Sab concentrations indicate chemotherapeutic susceptibility in ovarian cancer cell lines.

The occurrence of chemotherapy-resistant tumors makes ovarian cancer (OC) the most lethal gynecological malignancy. While many factors may contribute to chemoresistance, the mechanisms responsible for regulating tumor vulnerability are under investigation. Our analysis of gene expression data revealed that Sab, a mitochondrial outer membrane (MOM) scaffold protein, was down-regulated in OC patients. Sab-mediated signaling induces cell death, suggesting that this apoptotic pathway is diminished in OC. We examined Sab expression in a panel of OC cell lines and found that the magnitude of Sab expression correlated to chemo-responsiveness; wherein, OC cells with low Sab levels were chemoresistant. The Sab levels were reflected by a corresponding amount of stress-induced c-Jun N-terminal kinase (JNK) on the MOM. BH3 profiling and examination of Bcl-2 and BH3-only protein concentrations revealed that cells with high Sab concentrations were primed for apoptosis, as determined by the decrease in pro-survival Bcl-2 proteins and an increase in pro-apoptotic BH3-only proteins on mitochondria. Furthermore, overexpression of Sab in chemoresistant cells enhanced apoptotic priming and restored cellular vulnerability to a combination treatment of cisplatin and paclitaxel. Contrariwise, inhibiting Sab-mediated signaling or silencing Sab expression in a chemosensitive cell line resulted in decreased apoptotic priming and increased resistance. The effects of silencing on Sab on the resistance to chemotherapeutic agents were emulated by the silencing or inhibition of JNK, which could be attributed to changes in Bcl-2 protein concentrations induced by sub-chronic JNK inhibition. We propose that Sab may be a prognostic biomarker to discern personalized treatments for OC patients.

A rapid and sensitive high-throughput screening method to identify compounds targeting protein-nucleic acids interactions.

DNA-binding and RNA-binding proteins are usually considered 'undruggable' partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein-nucleic acids interactions based on protein-DNA or protein-RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2-DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2-DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2-DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.

Sub-chronic administration of LY294002 sensitizes cervical cancer cells to chemotherapy by enhancing mitochondrial JNK signaling.

Chemo-sensitization is used to improve the efficacy of chemotherapeutic agents against cancers, and understanding the precise molecular mechanisms of chemo-sensitization could lead to safer and more effective approaches to treat cancer. We have previously demonstrated that mitochondrial c-Jun N-terminal Kinase (JNK) signaling is a critical component of cell death. Mitochondrial JNK signaling is coordinated on the scaffold protein Sab. In this work, we developed a sub-chronic chemo-sensitization model by exposing HeLa cells to low-dose (2 µM) LY294002. We found that this treatment increased Sab expression on mitochondria, an effect not observed in acute exposures. To examine the role of Sab in chemo-sensitization, we ectopically expressed and silenced Sab in HeLa cells. We found that elevating Sab levels in HeLa cells increased the efficacy of chemotherapeutic agents, paclitaxel and cisplatin, while silencing Sab decreased the sensitivity of cells towards these agents. The effect of Sab-mediated signaling appeared to be dependent upon mitogen dependent protein kinases (MAPKs) as ablation of Sab's MAPK-binding motifs prevented chemo-sensitization. These results suggest that mitochondrial JNK signaling is an adaptable signaling pathway that can be enhanced or restored in cancer cells to improve therapeutic efficacy.

A trivalent approach for determining in vitro toxicology: Examination of oxime K027.

