Mitochondrial complex I is a central metabolic enzyme that uses the reducing potential of NADH to reduce ubiquinone-10 (Q10) and drive four protons across the inner mitochondrial membrane, powering oxidative phosphorylation. Although many complex I structures are now available, the mechanisms of Q10 reduction and energy transduction remain controversial. Here, we reconstitute mammalian complex I into phospholipid nanodiscs with exogenous Q10. Using cryo-EM, we reveal a Q10 molecule occupying the full length of the Q-binding site in the ‘active’ (ready-to-go) resting state together with a matching substrate-free structure, and apply molecular dynamics simulations to propose how the charge states of key residues influence the Q10 binding pose. By comparing ligand-bound and ligand-free forms of the ‘deactive’ resting state (that require reactivating to catalyse), we begin to define how substrate binding restructures the deactive Q-binding site, providing insights into its physiological and mechanistic relevance.
Many anticancer agents damage DNA and arrest cell cycle progression primarily in S or G2 phase of the cell cycle. Previous studies with the topoisomerase I inhibitor SN38 have demonstrated the efficacy of the Chk1 inhibitor UCN-01 to overcome this arrest and induce mitotic catastrophe. UCN-01 was limited in clinical trials by unfavorable pharmacokinetics. SCH900776 is a novel and more selective Chk1 inhibitor that potently inhibits Chk1 and abrogates cell cycle arrest induced by SN38. Like UCN-01, abrogation of SN38-induced arrest enhances the rate of cell death but does not increase overall cell death. In contrast, SCH900776 reduced the growth-inhibitory concentration of hydroxyurea by 20-70 fold. A similar magnitude of sensitization was observed with cytarabine, a 5-10 fold sensitization occurred with gemcitabine, but no sensitization occurred with cisplatin, 5-fluorouracil or 6-thioguanine. Sensitization occurred at hydroxyurea concentrations that marginally slowed DNA replication without apparent activation of Chk1, but this led to dependence on Chk1 that increased with time. For example, when added 18 h after hydroxyurea, SCH900776 induced DNA double-strand breaks consistent with rapid collapse of replication forks. In addition, some cell lines were highly sensitive to SCH900776 alone, and these cells required lower concentrations of SCH900776 to sensitize them to hydroxyurea. We conclude that some tumors may be very sensitive to the combination of SCH900776 and hydroxyurea. Delayed administration of SCH900776 may be more effective than concurrent treatment. SCH900776 is currently in Phase I clinical trials, and these results provide the rationale and schedule for future clinical trials.
BackgroundChk1 inhibitors have emerged as promising anticancer therapeutic agents particularly when combined with antimetabolites such as gemcitabine, cytarabine or hydroxyurea. Here, we address the importance of appropriate drug scheduling when gemcitabine is combined with the Chk1 inhibitor MK-8776, and the mechanisms involved in the schedule dependence.MethodsGrowth inhibition induced by gemcitabine plus MK-8776 was assessed across multiple cancer cell lines. Experiments used clinically relevant “bolus” administration of both drugs rather than continuous drug exposures. We assessed the effect of different treatment schedules on cell cycle perturbation and tumor cell growth in vitro and in xenograft tumor models.ResultsMK-8776 induced an average 7-fold sensitization to gemcitabine in 16 cancer cell lines. The time of MK-8776 administration significantly affected the response of tumor cells to gemcitabine. Although gemcitabine induced rapid cell cycle arrest, the stalled replication forks were not initially dependent on Chk1 for stability. By 18 h, RAD51 was loaded onto DNA indicative of homologous recombination. Inhibition of Chk1 at 18 h rapidly dissociated RAD51 leading to the collapse of replication forks and cell death. Addition of MK-8776 from 18–24 h after a 6-h incubation with gemcitabine induced much greater sensitization than if the two drugs were incubated concurrently for 6 h. The ability of this short incubation with MK-8776 to sensitize cells is critical because of the short half-life of MK-8776 in patients’ plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G2 phase 18 h after gemcitabine administration, but cells had started to recover by 42 h. Administration of MK-8776 18 h after gemcitabine caused significantly delayed tumor growth compared to either drug alone, or when the two drugs were administered with only a 30 min interval.ConclusionsThere are two reasons why delayed addition of MK-8776 enhances sensitivity to gemcitabine: first, there is an increased number of cells arrested in S phase; and second, the arrested cells have adequate time to initiate recombination and thereby become Chk1 dependent. These results have important implications for the design of clinical trials using this drug combination.
