Mechanistic analysis of actin-binding compounds that affect the kinetics of cardiac myosin-actin interaction.

Actin-myosin mediated contractile forces are crucial for many cellular functions, including cell motility, cytokinesis, and muscle contraction. We determined the effects of ten actin-binding compounds on the interaction of cardiac myosin subfragment 1 (S1) with pyrene-labeled F-actin (PFA). These compounds, previously identified from a small-molecule high-throughput screen (HTS), perturb the structural dynamics of actin and the steady-state actin-activated myosin ATPase activity. However, the mechanisms underpinning these perturbations remain unclear. Here we further characterize them by measuring their effects on PFA fluorescence, which is decreased specifically by the strong binding of myosin to actin. We measured these effects under equilibrium and steady-state conditions, and under transient conditions, in stopped-flow experiments following addition of ATP to S1-bound PFA. We observed that these compounds affect early steps of the myosin ATPase cycle to different extents. They increased the association equilibrium constant K1 for the formation of the strongly bound collision complex, indicating increased ATP affinity for actin-bound myosin, and decreased the rate constant k+2 for subsequent isomerization to the weakly bound ternary complex, thus slowing the strong-to-weak transition that actin-myosin interaction undergoes early in the ATPase cycle. The compounds' effects on actin structure allosterically inhibit the kinetics of the actin-myosin interaction in ways that may be desirable for treatment of hypercontractile forms of cardiomyopathy. This work helps to elucidate the mechanisms of action for these compounds, several of which are currently used therapeutically, and sets the stage for future HTS campaigns that aim to discover new drugs for treatment of heart failure.

Modulatory Role of Vitamin E on Proton Pump (ATPase) Activity of Cadmium Chloride-Induced Testicular Damage in Wistar Rats.

Proton pumps are membrane-bound enzymes important in generating gradients that help in maintaining cellular ion homeostasis, cell membrane potential, water, and solute transport across the cell surface. This study investigated the modulatory role of vitamin E on proton pump activity and reproductive parameters in cadmium-induced testicular damage. Twenty (20) male Wistar rats weighing between 180 and 200 g were sorted into 4 groups of five rats each. Group I served as the control and was given normal saline orally, Group II rats were treated with a single dose of 2 mg/kg BW cadmium chloride (CdCl2) intraperitoneally, Group III rats were given 100 mg/kg BW of vitamin E orally, and Group IV rats were given 100 mg/kg BW of vitamin E orally for 30 days prior to intraperitoneal administration of single dose of 2 mg/kg BW of cadmium chloride. The rats were anaesthetized with diethyl ether, and blood samples were obtained for sex hormonal analysis; caudal epididymis was dissected for sperm count, motility, and viability, and the testis were homogenized for lipid peroxidation and proton pump (Na+/K+ ATPase, Ca2+ ATPase, and Mg2+ ATPase) activity. Proton pump activity was assayed spectrophotometrically using the Stewart method to determine the inorganic phosphate level. Histopathological changes of the testis were also studied. The group treated with CdCl2 showed a significant (p < 0.05) decrease in proton pump activity, sperm count, and motility and a significant (p < 0.05) increase in malondialdehyde level when compared with the control group. The CdCl2-treated group also showed decrease reproductive organ weights and hormonal levels and cause necrosis of spermatogonia lining the seminiferous tubules. Rats treated with vitamin E orally for 30 days prior to CdCl2 exposure showed improvement in proton pump activity, a significant (p < 0.05) increase in sperm parameters and luteinizing hormonal level, and a decrease in the lipid peroxidation level as compared with the CdCl2 group. This study showed that vitamin E ameliorated the toxic effect of CdCl2 on proton pump activity in the testes, hence improving testicular integrity, structures, and functions.

A gene silencing of V-ATPase subunit A interferes with survival and development of the tomato leafminer, Tuta absoluta.

RNA interference (RNAi) technology is not only considered as a tool to analyze gene function, but it is also potentially considered as a strategy to develop novel biopesticide. In the current study, a double-stranded RNA specific to v-ATPase subunit A of the tomato leafminer, Tuta absoluta (Meyrick; Lepidoptera: Gelechiidae), was orally administered. A gradual decrease in the expression of the gene was observed from Day 1 to 3 and resulted in significant larval mortality. These results suggest that v-ATPases A can be considered as a promising target gene by RNAi technology to be used in the management of the tomato leafminer.