Unforeseen toxic effects contribute to compound attrition during preclinical evaluation and clinical trials. Consequently, there is a need to correlate in vitro toxicity to in vivo and clinical outcomes quickly and effectively. We propose an expedited evaluation of physiological parameters in vitro that will improve the ability to predict in vivo toxicity of potential therapeutics. By monitoring metabolism, mitochondrial physiology and cell viability, our approach provides insight to the extent of drug toxicity in vitro. To implement our approach, we used human hepatocellular carcinoma cells (HepG2) and neuroblastoma cells (SH-SY5Y) to monitor hepato- and neurotoxicity of the experimental oxime K027. We utilized a trivalent approach to measure metabolism, mitochondrial stress and induction of apoptosis in 96-well formats. Any change in these three areas may suggest drug-induced toxicity in vivo. K027 and pralidoxime, an oxime currently in clinical use, had no effect on glycolysis or oxygen consumption in HepG2 and SH-SY5Y cells. Similarly, these oximes did not induce oxidant generation nor alter mitochondrial membrane potential. Further, K027 and pralidoxime failed to activate effector caspases, and these oximes did not alter viability. The chemotherapeutic agent, docetaxel, negatively affected metabolism, mitochondrial physiology and viability. Our studies present a streamlined high-throughput trivalent approach for predicting toxicity in vitro, and this approach reveals that K027 has no measurable hepatotoxicity or neurotoxicity in vitro, which correlates with their in vivo data. This approach could eliminate toxic drugs from consideration for in vivo preclinical evaluation faster than existing toxicity prediction panels and ultimately prevent unnecessary experimentation.

Blocking c-Jun N-terminal kinase (JNK) translocation to the mitochondria prevents 6-hydroxydopamine-induced toxicity in vitro and in vivo.

Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-Sab(KIM1) protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-Sab(KIM1) via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-Sab(KIM1) decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-Sab(KIM1) at day 7 were 750 nm, which was ∼3.4-fold higher than the IC(50) for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.

Inhibition of JNK mitochondrial localization and signaling is protective against ischemia/reperfusion injury in rats.

To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.

Physiological JNK3 Concentrations Are Higher in Motor-related and Disease-implicated Brain Regions of C57BL6/J Mice.

The c-Jun N-terminal kinase 3 (JNK3) is a stress-responsive protein kinase primarily expressed in the central nervous system (CNS). JNK3 exhibits nuanced neurological activities, such as roles in behavior, circadian rhythms, and neurotransmission, but JNK3 is also implicated in cell death and neurodegeneration. Despite the critical role of JNK3 in neurophysiology and pathology, its localization in the brain is not fully understood due to a paucity of tools to distinguish JNK3 from other isoforms. While previous functional and histological studies suggest locales for JNK3 in the CNS, a comprehensive and higher resolution of JNK3 distribution and abundance remained elusive. Here, we sought to define the anatomical and cellular distribution of JNK3 in adult mouse brains. Data reveal the highest levels of JNK3 and pJNK3 were found in the cortex and the hippocampus. JNK3 possessed neuron-type selectivity as JNK3 was present in GABAergic, cholinergic, and dopaminergic neurons, but was not detectable in VGLUT-1-positive glutamatergic neurons and astrocytes in vivo . Intriguingly, higher JNK3 signals were found in motor neurons and relevant nuclei in the cortex, basal ganglia, brainstem, and spinal cord. While JNK3 was primarily observed in the cytosol of neurons in the cortex and the hippocampus, JNK3 appeared commonly within the nucleus in the brainstem. These distinctions suggest the potential for significant differences between JNK3 actions in distinct brain regions and cell types. Our results provide a significant improvement over previous reports of JNK3 spatial organization in the adult CNS and support continued investigation of JNK3's role in neurophysiology and pathophysiology.

Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long-term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2-associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45(-ve) /CD44(+ve) cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300-fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen-induced stress response was dependent upon p53 since siRNA-mediated knockdown of p53 in wild-type cells or exposure of p53(-/-) MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long-term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen-induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long-term expansion in vitro.

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation.

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.

Novel and Structurally Diversified Bacterial DNA Gyrase Inhibitors Discovered through a Fluorescence-Based High-Throughput Screening Assay.

