Small-molecule modulators of mitochondrial channels as chemotherapeutic agents

Ion channels residing in the inner (IMM) and outer (OMM) mitochondrial membranes are emerging as noteworthy pharmacological targets in oncology. While these aspects have not been investigated for all of them, a role in cancer growth and/or metastasis and/or drug resistance has been shown at least for the IMM-residing Ca2+ uniporter complex and K+- selective mtKV1.3, mtIKCa, mtSKCa and mtTASK-3, and for the OMM Voltage-Dependent Anion Channel (mitochondrial porin). A special case is that of the Mitochondrial Permeability Transition Pore, a large pore which forms in the IMM of severely stressed cells, and which may be exploited to precipitate the death of cancerous cells. Here we briefly discuss the oncological relevance of mitochondria and their channels, and summarize the methods that can be adopted to selectively target these intracellular organelles. We then present an updated list of known mitochondrial channels, and review the pharmacology of those with proven relevance for cancer

Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

Abstract Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS)-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. Methods Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle) by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP) activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. Results Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. Conclusions Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.http://deepblue.lib.umich.edu/bitstream/2027.42/78260/1/1465-9921-11-131.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78260/2/1465-9921-11-131.pdfPeer Reviewe

Pharmacological targeting of the mitochondrial calcium-dependent potassium channel KCa3.1 triggers cell death and reduces tumor growth and metastasis in vivo

Ion channels are non-conventional, druggable oncological targets. The intermediate-conductance calcium-dependent potassium channel (K(Ca)3.1) is highly expressed in the plasma membrane and in the inner mitochondrial membrane (mitoK(Ca)3.1) of various cancer cell lines. The role mitoK(Ca)3.1 plays in cancer cells is still undefined. Here we report the synthesis and characterization of two mitochondria-targeted novel derivatives of a high-affinity K(Ca)3.1 antagonist, TRAM-34, which retain the ability to block channel activity. The effects of these drugs were tested in melanoma, pancreatic ductal adenocarcinoma and breast cancer lines, as well as in vivo in two orthotopic models. We show that the mitochondria-targeted TRAM-34 derivatives induce release of mitochondrial reactive oxygen species, rapid depolarization of the mitochondrial membrane, fragmentation of the mitochondrial network. They trigger cancer cell death with an EC50 in the mu M range, depending on channel expression. In contrast, inhibition of the plasma membrane K(Ca)3.1 by membrane-impermeant Maurotoxin is without effect, indicating a specific role of mitoK(Ca)3.1 in determining cell fate. At sub-lethal concentrations, pharmacological targeting of mitoK(Ca)3.1 significantly reduced cancer cell migration by enhancing production of mitochondrial reactive oxygen species and nuclear factor-kappa B (NF-kappa B) activation, and by downregulating expression of Bcl-2 Nineteen kD-Interacting Protein (BNIP-3) and of Rho GTPase CDC-42. This signaling cascade finally leads to cytoskeletal reorganization and impaired migration. Overexpression of BNIP-3 or pharmacological modulation of NF-kappa B and CDC-42 prevented the migration-reducing effect of mitoTRAM-34. In orthotopic models of melanoma and pancreatic ductal adenocarcinoma, the tumors at sacrifice were 60% smaller in treated versus untreated animals. Metastasis of melanoma cells to lymph nodes was also drastically reduced. No signs of toxicity were observed. In summary, our results identify mitochondrial K(Ca)3.1 as an unexpected player in cancer cell migration and show that its pharmacological targeting is efficient against both tumor growth and metastatic spread in vivo

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing.This study was funded by ProQR. iPSC work in the Cheetham lab is also supported by the Wellcome Trust, Fight for Sight, RP Fighting Blindness, and Moorfields Eye Charity

Interactive 3D Digital Models for Anatomy and Medical Education

This chapter explores the creation and use of interactive, three-dimensional (3D), digital models for anatomy and medical education. Firstly, it looks back over the history and development of virtual 3D anatomy resources before outlining some of the current means of their creation; including photogrammetry, CT and surface scanning, and digital modelling, outlining advantages and disadvantages for each. Various means of distribution are explored, including; virtual learning environments, websites, interactive PDF’s, virtual and augmented reality, bespoke applications, and 3D printing, with a particular focus on the level of interactivity each method offers. Finally, and perhaps most importantly, the use of such models for education is discussed. Questions addressed include; How can such models best be used to enhance student learning? How can they be used in the classroom? How can they be used for selfdirected study? As well as exploring if they could one day replace human specimens, and how they complement the rise of online and e-learning