Evaluation of the acute toxic effect of azoxystrobin on non-target crayfish (Astacus leptodactylus Eschscholtz, 1823) by using oxidative stress enzymes, ATPases and cholinesterase as biomarkers.

Azoxystrobin is a broad-spectrum fungicide used worldwide. Since azoxystrobin spreads to large areas, its toxic effects on non-target organisms have aroused interest. In this study, the acute toxicity (96 h) of azoxystrobin on the crayfish (Astacus leptodactylus) was examined by using various biomarkers. The 96 h-LC50 dose (1656 mg L-) and its three sub-doses (828, 414, 207 mg L-1) were applied to crayfish. Superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were increased significantly compared to the control in hepatopancreas, gill and muscle tissues. The activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) increased, and glutathione reductase (GR) activity decreased significantly in hepatopancreas. Level of reduced glutathione (GSH) decreased significantly. The content of malondialdehyde (MDA) increased in a dose-dependent manner in all azoxystrobin treatments with the exception of the lowest dose (207 mg L-1)treatment. ATPases (Na+/K+ -ATPase, Mg2+ -ATPase, Ca2+ -ATPase, total ATPase) were significantly inhibited in gill and muscle tissues. The results of the present study indicate that azoxystrobin induces oxidative stress, and has adverse effects on activities of AChE and ATPases in crayfish.

Fully human anti-CD39 antibody potently inhibits ATPase activity in cancer cells via uncompetitive allosteric mechanism.

The extracellular ATP/adenosine axis in the tumor microenvironment (TME) has emerged as an important immune-regulatory pathway. Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), otherwise known as CD39, is highly expressed in the TME, both on infiltrating immune cells and tumor cells across a broad set of cancer indications. CD39 processes pro-inflammatory extracellular ATP to ADP and AMP, which is then processed by Ecto-5'-nucleotidase/CD73 to immunosuppressive adenosine. Directly inhibiting the enzymatic function of CD39 via an antibody has the potential to unleash an immune-mediated anti-tumor response via two mechanisms: 1) increasing the availability of immunostimulatory extracellular ATP released by damaged and/or dying cells, and 2) reducing the generation and accumulation of suppressive adenosine within the TME. Tizona Therapeutics has engineered a novel first-in-class fully human anti-CD39 antibody, TTX-030, that directly inhibits CD39 ATPase enzymatic function with sub-nanomolar potency. Further characterization of the mechanism of inhibition by TTX-030 using CD39+ human melanoma cell line SK-MEL-28 revealed an uncompetitive allosteric mechanism (α < 1). The uncompetitive mechanism of action enables TTX-030 to inhibit CD39 at the elevated ATP concentrations reported in the TME. Maximal inhibition of cellular CD39 ATPase velocity was 85%, which compares favorably to results reported for antibody inhibitors to other enzyme targets. The allosteric mechanism of TTX-030 was confirmed via mapping the epitope to a region of CD39 distant from its active site, which suggests possible models for how potent inhibition is achieved. In summary, TTX-030 is a potent allosteric inhibitor of CD39 ATPase activity that is currently being evaluated in clinical trials for cancer therapy.

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

OBJECTIVES: To evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches. METHODS: Redx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863. RESULTS: Both compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive. CONCLUSIONS: Redx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

