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 ;-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 top2B À/À mouse embryonic fibroblasts (MEF) compared with TOP2B +/+ MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIB (Top2B). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2B, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2B, which could implicate Top2B in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2B in doxorubicininduced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2B-DNA covalent complexes exposes the Top2B-concealed DNA doublestrand breaks.
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.
2-Deoxystreptamine (2-DOS) aminoglycosides are a family of structurally related broad-spectrum antibiotics that are used widely in the treatment of infections caused by aerobic Gram-negative bacilli. Their antibiotic activities are ascribed to their abilities to bind a highly conserved A site in the 16 S rRNA of the 30 S ribosomal subunit and interfere with protein synthesis. The abilities of the 2-DOS aminoglycosides to recognize a specific subdomain of a large RNA molecule make these compounds archetypical models for RNA-targeting drugs. This article presents a series of calorimetric, spectroscopic, osmotic stress, and computational studies designed to evaluate the thermodynamics (DeltaG, DeltaH, DeltaS, DeltaCp) of aminoglycoside-rRNA interactions, as well as the hydration changes that accompany these interactions. In conjunction with the current structural database, the results of these studies provide important insights into the molecular forces that dictate and control the rRNA binding affinities and specificities of the aminoglycosides. Significantly, identification of these molecular driving forces [which include binding-linked drug protonation reactions, polyelectrolyte contributions from counterion release, conformational changes, hydration effects, and molecular interactions (e.g., hydrogen bonds and van der Waals interactions)], as well as the relative magnitudes of their contributions to the binding free energy, could not be achieved by consideration of structural data alone, highlighting the importance of acquiring both thermodynamic and structural information for developing a complete understanding of the drug-RNA binding process. The results presented here begin to establish a database that can be used to predict, over a range of conditions, the relative affinity of a given aminoglycoside or aminoglycoside mimetic for a targeted RNA site vs binding to potential competing secondary sites. This type of predictive capability is essential for establishment of a rational design approach to the development of new RNA-targeted drugs.
Rapidly proliferating tumors attempt to meet the demands for nucleotide biosynthesis by up regulating folate pathways that provide the building blocks for pyrimidine and purine biosynthesis. In particular, the key role of mitochondrial folate enzymes in providing formate for de novo purine synthesis and for providing the one carbon moiety for thymidylate synthesis has been recognized in recent studies. We have shown a significant correlation between the up regulation of the mitochondrial folate enzymes, high proliferation rates and sensitivity to the folate antagonist, methotrexate (MTX). Burkitt’s lymphoma and Diffuse Large cell lymphoma tumor specimens have the highest levels of mitochondrial folate enzyme expression and are known to be sensitive to treatment with MTX. A key enzyme up regulated in rapidly proliferating tumors but not in normal adult cells is the mitochondrial enzyme, methylene tetrahydrofolate dehydrogenase, MTHFD2. This prospective outlines the rationale for specific targeting of MTHFD2 and compares known and generated crystal structures of MTHFD2 and closely related enzymes as a molecular basis for developing therapeutic agents against MTHFD2. Importantly, the development of selective inhibitors of mitochondrial methylene tetrahydrofolate dehydrogenase is expected to have substantial activity and this perspective supports the investigation and development of MTHFD2 inhibitors for anticancer therapy.
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