Sensitization of Human Cancer Cells to Gemcitabine by the Chk1 Inhibitor MK-8776: Cell Cycle Perturbation and Impact of Administration Schedule in Vitro and in Vivo

Chk1 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

Preserved respiratory chain capacity and physiology in mice with profoundly reduced levels of mitochondrial respirasomes

The mammalian respiratory chain complexes I, III 2, and IV (CI, CIII 2, and CIV) are critical for cellular bioenergetics and form a stable assembly, the respirasome (CI-CIII 2-CIV), that is biochemically and structurally well documented. The role of the respirasome in bioenergetics and the regulation of metabolism is subject to intense debate and is difficult to study because the individual respiratory chain complexes coexist together with high levels of respirasomes. To critically investigate the in vivo role of the respirasome, we generated homozygous knockin mice that have normal levels of respiratory chain complexes but profoundly decreased levels of respirasomes. Surprisingly, the mutant mice are healthy, with preserved respiratory chain capacity and normal exercise performance. Our findings show that high levels of respirasomes are dispensable for maintaining bioenergetics and physiology in mice but raise questions about their alternate functions, such as those relating to the regulation of protein stability and prevention of age-associated protein aggregation

Identification of a novel toxicophore in anti-cancer chemotherapeutics that targets mitochondrial respiratory complex I

Disruption of mitochondrial function selectively targets tumour cells that are dependent on oxidative phosphorylation. However, due to their high energy demands, cardiac cells are disproportionately targeted by mitochondrial toxins resulting in a loss of cardiac function. An analysis of the effects of mubritinib on cardiac cells showed that this drug did not inhibit HER2 as reported, but directly inhibits mitochondrial respiratory complex I, reducing cardiac-cell beat rate, with prolonged exposure resulting in cell death. We used a library of chemical variants of mubritinib and showed that modifying the 1H-1,2,3-triazole altered complex I inhibition, identifying the heterocyclic 1,3-nitrogen motif as the toxicophore. The same toxicophore is present in a second anti-cancer therapeutic carboxyamidotriazole (CAI) and we demonstrate that CAI also functions through complex I inhibition, mediated by the toxicophore. Complex I inhibition is directly linked to anti-cancer cell activity, with toxicophore modification ablating the desired effects of these compounds on cancer cell proliferation and apoptosis

Cryo-EM studies of substrate and inhibitor binding to mammalian respiratory complex I

Mammalian respiratory complex I (NADH:ubiquinone oxidoreductase) is an intricate multi-subunit, energy-transducing membrane protein that is essential for aerobic energy metabolism and NADH/NAD⁺ homeostasis. It couples the energy released from NADH oxidation and ubiquinone (Q) reduction to pump four protons across the inner mitochondrial membrane, contributing to the proton motive force used to synthesise ATP. Despite recent advances in structural knowledge and decades of biochemical investigations, the mechanism of redox-coupled proton translocation by complex I is still unknown. In the work presented in this thesis, electron cryomicroscopy (cryo-EM) was used as a primary tool to provide fundamental insight on this mechanism by generating three-dimensional reconstructions of complex I bound to substrates, ligands or inhibitors. Analyses of the structural data and further interrogation by complementary biochemical, biophysical, and computational approaches were used to develop an integrated understanding of substrate and inhibitor binding to complex I. First, to investigate the mechanism of action of a drug in phase 1 clinical trials against cancers reliant on oxidative phosphorylation (IACS-010759), the structure of mouse complex I inhibited by IACS-2858 – a tighter binding derivative – was resolved to a global resolution of 3.0 Å. The inhibitor, which bears little resemblance to ubiquinone-10 (Q₁₀), occupies the entrance to the Q-binding channel in a ‘cork-in-bottle’ binding mode not previously observed for complex I. Key inhibitor-enzyme interactions were identified, providing a molecular basis for understanding cross-species differences in binding affinities. Modelling of kinetic data showed that IACS-2858 is a simple one-site competitive inhibitor, and the structural motif of a ‘chain’ of aromatic rings was proposed as a characteristic that promotes complex I inhibition. Next, a strategy for reconstituting bovine complex I into lipid nanodiscs supplemented with exogenous Q₁₀ was devised to probe how the native substrate Q₁₀ binds to the ‘reactive site’ of the Q-binding channel. Five structurally and biochemically distinct conformational classes were identified at global resolutions up to 2.3 Å. These structures fall into three major states: an ‘active’ ready-to-catalyse state, a ‘deactive’ pronounced resting state, and a ‘slack’ state that appears partially disrupted and is of uncertain physiological and biochemical relevance. Comparisons of the deactive structures suggested how substrate/ligand binding restructures the Q-binding site and why both Q and NADH are required for reactivation. Importantly, a Q₁₀ molecule spanning the entirety of the Q-binding site was observed with the Q-headgroup close to its proposed ligating partners NDUFS2-His59 and NDUFS2-Tyr108. Combined with results from molecular dynamics simulations, these structures reveal how the charge states of key active-site residues influence the Q₁₀ binding pose. The bound Q₁₀ species is attributed to a quinone paused in a ‘pre-reactive’ conformation

Sensitization of human cancer cells to gemcitabine by the Chk1 inhibitor MK-8776: cell cycle perturbation and impact of administration schedule in vitro and in vivo

Background: Chk1 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. Methods: Growth 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. Results: MK-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\u27 plasma. Cell cycle perturbation was also assessed in human pancreas tumor xenografts in mice. There was a dramatic accumulation of cells in S/G(2) 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. Conclusions: There 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

Making the leap from structure to mechanism: are the open states of mammalian complex I identified by cryoEM resting states or catalytic intermediates?