Bacterial DNA gyrase, a type IIA DNA topoisomerase that plays an essential role in bacterial DNA replication and transcription, is a clinically validated target for discovering and developing new antibiotics. In this article, based on a supercoiling-dependent fluorescence quenching (SDFQ) method, we developed a high-throughput screening (HTS) assay to identify inhibitors targeting bacterial DNA gyrase and screened the National Institutes of Health's Molecular Libraries Small Molecule Repository library containing 370,620 compounds in which 2891 potential gyrase inhibitors have been identified. According to these screening results, we acquired 235 compounds to analyze their inhibition activities against bacterial DNA gyrase using gel- and SDFQ-based DNA gyrase inhibition assays and discovered 155 new bacterial DNA gyrase inhibitors with a wide structural diversity. Several of them have potent antibacterial activities. These newly discovered gyrase inhibitors include several DNA gyrase poisons that stabilize the gyrase-DNA cleavage complexes and provide new chemical scaffolds for the design and synthesis of bacterial DNA gyrase inhibitors that may be used to combat multidrug-resistant bacterial pathogens. Additionally, this HTS assay can be applied to screen inhibitors against other DNA topoisomerases.

Radiolabeled Aminopyrazoles as Novel Isoform Selective Probes for pJNK3 Quantification.

The c-Jun N-terminal kinase 3 (JNK3) is a stress-activated kinase primarily expressed in the brain and implicated as an early mediator of neuronal apoptosis. We sought to develop a PET tracer to visualize pathological JNK3 activation. Because regional JNK3 activation precedes apoptosis, such an imaging agent might enable the detection of "at risk" brain regions prior to neuronal death. We prepared a set of 19F-containing compounds on the basis of the reported aminopyrazoles. The candidate, F3, was tritiated and used in autoradiography experiments to demonstrate regional and temporal changes in JNK3 activation in a mouse model of Parkinson's disease. A significant increase in pJNK3 B max versus control animals in multiple brain regions was observed at 8 months, including the ventral midbrain. Pathological activation of JNK3 in these regions preceded statistically significant neuron loss. Analyses of brain concentrations of [18F]-F3 in naïve rats following intravenous injection revealed a small but detectable signal over the background, but was likely not sufficient to support PET imaging.

Glutamine metabolism is essential for human cytomegalovirus infection.

Human fibroblasts infected with human cytomegalovirus (HCMV) were more viable than uninfected cells during glucose starvation, suggesting that an alternate carbon source was used. We have determined that infected cells require glutamine for ATP production, whereas uninfected cells do not. This suggested that during infection, glutamine is used to fill the tricarboxylic acid (TCA) cycle (anaplerosis). In agreement with this, levels of glutamine uptake and ammonia production increased in infected cells, as did the activities of glutaminase and glutamate dehydrogenase, the enzymes needed to convert glutamine to alpha-ketoglutarate to enter the TCA cycle. Infected cells starved for glutamine beginning 24 h postinfection failed to produce infectious virions. Both ATP and viral production could be rescued in glutamine-starved cells by the TCA intermediates alpha-ketoglutarate, oxaloacetate, and pyruvate, confirming that in infected cells, a program allowing glutamine to be used anaplerotically is induced. Thus, HCMV infection activates the mechanisms needed to switch the anaplerotic substrate from glucose to glutamine to accommodate the biosynthetic and energetic needs of the viral infection and to allow glucose to be used biosynthetically.

Quercetin, a fluorescent bioflavanoid, inhibits Trypanosoma brucei hexokinase 1.

Hexokinases from the African trypanosome, Trypanosoma brucei, are attractive targets for the development of anti-parasitic drugs, in part because the parasite utilizes glycolysis exclusively for ATP production during the mammalian infection. Here, we have demonstrated that the bioflavanoid quercetin (QCN), a known trypanocide, is a mixed inhibitor of Trypanosoma brucei hexokinase 1 (TbHK1) (IC(50) = 4.1 ± 0.8µM). Spectroscopic analysis of QCN binding to TbHK1, taking advantage of the intrinsically fluorescent single tryptophan (Trp177) in TbHK1, revealed that QCN quenches emission of Trp177, which is located near the hinge region of the enzyme. ATP similarly quenched Trp177 emission, while glucose had no impact on fluorescence. Supporting the possibility that QCN toxicity is a consequence of inhibition of the essential hexokinase, in live parasites QCN fluorescence localizes to glycosomes, the subcellular home of TbHK1. Additionally, RNAi-mediated silencing of TbHK1 expression expedited QCN induced death, while over-expressing TbHK1 protected trypanosomes from the compound. In summary, these observations support the suggestion that QCN toxicity is in part attributable to inhibition of the essential TbHK1.

