A High-Throughput Screening Assay for Pyruvate Carboxylase

Pyruvate carboxylase (PC) catalyzes the conversion of pyruvate to oxaloacetate (OAA), an important metabolic reaction in a wide range of organisms. Small molecules directed against PC would enable detailed studies on the metabolic role of this enzyme and would have the potential to be developed into pharmacological agents. Currently, specific and potentsmall molecule regulators of PC are unavailable. To assist in efforts to find, develop, and characterize small molecule effectors of PC, a novel fixed-time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored adduct with an absorbance maximum at 530 nm. This fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC, Staphylococcus aureus PC, and Listeria monocytogenes PC, and is highly amenable to high-throughput screening. The assay was validated using a plate uniformity assessment test and a pilot screen of a library of 1280 compounds. The results indicate that the assay is suitable for screening small molecule libraries to find novel small molecule effectors of PC

Development of High-Throughput Screening Assay for Antihantaviral Therapeutics

Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. There is no treatment for hantavirus diseases at present. Therapeutic intervention during early stages of viral infection can improve the outcome of this zoonotic viral illness. The interaction between an evolutionary conserved sequence at the 5′ terminus of hantaviral genomic RNA and hantavirus nucleocapsid protein plays a critical role in the hantavirus replication cycle. This unique interaction is a novel target for therapeutic intervention of hantavirus disease. We developed a very sensitive, tractable, and cost-effective fluorescence-based assay to monitor the interaction between the nucleocapsid protein and the target RNA sequence. The assay was optimized for high-throughput screening of chemical libraries to identify molecules that interrupt this RNA–protein interaction. The assay was validated using a library of 6880 chemical compounds. This validation screen demonstrated the reproducibility and validity of required statistical criteria for high-throughput screening. The assay is ready to use for high-throughput screening of large chemical libraries to identify antihantaviral therapeutic molecules and can be amenable to similar targets in other viruses

A high-throughput screening assay for Mst1 and Mst2 kinase inhibitors

Hippo信号通路是一条在进化上高度保守的激酶级联信号通路，主要通过调节细胞凋亡和细胞增殖控制器官大小、组织稳态和组织再生。丝/苏氨酸蛋白激酶Mst1/2是Hippo信号通路的重要组成成员，在生物体内广泛表达，能诱导细胞凋亡，在细胞增殖、存活、形态建成和运动方面都具有重要作用。在一定的刺激下Mst1/2发生自磷酸化而活化，活化的Mst1/2磷酸化Mob1以及在WW45的协助下磷酸化Lats1/2而使其活化，活化的Lats1/2进一步磷酸化转录共激活因子YAP使其与14-3-3蛋白结合而滞留在细胞质中。在Hippo信号通路失活的的情况下，YAP不能被磷酸化而入核与转录因子TEAD结合促进下游与细...The Hippo signaling pathway plays an important role in organ size control, tissue regeneration and stem cell self-renewal. The Ste20-like kinases Mst1 and 2 (Mst1/2), the mammalian Hippo orthologs, are key components of the Hippo signaling pathway along with a scaffolding protein Salvador/WW45 (Sav), NDR family kinases NDR1/2 and LATS1/2, and an adaptor protein MOB1. Mst1/2 phosphorylate and activ...学位：理学硕士院系专业：生命科学学院_细胞生物学学号：2162014115250

An improved high-throughput screening assay for tunicamycin sensitivity in Arabidopsis seedlings

Tunicamycin sensitivity assays are a useful method for studies of endoplasmic reticulum stress and the unfolded protein response in eukaryotic cells. While tunicamycin sensitivity and tunicamycin recovery assays have been previously described, these existing methods are time-consuming, labor intensive and subjected to mechanical wounding. This study shows an improved method of testing tunicamycin sensitivity in Arabidopsis using liquid Murashige and Skoog medium versus the traditional solid agar plates. Liquid medium bypasses the physical manipulation of seedlings, thereby eliminating the risk of potential mechanical damage and additional unwanted stress to seedlings. Seedlings were subjected to comparative treatments with various concentrations of tunicamycin on both solid and liquid media and allowed to recover. Determination of fresh weight, chlorophyll contents analysis and qRT-PCR results confirm the efficacy of using liquid medium to perform quantitative tunicamycin stress assays

Development of a High-throughput Screening Assay for Potential Inhibitors Against DNA Polymerase Eta