α1-Na/K-ATPase inhibition rescues aberrant dendritic calcium dynamics and memory deficits in the hippocampus of an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of the maternal copy of the UBE3A gene. Previous studies reported an increase in α1-Na/K-ATPase (α1-NaKA) expression in the AS hippocampus at the age of 2 weeks as the initial and isolated molecular alteration. This increase was further implied upon actuating much of the hippocampal-related deficits in an AS mouse model, although the underlying mechanism was never investigated. Here, we showed that enhanced α1-NaKA expression resulted in increased pump activity that reduced activity-dependent dendritic Ca2+ dynamics in the AS hippocampus, as well as selective inhibition of α1-NaKA by marinobufagenin (MBG) to normalize these aberrant Ca2+ dynamics. In addition, we demonstrated that selective α1-NaKA inhibition corrected impaired hippocampal synaptic plasticity and hippocampal-dependent cognitive deficits. Furthermore, we showed that the isolated increase in hippocampal α1-NaKA expression in AS mice at 2 weeks of age was accompanied by an unexpected enhancement in excitability. Altogether, our study implicates the modification of Ca2+ dynamics as one of the major underlying mechanisms by which enhanced α1-NaKA expression induces deleterious effects in the hippocampus of AS model mice. Finally, we propose a therapeutic approach for AS and possibly other neurodevelopmental disorders that entail aberrant NaKA expression or abnormal Ca2+ dynamics.

Modeling Host-Pathogen Interaction to Elucidate the Metabolic Drug Response of Intracellular Mycobacterium tuberculosis.

Little is known about the metabolic state of Mycobacterium tuberculosis (Mtb) inside the phagosome, a compartment inside phagocytes for killing pathogens and other foreign substances. We have developed a combined model of Mtb and human metabolism, sMtb-RECON and used this model to predict the metabolic state of Mtb during infection of the host. Amino acids are predicted to be used for energy production as well as biomass formation. Subsequently we assessed the effect of increasing dosages of drugs targeting metabolism on the metabolic state of the pathogen and predict resulting metabolic adaptations and flux rerouting through various pathways. In particular, the TCA cycle becomes more important upon drug application, as well as alanine, aspartate, glutamate, proline, arginine and porphyrin metabolism, while glycine, serine, and threonine metabolism become less important. We modeled the effect of 11 metabolically active drugs. Notably, the effect of eight could be recreated and two major profiles of the metabolic state were predicted. The profiles of the metabolic states of Mtb affected by the drugs BTZ043, cycloserine and its derivative terizidone, ethambutol, ethionamide, propionamide, and isoniazid were very similar, while TMC207 is predicted to have quite a different effect on metabolism as it inhibits ATP synthase and therefore indirectly interferes with a multitude of metabolic pathways.

Single-molecule observation of nucleotide induced conformational changes in basal SecA-ATP hydrolysis.

SecA is the critical adenosine triphosphatase that drives preprotein transport through the translocon, SecYEG, in Escherichia coli. This process is thought to be regulated by conformational changes of specific domains of SecA, but real-time, real-space measurement of these changes is lacking. We use single-molecule atomic force microscopy (AFM) to visualize nucleotide-dependent conformations and conformational dynamics of SecA. Distinct topographical populations were observed in the presence of specific nucleotides. AFM investigations during basal adenosine triphosphate (ATP) hydrolysis revealed rapid, reversible transitions between a compact and an extended state at the ~100-ms time scale. A SecA mutant lacking the precursor-binding domain (PBD) aided interpretation. Further, the biochemical activity of SecA prepared for AFM was confirmed by tracking inorganic phosphate release. We conclude that ATP-driven dynamics are largely due to PBD motion but that other segments of SecA contribute to this motion during the transition state of the ATP hydrolysis cycle.

Reconstitution and functional studies of hamster P-glycoprotein in giant liposomes.