Simultaneous Ca2+ Imaging and Optogenetic Stimulation of Cortical Astrocytes in Adult Murine Brain Slices.

Astrocytes are actively involved in a neuroprotective role in the brain, which includes scavenging reactive oxygen species to minimize tissue damage. They also modulate neuroinflammation and reactive gliosis prevalent in several brain disorders like epilepsy, Alzheimer's, and Parkinson's disease. In animal models, targeted manipulation of astrocytic function via modulation of their calcium (Ca2+ ) oscillations by incorporating light-sensitive cation channels like Channelrhodopsin-2 (ChR2) offers a promising avenue in influencing the long-term progression of these disorders. However, using adult animals for Ca2+ imaging poses major challenges, including accelerated deterioration of in situ slice health and age- related changes. Additionally, optogenetic preparations necessitate usage of a red-shifted Ca2+ indicator like Rhod-2 AM to avoid overlapping light issues between ChR2 and the Ca2+ indicator during simultaneous optogenetic stimulation and imaging. In this article, we provide an experimental setting that uses live adult murine brain slices (2-5 months) from a knock-in model expressing Channelrhodopsin-2 (ChR2(C128S)) in cortical astrocytes, loaded with Rhod-2 AM to elicit robust Ca2+ response to light stimulation. We have developed and standardized a protocol for brain extraction, sectioning, Rhod-2 AM loading, maintenance of slice health, and Ca2+ imaging during light stimulation. This has been successfully applied to optogenetically control adult cortical astrocytes, which exhibit synchronous patterns of Ca2+ activity upon light stimulation, drastically different from resting spontaneous activity. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Experimental preparation, setup, slice preparation and Rhod-2 AM staining Basic Protocol 2: Image acquisition and analysis.

A Novel Interaction of Translocator Protein 18 kDa (TSPO) with NADPH Oxidase in Microglia.

In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.

Phosphoregulation on mitochondria: Integration of cell and organelle responses.

Mitochondria are highly integrated organelles that are crucial to cell adaptation and mitigating adverse physiology. Recent studies demonstrate that fundamental signal transduction pathways incorporate mitochondrial substrates into their biological programs. Reversible phosphorylation is emerging as a useful mechanism to modulate mitochondrial function in accordance with cellular changes. Critical serine/threonine protein kinases, such as the c-Jun N-terminal kinase (JNK), protein kinase A (PKA), PTEN-induced kinase-1 (PINK1), and AMP-dependent protein kinase (AMPK), readily translocate to the outer mitochondrial membrane (OMM), the interface of mitochondria-cell communication. OMM protein kinases phosphorylate diverse mitochondrial substrates that have discrete effects on organelle dynamics, protein import, respiratory complex activity, antioxidant capacity, and apoptosis. OMM phosphorylation events can be tempered through the actions of local protein phosphatases, such as mitogen-activated protein kinase phosphatase-1 (MKP-1) and protein phosphatase 2A (PP2A), to regulate the extent and duration of signaling. The central mediators of OMM signal transduction are the scaffold proteins because the relative abundance of these accessory proteins determines the magnitude and duration of a signaling event on the mitochondrial surface, which dictates the biological outcome of a local signal transduction pathway. The concentrations of scaffold proteins, such as A-kinase anchoring proteins (AKAPs) and Sab (or SH3 binding protein 5-SH3BP5), have been shown to influence neuronal survival and vulnerability, respectively, in models of Parkinson's disease (PD), highlighting the importance of OMM signaling to health and disease. Despite recent progress, much remains to be discovered concerning the mechanisms of OMM signaling. Nonetheless, enhancing beneficial OMM signaling events and inhibiting detrimental protein-protein interactions on the mitochondrial surface may represent highly selective approaches to restore mitochondrial health and homeostasis and mitigate organelle dysfunction in conditions such as PD.