Respiratory complex I (NADH:ubiquinone oxidoreductase) is a multi-subunit, energy-transducing mitochondrial enzyme that is essential for oxidative phosphorylation and regulating NAD+/NADH pools. Despite recent advances in structural knowledge and a long history of biochemical analyses, the mechanism of redox-coupled proton translocation by complex I remains unknown. Due to its ability to separate molecules in a mixed population into distinct classes, single-particle electron cryomicroscopy has enabled identification and characterisation of different complex I conformations. However, deciding on their catalytic and/or regulatory properties to underpin mechanistic hypotheses, especially without detailed biochemical characterisation of the structural samples, has proven challenging. In this review we explore different mechanistic interpretations of the closed and open states identified in cryoEM analyses of mammalian complex I

Investigation of hydrated channels and proton pathways in a high-resolution cryo-EM structure of mammalian complex I.

Respiratory complex I, a key enzyme in mammalian metabolism, captures the energy released by reduction of ubiquinone by NADH to drive protons across the inner mitochondrial membrane, generating the proton-motive force for ATP synthesis. Despite remarkable advances in structural knowledge of this complicated membrane-bound enzyme, its mechanism of catalysis remains controversial. In particular, how ubiquinone reduction is coupled to proton pumping and the pathways and mechanisms of proton translocation are contested. We present a 2.4-Å resolution cryo-EM structure of complex I from mouse heart mitochondria in the closed, active (ready-to-go) resting state, with 2945 water molecules modeled. By analyzing the networks of charged and polar residues and water molecules present, we evaluate candidate pathways for proton transfer through the enzyme, for the chemical protons for ubiquinone reduction, and for the protons transported across the membrane. Last, we compare our data to the predictions of extant mechanistic models, and identify key questions to answer in future work to test them

Cryo-EM structures of mitochondrial respiratory complex I from Drosophila melanogaster.

Peer reviewed: TrueAcknowledgements: We thank D Chirgadze (University of Cambridge Cryo-EM facility) for assistance with grid screening and cryo-EM data collection; T Croll (Cambridge Institute for Medical Research) for assistance with ISOLDE and I M Fearnley and S Ding (MRC MBU) for mass spectrometry analyses. This work was supported by the Medical Research Council (MC_UU_00015/6 and MC_UU_00028/6 to AJW and MC_UU_00015/2 and MC_UU_00028/1 to JH). Drosophila were obtained from the Bloomington Drosophila Stock Center, which is supported by grant NIH P40OD018537.Respiratory complex I powers ATP synthesis by oxidative phosphorylation, exploiting the energy from NADH oxidation by ubiquinone to drive protons across an energy-transducing membrane. Drosophila melanogaster is a candidate model organism for complex I due to its high evolutionary conservation with the mammalian enzyme, well-developed genetic toolkit, and complex physiology for studies in specific cell types and tissues. Here, we isolate complex I from Drosophila and determine its structure, revealing a 43-subunit assembly with high structural homology to its 45-subunit mammalian counterpart, including a hitherto unknown homologue to subunit NDUFA3. The major conformational state of the Drosophila enzyme is the mammalian-type 'ready-to-go' active resting state, with a fully ordered and enclosed ubiquinone-binding site, but a subtly altered global conformation related to changes in subunit ND6. The mammalian-type 'deactive' pronounced resting state is not observed: in two minor states, the ubiquinone-binding site is unchanged, but a deactive-type π-bulge is present in ND6-TMH3. Our detailed structural knowledge of Drosophila complex I provides a foundation for new approaches to disentangle mechanisms of complex I catalysis and regulation in bioenergetics and physiology

Reverse Electron Transfer by Respiratory Complex I Catalyzed in a Modular Proteoliposome System.

Respiratory complex I is an essential metabolic enzyme that uses the energy from NADH oxidation and ubiquinone reduction to translocate protons across an energy transducing membrane and generate the proton motive force for ATP synthesis. Under specific conditions, complex I can also catalyze the reverse reaction, Δp-linked oxidation of ubiquinol to reduce NAD+ (or O2), known as reverse electron transfer (RET). Oxidative damage by reactive oxygen species generated during RET underpins ischemia reperfusion injury, but as RET relies on several converging metabolic pathways, little is known about its mechanism or regulation. Here, we demonstrate Δp-linked RET through complex I in a synthetic proteoliposome system for the first time, enabling complete kinetic characterization of RET catalysis. We further establish the capability of our system by showing how RET in the mammalian enzyme is regulated by the active-deactive transition and by evaluating RET by complex I from several species in which direct assessment has not been otherwise possible. We thus provide new insights into the reversibility of complex I catalysis, an important but little understood mechanistic and physiological feature