This paper describes the preparation of giant unilamellar vesicles with reconstituted hamster P-glycoprotein (Pgp, ABCB1) for studying the transport activity of this efflux pump in individual liposomes using optical microscopy. Pgp, a member of ABC (ATP-binding cassette) transporter family, is known to contribute to the cellular multidrug resistance (MDR) against variety of drugs. The efficacy of many therapeutics is, thus, hampered by this efflux pump, leading to a high demand for simple and effective strategies to monitor the interactions of candidate drugs with this protein. Here, we applied small Pgp proteoliposomes to prepare giant Pgp-bearing liposomes via modified electroformation techniques. The presence of Pgp in the membrane of giant proteoliposomes was confirmed using immunohistochemistry. Assessment of Pgp ATPase activity suggested that this transporter retained its activity upon reconstitution into giant liposomes, with an ATPase specific activity of 439 ± 103 nmol/mg protein/min. For further confirmation, we assessed the transport activity of Pgp in these proteoliposomes by monitoring the translocation of rhodamine 123 (Rho123) across the membrane using confocal microscopy at various ATP concentrations (0-2 mM) and in the presence of Pgp inhibitors. Rate of change in Rho123 concentration inside the liposomal lumen was used to estimate the Rho123 transport rates (1/s) for various ATP concentrations, which were then applied to retrieve the Michaelis-Menten constant (Km) of ATP in Rho123 transport (0.42 ± 0.75 mM). Similarly, inhibitory effects of verapamil, colchicine, and cyclosporin A on Pgp were studied in this system and the IC50 values for these Pgp inhibitors were found 26.6 ± 6.1 µM, 94.6 ± 47.6 µM, and 0.21 ± 0.07 µM, respectively. We further analyzed the transport data using a kinetic model that enabled dissecting the passive diffusion of Rho123 from its Pgp-mediated transport across the membrane. Based on this model, the permeability coefficient of Rho123 across the liposomal membrane was approximately 1.25×10-7 cm/s. Comparing the membrane permeability in liposomes with and without Pgp revealed that the presence of this protein did not have a significant impact on membrane integrity and permeability. Furthermore, we used this model to obtain transport rate constants for the Pgp-mediated transport of Rho123 (m3/mol/s) at various ATP and inhibitor concentrations, which were then applied to estimate values of 0.53 ± 0.66 mM for Km of ATP and 25.2 ± 5.0 µM for verapamil IC50, 61.8 ± 34.8 µM for colchicine IC50, and 0.23 ± 0.09 µM for cyclosporin A IC50. The kinetic parameters obtained from the two analyses were comparable, suggesting a minimal contribution from the passive Rho123 diffusion across the membrane. This approach may, therefore, be applied for screening the transport activity of Pgp against potential drug candidates.

Antibacterial mechanisms of cinnamon and its constituents: A review.

BACKGROUND: In the current healthcare environment, an alarming rise in multi-drug resistant bacterial infections has led to a global health threat. The lack of new antibiotics has created a need for developing alternative strategies. OBJECTIVE: Understanding the antibacterial mechanisms of cinnamon and its constituents is crucial to enhance it as a potential new source of antibiotic. The objective of this review is to provide a compilation of all described mechanisms of antibacterial action of cinnamon and its constituents and synergism with commercial antibiotics in order to better understand how cinnamon and its constituents can collaborate as alternative treatment to multi-drug resistant bacterial infections. METHODS: The relevant references on antibacterial activities of cinnamon and its constituents were searched. Meanwhile, the references were classified according to the type of mechanism of action against bacteria. Relationships of cinnamon or its constituents and antibiotics were also analyzed and summarized. RESULTS: Cinnamon extracts, essential oils, and their compounds have been reported to inhibit bacteria by damaging cell membrane; altering the lipid profile; inhibiting ATPases, cell division, membrane porins, motility, and biofilm formation; and via anti-quorum sensing effects. CONCLUSION: This review describes the antibacterial effects of cinnamon and its constituents, such as cinnamaldehyde and cinnamic acid, against pathogenic Gram-positive and Gram-negative bacteria. The review also provides an overview of the current knowledge of the primary modes of action of these compounds as well as the synergistic interactions between cinnamon or its constituents with known antibacterial agents. This information will be useful in improving the effectiveness of therapeutics based on these compounds.

Structural model, functional modulation by ivermectin and tissue localization of Haemonchus contortus P-glycoprotein-13.

Haemonchus contortus, one of the most economically important parasites of small ruminants, has become resistant to the anthelmintic ivermectin. Deciphering the role of P-glycoproteins in ivermectin resistance is desirable for understanding and overcoming this resistance. In the model nematode, Caenorhabditis elegans, P-glycoprotein-13 is expressed in the amphids, important neuronal structures for ivermectin activity. We have focused on its ortholog in the parasite, Hco-Pgp-13. A 3D model of Hco-Pgp-13, presenting an open inward-facing conformation, has been constructed by homology with the Cel-Pgp-1 crystal structure. In silico docking calculations predicted high affinity binding of ivermectin and actinomycin D to the inner chamber of the protein. Following in vitro expression, we showed that ivermectin and actinomycin D modulated Hco-Pgp-13 ATPase activity with high affinity. Finally, we found in vivo Hco-Pgp-13 localization in epithelial, pharyngeal and neuronal tissues. Taken together, these data suggest a role for Hco-Pgp-13 in ivermectin transport, which could contribute to anthelmintic resistance.