All living cells perform DNA replication through a group of enzymes called DNA polymerases. Unfortunately, DNA is constantly exposed to external and internal agents capable of causing damage. DNA damage causes blocks to replication that can be overcome through a special class of DNA polymerases, called the Y-family polymerases; which are able to traverse DNA lesions. Cisplatin is a common chemotherapeutic agent that damages DNA and has been used to treat cancer. However, many patients become resistant to cisplatin. The chemoresistance to cisplatin treatment is due in part to damage bypass performed by the specific Y-family polymerase, polymerase eta. The potential role of Y-family DNA polymerases in the pathway of chemotherapeutic resistance offers a novel target for inhibition. To cultivate this thesis, I will provide a brief introduction of cancer and how DNA plays a role in the development of the disease. I will then discuss the various types of DNA damage and how they affect the replication process resulting in mutagenesis. Following that, I will begin discussing several types of repair processes that our cells have adopted to tolerate DNA damage. This will lead me to hone in one specific mechanism called translesion synthesis, and discuss how through the recruitment of a special class of DNA polymerases called the Y family polymerases allows for DNA replication to continue without fixing the damage, and how this plays a role in chemotherapeutic resistance. From this I will get into the specifics of my project, centered on DNA polymerase eta’s role in cisplatin resistance which led to implementation of a high-throughput screening assay to identify potential small molecule targets against Y-family polymerase eta. I will conclude by summarizing the overall significance of the work done, and discuss research work that is currently being done on the inhibition of Y-family polymerases, as well as conclude with what future work can be done regarding this project

RyR1-targeted drug discovery pipeline integrating FRET-based high-throughput screening and human myofiber dynamic Ca2+ assays.

Elevated cytoplasmic [Ca2+] is characteristic in severe skeletal and cardiac myopathies, diabetes, and neurodegeneration, and partly results from increased Ca2+ leak from sarcoplasmic reticulum stores via dysregulated ryanodine receptor (RyR) channels. Consequently, RyR is recognized as a high-value target for drug discovery to treat such pathologies. Using a FRET-based high-throughput screening assay that we previously reported, we identified small-molecule compounds that modulate the skeletal muscle channel isoform (RyR1) interaction with calmodulin and FK506 binding protein 12.6. Two such compounds, chloroxine and myricetin, increase FRET and inhibit [3H]ryanodine binding to RyR1 at nanomolar Ca2+. Both compounds also decrease RyR1 Ca2+ leak in human skinned skeletal muscle fibers. Furthermore, we identified compound concentrations that reduced leak by > 50% but only slightly affected Ca2+ release in excitation-contraction coupling, which is essential for normal muscle contraction. This report demonstrates a pipeline that effectively filters small-molecule RyR1 modulators towards clinical relevance

Topoisomerase II inhibitors induce DNA damage-dependent interferon responses circumventing Ebola virus immune evasion

Ebola virus (EBOV) protein VP35 inhibits production of interferon alpha/beta (IFN) by blocking RIG-I-like receptor signaling pathways, thereby promoting virus replication and pathogenesis. A high-throughput screening assay, developed to identify compounds that either inhibit or bypass VP35 IFN-antagonist function, identified five DNA intercalators as reproducible hits from a library of bioactive compounds. Four, including doxorubicin and daunorubicin, are anthracycline antibiotics that inhibit topoisomerase II and are used clinically as chemotherapeutic drugs. These compounds were demonstrated to induce IFN responses in an ATM kinase-dependent manner and to also trigger the DNA-sensing cGAS-STING pathway of IFN induction. These compounds also suppress EBOV replication in vitro and induce IFN in the presence of IFN-antagonist proteins from multiple negative-sense RNA viruses. These findings provide new insights into signaling pathways activated by important chemotherapy drugs and identify a novel therapeutic approach for IFN induction that may be exploited to inhibit RNA virus replication

Cell-based high-throughput screening assay for identification of G-protein-coupled receptors agonists and antagonists

The GPCRs are the target of a significant portion of all approved drugs and also represent an important parcel of potential drug targets for new indications. Activation of GPCRs can be detected by the utilization of functional cell-based assays like measuring transient intracellular calcium mobilization. The emergence of the fluorescent imaging plate reader and Ca2+-sensitive fluorescent dyes has made the high-content screening of GPCR possible. Here was applied a dual automated FLIPR platform to testing a large collection of compounds to identification of both agonist and antagonists form a single screen of GPC