Microbiota plays an important role in regulating immune responses associated with atopic diseases. We sought to evaluate relationships among airway microbiota, serum IgE levels, allergic sensitization and their relevance to rhinitis and asthma. Microbial characterization was performed using Illumina-based 16S rRNA gene sequencing of 87 throat swabs collected from children with asthma (n = 32) and rhinitis (n = 23), and from healthy controls (n = 32). Data analysis was performed using QIIME (Quantitative Insights Into Microbial Ecology) v1.8. Significantly higher abundance of Proteobacteria was found in children with rhinitis than in the healthy controls (20.1% vs. 16.1%, P = 0.009). Bacterial species richness (Chao1 index) and diversity (Shannon index) were significantly reduced in children with mite sensitization but not in those with food or IgE sensitization. Compared with healthy children without mite sensitization, the mite-sensitized children with rhinitis and asthma showed significantly lower Chao1 and Shannon indices. Moraxella and Leptotrichia species were significantly found in the interaction of mite sensitization with rhinitis and asthma respectively. Airway microbial diversity appears to be inversely associated with sensitization to house dust mites. A modulation between airway dysbiosis and responses to allergens may potentially cause susceptibility to rhinitis and asthma in early childhood.
Mitochondrial complex I (NADH:ubiquinone oxidoreductase), a major contributor of free energy for oxidative phosphorylation, is increasingly recognized as a promising drug target for ischemia-reperfusion injury, metabolic disorders, and various cancers. Several pharmacologically relevant but structurally unrelated small molecules have been identified as specific complex I inhibitors, but their modes of action remain unclear. Here, we present a 3.0-Å resolution cryo–electron microscopy structure of mammalian complex I inhibited by a derivative of IACS-010759, which is currently in clinical development against cancers reliant on oxidative phosphorylation, revealing its unique cork-in-bottle mechanism of inhibition. We combine structural and kinetic analyses to deconvolute cross-species differences in inhibition and identify the structural motif of a “chain” of aromatic rings as a characteristic that promotes inhibition. Our findings provide insights into the importance of π-stacking residues for inhibitor binding in the long substrate-binding channel in complex I and a guide for future biorational drug design.
The molecular mode of action of biguanides, including the drug metformin, which is widely used in the treatment of diabetes, is incompletely characterized. Here, we define the inhibitory drug-target interaction(s) of a model biguanide with mammalian respiratory complex I by combining cryo–electron microscopy and enzyme kinetics. We interpret these data to explain the selectivity of biguanide binding to different enzyme states. The primary inhibitory site is in an amphipathic region of the quinone-binding channel, and an additional binding site is in a pocket on the intermembrane-space side of the enzyme. An independent local chaotropic interaction, not previously described for any drug, displaces a portion of a key helix in the membrane domain. Our data provide a structural basis for biguanide action and enable the rational design of medicinal biguanides.
The objective of this study was to develop an adenoviral vector system that would generate a pattern of expression of exogenous therapeutic genes appropriate for the treatment of ovarian cancer. For this purpose, we have generated a replication-deficient recombinant adenoviral vector, AdLPLacZ, which contains the human L-plastin (LP) promoter (LP-P) driving the Escherichia coli LacZ gene. LP is constitutively expressed at high levels in malignant epithelial cells but is not expressed in normal tissues, except at low levels in mature hematopoietic cells. Because adenoviral vectors infect early hematopoietic multilineage precursor cells only poorly or not at all, this vector would be of use in the peritoneal cavity and in vitro for marrow purging. We first analyzed the expression of the LacZ reporter gene in ovarian and breast cancer cell lines, normal fibroblasts, and leukemia cell lines using the adenoviral vector in which the LacZ gene is governed by the LP-P promoter (AdLPLacZ) or in which the LacZ gene is governed by the cytomegalovirus (CMV) promoter (AdCMVLacZ). We found equivalent and high levels of expression of -galactosidase (-gal) by AdLPLacZ and AdCMVLacZ vectors in the breast or ovarian cancer cell lines as well as in a fibrosarcoma cell line, indicating that the adenoviral vectors infected these cells and expressed their transgenes equally with the LP and CMV promoters. Expression of the LacZ gene with the CMV vector but not with the LP-P vector was observed in experiments with normal fibroblasts, indicating that the vectors infected the cells, but that the LP-P was not active within them. In hematopoietic cells such as U937 cells, no measurable -gal activity was detected in cells infected either by AdLPLacZ or by AdCMVLacZ, indicating that the adenoviral vectors were not infecting the cells. Although -gal activity was observed in fresh ascitic ovarian cancer cells after infection with adenoviral vectors containing CMV or the LP promoters, -gal activity was detected in a portion of a biopsy of normal peritoneum when the tissues were exposed to the AdCMVLacZ vector, but not when tissues were exposed to the AdLPLacZ vector. These results suggest that the transcription of therapeutic genes in cells infected by the AdLP vectors would be restricted to LP expression-positive ovarian carcinoma cells but would not be seen in the normal mesothelial cells of the peritoneal cavity. This possibility implies that adenoviral vectors carrying therapeutic genes driven by the LP-P would be of use for the intracavitary treatment ovarian cancer.
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