Perturbation of the yeast mitochondrial lipidome and associated membrane proteins following heterologous expression of Artemia-ANT

Heterologous expression is a landmark technique for studying a protein itself or its effect on the expression host, in which membrane-embedded proteins are a common choice. Yet, the impact of inserting a foreign protein to the lipid environment of host membranes, has never been addressed. Here we demonstrated that heterologous expression of the Artemia franciscana adenine nucleotide translocase (ANT) in yeasts altered lipidomic composition of their inner mitochondrial membranes. Along with this, activities of complex II, IV and ATP synthase, all membrane-embedded components, were significantly decreased while their expression levels remained unaffected. Although the results represent an individual case of expressing a crustacean protein in yeast inner mitochondrial membranes, it cannot be excluded that host lipidome alterations is a more widespread epiphenomenon, potentially biasing heterologous expression experiments. Finally, our results raise the possibility that not only lipids modulate protein function, but also membrane-embedded proteins modulate lipid composition, thus revealing a reciprocal mode of regulation for these two biomolecular entities

Cancer stem cell metabolism

Cancer is now viewed as a stem cell disease. There is still no consensus on the metabolic characteristics of cancer stem cells, with several studies indicating that they are mainly glycolytic and others pointing instead to mitochondrial metabolism as their principal source of energy. Cancer stem cells also seem to adapt their metabolism to microenvironmental changes by conveniently shifting energy production from one pathway to another, or by acquiring intermediate metabolic phenotypes. Determining the role of cancer stem cell metabolism in carcinogenesis has become a major focus in cancer research, and substantial efforts are conducted towards discovering clinical targets

Structural insights into human SNF2/SWI2 chromatin remodeler SMARCAD1 and its role in DNA repair

ATP-dependent chromatin remodelers have been proposed to act sequentially, and to a certain extent non-redundantly, in the priming stages of the DNA Damage Response pathways by establishing chromatin in lesion sites ready to act as a scaffold for repair factors or to be displaced in order to allow DNA repair. Among remodeling factors proposed to play a role in DNA repair is SMARCAD1, a poorly characterized, non-canonical member of the SWR1-like family of SNF2/SWI2 superfamily of ATPases, which has recently been identified as a potential target for ATM/ATR phosphorylation at canonical and non-canonical sites upon DNA damage. The actual mechanism for SMARCAD1 recruitment and involvement in DNA remodeling is still unknown, and unlike most other chromatin remodelers, SMARCAD1 does not contain DNA- or histone-binding domains frequently accompanying such proteins. Instead, in addition to the core ATPase domain, only two CUE domains (a type of helical ubiquitin-binding domain) have been identified. This thesis presents the findings of an investigation intended to structurally characterize SMARCAD1 by dissecting and identifying its domain architecture, and examining the activity and ligand selectivity of its binding domains in the functional context of DNA damage repair. The solution NMR structure of the CUE1 domain is presented, describing a triple helix bundle consistent with other members of the family. Furthermore, a novel SUMO interacting motif was identified and through a combination of NMR titrations and phospho-proteomics analysis, shown to be constitutively phosphorylated which excludes the possibility of DNA damage dependent ATM targeting as the recruitment mechanism for DNA repair. Additionally, it is demonstrated that both CUE domains are poor binders of mono-ubiquitin, however CUE1 specifically mediates the high affinity binary interaction with the transcriptionally repressive master regulator KAP1. This interaction was shown to be independent of post-translational ubiquitylation but rather sustained through direct interaction with the dimeric RBCC domain of KAP1. Finally, mass spectrometry profiling of domain-dependent interactions (based on differential abundance relative to changes due to chemically induced DNA damage) suggests SMARCAD1 may be involved in p53 transcriptional regulation through interactions maintained with CUE1 prior to DNA damage, whereas the SIM domain selectively targets protein interactions upon DNA damage that simultaneously activate p53 transcriptional control and recruit SMARCAD1 to DNA damage repair pathways.</p