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

Mapping the Binding Interactions between Human Gasdermin D and Human Caspase-1 Using Carbene Footprinting

Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 (C285A) active-site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter, consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type hCaspase-1, VRT-043198 modifies the active-site Cys285 through the formation of a S,O-hemiacetal. Here, we showed by carbene labeling that this inhibitor can noncovalently occupy the active site of a C285A mutant. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system

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

PCNA dependent cellular activities tolerate dramatic perturbations in PCNA client interactions

Proliferating cell nuclear antigen (PCNA) is an essential cofactor for DNA replication and repair, recruiting multiple proteins to their sites of action. We examined the effects of the PCNA(S228I) mutation that causes PCNA-associated DNA repair disorder (PARD). Cells from individuals affected by PARD are sensitive to the PCNA inhibitors T3 and T2AA, showing that the S228I mutation has consequences for undamaged cells. Analysis of the binding between PCNA and PCNA-interacting proteins (PIPs) shows that the S228I change dramatically impairs the majority of these interactions, including that of Cdt1, DNMT1, PolD3(p66) and PolD4(p12). In contrast p21 largely retains the ability to bind PCNA(S228I). This property is conferred by the p21 PIP box sequence itself, which is both necessary and sufficient for PCNA(S228I) binding. Ubiquitination of PCNA is unaffected by the S228I change, which indirectly alters the structure of the inter-domain connecting loop. Despite the dramatic in vitro effects of the PARD mutation on PIP-degron binding, there are only minor alterations to the stability of p21 and Cdt1 in cells from affected individuals. Overall our data suggests that reduced affinity of PCNA(S228I) for specific clients causes subtle cellular defects in undamaged cells which likely contribute to the etiology of PARD

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

Mitochondrial effects of plant-made compounds.

SIGNIFICANCE: Plants produce many small molecules with biomedical potential. Their absorption from foods, metabolism, their effects on physiological and pathological processes, and the mechanisms of action are intensely investigated. Many are known to affect multiple cellular functions. Mitochondria are coming to be recognized as a major target for these compounds, especially redox-active ones, but the mechanisms involved still need clarification. At the same time, frontline research is uncovering the importance of processes involving these organelles for the cell and for an array of physiological and pathological processes. We review the major functions and possible dysfunctions of mitochondria, identify signaling pathways through which plant-derived molecules have an impact, and show how this may be relevant for major pathologies. RECENT ADVANCES: Antioxidant, protective effects may arise as a reaction to a low-level pro-oxidant activity, largely taking place at mitochondria. Some plant-derived molecules can activate AMP-dependent kinase, with a consequent upregulation of mitochondrial biogenesis and a potential favorable impact on aging, pathologies like diabetes and neurodegeneration, and on ischemic damage. CRITICAL ISSUES: The extrapolation of in vitro results and the verification of paradigms in vivo is a key issue for current research on both plant-derived compounds and mitochondria. The low bioavailability of many of these molecules poses a problem for both the study of their activities and their utilization. FUTURE DIRECTIONS: The further clarification of the role of mitochondria in the activities of plant dietary compounds and their metabolites, mitochondrial targeting, the development of analogs and pro-drugs are all topics for promising research

Quercetin mitochondriotropic derivatives antagonize nitrate tolerance and endothelial dysfunction of isolated rat aorta rings

Chronic use of glyceryl trinitrate is limited by serious side effects, inter alia tolerance and endothelial dysfunction of coronary and resistance arteries. The natural flavonoid quercetin has been shown to counteract the development of glyceryl trinitrate tolerance in vitro. Two mitochondriotropic, 4-O-triphenylphosphoniumbutyl derivatives of quercetin (QTA-3BTPI and Q-3BTPI) were compared to quercetin for protection against glyceryl trinitrate-induced tolerance and endothelial dysfunction of isolated rat aorta rings. Both QTA-3BTPI and Q-3BTPI significantly counteracted the reduced vascular responsiveness to both glyceryl trinitrate and acetylcholine caused by prolonged exposure of the vessel to glyceryl trinitrate itself, their potency being much greater than that of quercetin. QTA-3BTPI, however, turned out to cause endothelial dysfunction per se. Since Q-3BTPI antagonized in vitro nitrate tolerance and endothelial dysfunction of vessels, this encourages assessing whether this effect is displayed also in vivo during long-term glyceryl trinitrate treatment