Antifungal mechanism of sodium dehydroacetate against Geotrichum citri-aurantii.

This study investigated the potential anti-fungal mechanisms of sodium dehydroacetate (SD) against Geotrichum citri-aurantii. The results showed that the cell wall integrity of G. citri-aurantii was not affected, whereas the membrane permeability of G. citri-aurantii mycelia was visibly altered by SD. Dramatic morphological changes of the mycelia, such as loss of cytoplasm, plasmolysis, and dissolution of intracellular substances, were observed by scanning electron microscopy and transmission electron microscopy analyses, indicating that the mycelium is severely damaged by the SD treatment. Furthermore, SD apparently induced a decrease in the intracellular ATP content before 30 min of exposure. An increase in the activity of the Na+/K+-ATPase was also observed, indicating that Na+ ions might enter the cell and thus disturb the energy supply. Taken together, this study's findings suggest that the anti-fungal activity of SD against G. citri-aurantii can be attributed to the disruption of cell membrane permeability and energy metabolism.

Listeria monocytogenes behaviour and quality attributes during sausage storage affected by sodium nitrite, sodium lactate and thyme essential oil.

The effects of the addition of nitrite at 200 ppm (N), sodium lactate 1.5% (L) and thyme essential oil at 100 ppm (T1) on Listeria monocytogenes behaviour and ATPase activity inhibition were evaluated, as well as lipid oxidation through the quantification of malonaldehydes, in sausage stored at 8 ℃ for 41 days and at 30 ℃ for 14 days. The changes in the colour profile were performed during storage time at 8 ℃. Quantitative descriptive sensory analyses were performed after two days at 4 ℃. At 8 ℃, the treatments with the highest inhibition on L. monocytogenes were L and N, without significant differences. In turn, at 30 ℃, the bacterium was most inhibited with treatment L, followed by T1 and N, without significant differences. A 44.1% and 19% inhibition of ATPase activity was detected in L and T1 treatments, respectively. At 8 ℃ and 30 ℃, malonaldehydes content was not different between the treatments. N presented the highest values of a* and concentration of metmyoglobin after 41 days at 8 ℃. The panel detected differences between T1 and N for the aroma in the descriptors spices and herbal.

Methylamine irisolidone, a novel compound, increases total ATPase activity and inhibits apoptosis in vivo and in vitro.

We propose to further research the protective effect of MMI on myocardium ischemic rat model and H9c2 cells that underwent cell apoptosis induced by hypoxia. We established the myocardium ischemic rat model via the cardiac surgical procedures in vivo and treated the model rats with different concentration of MMI. In vitro, with the pretreatment of MMI for 12 h in the model of Na2S2O4-induced hypoxia injury, the H9c2 cells viability was determined by MTT assay. We found that MMI had significantly improved cardiac function of the myocardial ischemia, and significantly decreased the reactive oxygen species level. The expression of P53, Bcl-2, Bax, and caspase-9 was also induced by MMI. In vitro study revealed a concentration-dependent increase in cell viability associated with MMI pretreatment. Annexin V-FITC and PI staining results showed that MMI had a preventive effect on hypoxia-induced apoptosis in H9c2 cells. MMI also inhibited the mitochondrial membrane potential decrease and increased total ATPase activity during hypoxia in H9c2 cells. In conclusion, MMI can enhance the cardiac function in myocardial ischemic rat and increase cell viability and attenuate the apoptosis in H9c2 cells induced by hypoxia, which was associated with inhibiting MMP decreasion and increasing total ATPase activity.

Structure-Guided Discovery of Antitubercular Agents That Target the Gyrase ATPase Domain.

