the resin-bound amidoxime functionality to an amidine via treatment with 1M SnCl2•2H2O in DMF before cleavage of final product from the resin. The synthetic route developed and employed was simple and straightforward except for the final reduction that proved to be very arduous. All target compounds were obtained in good yield (based upon weight), averaging 73% mono-amino acid and 78% di-amino acid final compound upon cleavage from resin. Ultimately, the DNA binding activities of the amino acid-benzimidazole-amidine conjugates were analyzed using a fluorescent intercalator displacement (FID) assay and calf thymus DNA as a substrate. The relative DNA binding affinities of both the mono- and di-amino acid-benzimidazole-amidine conjugates were generally weaker than that of netropsin and distamycin with the dipeptide conjugates showing stronger binding affinities than the mono-amino acid conjugates. The dipeptide conjugates containing amino acids with positively charged side chains, Lys-Gly-BI-(+) and Arg-Gly-BI-(+), showed the strongest DNA binding affinities amongst all our synthesized conjugates.
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    Inhibition of CaMKK2 reverses age-associated decline in bone mass
    (Elsevier, 2015-06) Pritchard, Zachary J.; Cary, Rachel L.; Yang, Chang; Novack, Deborah V.; Voor, Michael J.; Sankar, Uma; Department of Anatomy & Cell Biology, IU School of Medicine
    Decline in bone formation is a major contributing factor to the loss of bone mass associated with aging. We previously showed that the genetic ablation of the tissue-restricted and multifunctional Ca(2+)/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) stimulates trabecular bone mass accrual, mainly by promoting anabolic pathways and inhibiting catabolic pathways of bone remodeling. In this study, we investigated whether inhibition of this kinase using its selective cell-permeable inhibitor STO-609 will stimulate bone formation in 32 week old male WT mice and reverse age-associated of decline in bone volume and strength. Tri-weekly intraperitoneal injections of saline or STO-609 (10 μM) were performed for six weeks followed by metabolic labeling with calcein and alizarin red. New bone formation was assessed by dynamic histomorphometry whereas micro-computed tomography was employed to measure trabecular bone volume, microarchitecture and femoral mid-shaft geometry. Cortical and trabecular bone biomechanical properties were assessed using three-point bending and punch compression methods respectively. Our results reveal that as they progress from 12 to 32 weeks of age, WT mice sustain a significant decline in trabecular bone volume, microarchitecture and strength as well as cortical bone strength. However, treatment of the 32 week old WT mice with STO-609 stimulated apposition of new bone and completely reversed the age-associated decrease in bone volume, quality, as well as trabecular and cortical bone strength. We also observed that regardless of age, male Camkk2(-/-) mice possessed significantly elevated trabecular bone volume, microarchitecture and compressive strength as well as cortical bone strength compared to age-matched WT mice, implying that the chronic loss of this kinase attenuates age-associated decline in bone mass. Further, whereas STO-609 treatment and/or the absence of CaMKK2 significantly enhanced the femoral mid-shaft geometry, the mid-shaft cortical wall thickness and material bending stress remained similar among the cohorts, implying that regardless of treatment, the material properties of the bone remain similar. Thus, our cumulative results provide evidence for the pharmacological inhibition of CaMKK2 as a bone anabolic strategy in combating age-associated osteoporosis.
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    Selective ALDH3A1 Inhibition by Benzimidazole Analogues Increase Mafosfamide Sensitivity in Cancer Cells
    (American Chemical Society, 2014-01-23) Parajuli, Bibek; Fishel, Melissa L.; Hurley, Thomas D.; Department of Biochemistry & Molecular Biology, IU School of Medicine
    Aldehyde dehydrogenase enzymes irreversibly oxidize aldehydes generated from metabolism of amino acids, fatty acids, food, smoke, additives, and xenobiotic drugs. Cyclophosphamide is one such xenobiotic used in cancer therapies. Upon activation, cyclophosphamide forms an intermediate, aldophosphamide, which can be detoxified to carboxyphosphamide by aldehyde dehydrogenases (ALDH), especially ALDH1A1 and ALDH3A1. Consequently, selective inhibition of ALDH3A1 could increase chemosensitivity toward cyclophosphamide in ALDH3A1 expressing tumors. Here, we report detailed kinetics and structural characterization of a highly selective submicromolar inhibitor of ALDH3A1, 1-[(4-fluorophenyl)sulfonyl]-2-methyl-1H-benzimidazole (CB7, IC50 of 0.2 μM). CB7 does not inhibit ALDH1A1, ALDH1A2, ALDH1A3, ALDH1B1, or ALDH2 activity. Structural, kinetics, and mutagenesis studies show that CB7 binds to the aldehyde binding pocket of ALDH3A1. ALDH3A1-expressing lung adenocarcinoma and glioblastoma cell lines are sensitized toward mafosfamide (MF) treatment in the presence analogues of CB7, whereas primary lung fibroblasts lacking ALDH3A1 expression, are not.