Kinetic Study of DNA Topoisomerases by Supercoiling-Dependent Fluorescence Quenching.

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes (Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes (E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M, V max, and k cat. We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors.

Tyrosyl-DNA Phosphodiesterase 1 and Topoisomerase I Activities as Predictive Indicators for Glioblastoma Susceptibility to Genotoxic Agents.

Glioblastoma (GBM) patients have an estimated survival of ~15 months with treatment, and the standard of care only modestly enhances patient survival. Identifying biomarkers representing vulnerabilities may allow for the selection of efficacious chemotherapy options to address personalized variations in GBM tumors. Irinotecan targets topoisomerase I (TOP1) by forming a ternary DNA-TOP1 cleavage complex (TOP1cc), inducing apoptosis. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a crucial repair enzyme that may reduce the effectiveness of irinotecan. We treated GBM cell lines with increasing concentrations of irinotecan and compared the IC50 values. We found that the TDP1/TOP1 activity ratio had the strongest correlation (Pearson correlation coefficient R = 0.972, based on the average from three sets of experiments) with IC50 values following irinotecan treatment. Increasing the TDP1/TOP1 activity ratio by the ectopic expression of wild-type TDP1 increased in irinotecan IC50, while the expression of the TDP1 catalytic-null mutant did not alter the susceptibility to irinotecan. The TDP1/TOP1 activity ratio may be a new predictive indicator for GBM vulnerability to irinotecan, allowing for the selection of individual patients for irinotecan treatment based on risk-benefit. Moreover, TDP1 inhibitors may be a novel combination treatment with irinotecan to improve GBM patient responsiveness to genotoxic chemotherapies.

Residues in an ATP binding domain influence sugar binding in a trypanosome hexokinase.

Trypanosoma brucei harbors two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. We previously found that recombinant TbHK1 (rTbHK1) has hexokinase activity, while rTbHK2 has not, a finding attributed to differences in the C-termini of the proteins. Sequence analysis suggests that the C-termini of TbHKs are part of a newly identified conserved motif found in other eukaryotic hexokinases. Here, we have explored the role of tail residues in the differences in catalytic activity between TbHK1 and TbHK2. Our studies reveal that tail residues D454, F462, M466, and N469 are essential for HK activity while both I458 and V468 are required for catalysis and substrate specificity. To activate rTbHK2, all of the residues important for activity in rTbHK1 (D454, V458, F462, M466, V468, and N469) were required. These results indicate that the overall structure of the C-terminal tail influences the HK activity of rTbHK1.

The anti-trypanosomal agent lonidamine inhibits Trypanosoma brucei hexokinase 1.

Glycolysis is essential to the parasitic protozoan Trypanosoma brucei. The first step in this metabolic pathway is mediated by hexokinase, an enzyme that transfers the gamma-phosphate of ATP to a hexose. The T. brucei genome (TREU927/4 GUTat10.1) encodes two hexokinases (TbHK1 and TbHK2) that are 98% identical at the amino acid level. Our previous efforts have revealed that TbHK2 is an important regulator of TbHK1 in procyclic form parasites. Here, we have found through RNAi that TbHK1 is essential to the bloodstream form parasite. Silencing the gene for 4 days reduces cellular hexokinase approximately 60% and leads to parasite death. Additionally, we have found that the recombinant enzyme is inhibited by lonidamine (IC(50)=850 microM), an anti-cancer drug that targets tumor hexokinases. This agent also inhibits HK activity from whole parasite lysate (IC(50)=965 microM). Last, lonidamine is toxic to cultured bloodstream form parasites (LD(50)=50 microM) and procyclic form parasites (LD(50)=180 microM). Interestingly, overexpression of TbHK1 protects PF parasites from lonidamine. These studies provide genetic evidence that TbHK1 is a valid therapeutic target while identifying a potential molecular target of the anti-trypanosomal agent lonidamine.