In this study we explored the pharmaceutically underexploited ATPase domain of DNA gyrase (GyrB) as a potential platform for developing novel agents that target Mycobacterium tuberculosis. In this effort a combination of ligand- and structure-based pharmacophore modeling was used to identify structurally diverse small-molecule inhibitors of the mycobacterial GyrB domain based on the crystal structure of the enzyme with a pyrrolamide inhibitor (PDB ID: 4BAE). Pharmacophore modeling and subsequent in vitro screening resulted in an initial hit compound 5 [(E)-5-(5-(2-(1H-benzo[d]imidazol-2-yl)-2-cyanovinyl)furan-2-yl)isophthalic acid; IC50 =4.6±0.1 µm], which was subsequently tailored through a combination of molecular modeling and synthetic chemistry to yield the optimized lead compound 24 [(E)-3-(5-(2-cyano-2-(5-methyl-1H-benzo[d]imidazol-2-yl)vinyl)thiophen-2-yl)benzoic acid; IC50 =0.3±0.2 µm], which was found to display considerable in vitro efficacy against the purified GyrB enzyme and potency against the H37 Rv strain of M. tuberculosis. Structural handles were also identified that will provide a suitable foundation for further optimization of these potent analogues.

Targeting of nucleotide-binding proteins by HAMLET--a conserved tumor cell death mechanism.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills tumor cells broadly suggesting that conserved survival pathways are perturbed. We now identify nucleotide-binding proteins as HAMLET binding partners, accounting for about 35% of all HAMLET targets in a protein microarray comprising 8000 human proteins. Target kinases were present in all branches of the Kinome tree, including 26 tyrosine kinases, 10 tyrosine kinase-like kinases, 13 homologs of yeast sterile kinases, 4 casein kinase 1 kinases, 15 containing PKA, PKG, PKC family kinases, 15 calcium/calmodulin-dependent protein kinase kinases and 13 kinases from CDK, MAPK, GSK3, CLK families. HAMLET acted as a broad kinase inhibitor in vitro, as defined in a screen of 347 wild-type, 93 mutant, 19 atypical and 17 lipid kinases. Inhibition of phosphorylation was also detected in extracts from HAMLET-treated lung carcinoma cells. In addition, HAMLET recognized 24 Ras family proteins and bound to Ras, RasL11B and Rap1B on the cytoplasmic face of the plasma membrane. Direct cellular interactions between HAMLET and activated Ras family members including Braf were confirmed by co-immunoprecipitation. As a consequence, oncogenic Ras and Braf activity was inhibited and HAMLET and Braf inhibitors synergistically increased tumor cell death in response to HAMLET. Unlike most small molecule kinase inhibitors, HAMLET showed selectivity for tumor cells in vitro and in vivo. The results identify nucleotide-binding proteins as HAMLET targets and suggest that dysregulation of the ATPase/kinase/GTPase machinery contributes to cell death, following the initial, selective recognition of HAMLET by tumor cells. The findings thus provide a molecular basis for the conserved tumoricidal effect of HAMLET, through dysregulation of kinases and oncogenic GTPases, to which tumor cells are addicted.

High-throughput screening for small molecule modulators of motor protein Kinesin.

The kinesin superfamily of motor proteins are involved in the active transport of a large number of cargos such as organelles, proteins, and RNAs from the neuronal cell body to distal neuronal processes. Previously, we have shown that kinesin-mediated axonal transport of proteins and RNAs are important for long-term memory storage. Identification of small molecules that can activate or inhibit kinesins is of specific interest due to the significance of kinesin-mediated functions in neuronal health and plasticity. Here, we describe a high-throughput screening assay designed to specifically identify compounds that inhibit or activate adenosine triphosphatase activity of the kinesin 5B of humans. The luminescence-based assay that we developed is highly reproducible and robust. Using this approach, we screened a pharmacologically characterized compound collection and have identified small molecules with either activator or inhibitor-like activity. To further characterize screening hits, we also developed an orthogonal assay based on absorbance and a counter screen assay based on luminescence. Development of such assays is important to help identify small molecules that can be used in potential drug development efforts targeted at modulating the function of kinesin.