Johnston Independent Research Projects

NIH R21NS073889-01 Johnston (PI) 2011-2012 HCS Assay to Identify Disruptors of AR-TIF2 Protein-Protein Interactions.

Prostate cancer (CaP) is the most common cancer and the second leading cause of cancer death among men in western countries, and approximately 30,000 patients die annually in the US due to androgen independent (A-I), also called castration-resistant, CaP. The most prescribed treatment is androgen ablation therapy (AAT), which targets the production or action of testicular androgens that bind to the androgen receptor (AR) to provide the critical growth and survival signals to prostate cells. While initial responses to AAT are typically good, the progression to hormone refractory or A-I CaP is almost inevitable. Studies suggest a relationship between cellular AR levels in primary and metastatic CaP lesions and subsequent disease progression. The AR is a member of the nuclear hormone receptor family of ligand-dependent and DNA-sequence specific transcription factors, that is required for the normal growth, terminal differentiation and function of the prostate gland. Our hypothesis is that CaP growth requires abrogation of normal AR function, either directly through changes in AR structure, sub-cellular localization and function, or indirectly via changes to the AR coactivator recruitment interactions.

Aberrant modulation of AR function by coactivators may contribute to the development of A-I CaP. Cumulative evidence from CaP patient samples indicates that Transcription Intermediary Factor 2 (TIF2/SRC-2), a member of the steroid receptor coactivator family, may be involved in the development and progression of A-I CaP: TIF2 levels were significantly higher in recurrent CaP after AAT compared to androgen-dependent CaP or benign prostatic hyperplasia tissue; increased TIF2 mRNA levels found in clinically localized CaP was associated with early recurrence and the highest TIF2 expression was found in patients with relapsed CaP after AAT; and there is a significant correlation between tumor TIF2 expression and CaP aggressiveness. TIF2 over-expression increased AR-TA responses to adrenal androgens and the non-AR ligands estradiol and progesterone. TIF2 expression was associated with increased CaP cell proliferation and decreased apoptosis, and over-expression of TIF2 could compensate for reduced androgen levels. Depletion of TIF2 by antisense oligonucleotides or siRNAs reduced AR target gene expression and slowed the proliferation of dependent and A-I CaP cells. Down-regulation of TIF2 by shTIF2 RNAi dramatically reduced the binding of AR to the prostate specific antigen promoter and induced significant growth inhibition in the absence of androgens. Prolonged AR localization on the promoters of AR target genes together with the recruitment of TIF2 has been proposed as a potential mechanism for the development of A-I CaP.

To quantify AR-TIF2 interactions we have generated a novel protein-protein interaction biosensor (PPIB) assay using recombinant adenovirus constructs to express chimeric proteins containing AR and TIF2 functional domains, targeting sequences and fluorescent reporters. The residues of TIF2 responsible for interacting with ligand-bound AR are expressed as a GFP fusion protein targeted and anchored in the nucleolus by a nuclear localization (NLS/NoLS) sequence. The residues of AR that encompass the androgen ligand binding domain and AF2 surface that interacts with TIF2 are expressed as a RFP fusion protein with both a nuclear localization and a nuclear export sequence. The AR-RFP protein interaction partner can shuttle between the cytoplasm and nucleus in a ligand dependent manner. TIF2-GFP expression remains localized to bright fluorescent puncta within the nucleolus and its distribution does not change upon exposure to DHT ligand or by co-infection with the AR-RFP biosensor. In cells co-infected with both AR and TIF2 biosensors, AR-RFP expression remains localized to the cytoplasm, but upon exposure to DHT the AR-RFP translocates to the nucleus and forms bright fluorescent puncta co-localized with the TIF2-GFP partner in the nucleolus. Without the TIF2-GFP partner, DHT treatment induces AR-RFP translocation to the nucleus where it exhibits a diffuse AR-RFP phenotype throughout the nucleus. The novel AR-TIF2 PPIB assay therefore recapitulates the ligand-induced translocation of AR into the nucleus and the recruitment interactions with the TIF2 coactivator, and we propose to use this assay to identify probes that disrupt AR-TIF2 interactions and to explore the role of these interactions in the development and progression of A-I CaP.

Specific Aim 1: Optimization of a novel AR-TIF2 PPIB high content image-based screening (HCS) assay to identify small molecule probes that block the formation of and/or disrupt interactions between AR and TIF2.

Specific Aim 2: Adapt the AR-TIF2 PPIB assay for HCS and validate the performance of the assay by screening the 1280 compound LOPAC and 446 compound NIH Clinical collections.

NIH R01 CA 160423-01 - Pending, Development of a Novel HCS Assay to Screen for Disruptors of AR-TIF2 Interactions. Johnston (PI), Zhou Wang (UPCI) and Billy Day (Pitt) (co-investigators).

Our novel AR-TIF2 protein-protein interaction (PPI) biosensor assay targets the ligand-induced translocation of AR into the nucleus and its physical interactions with TIF2, a key AR coactivator. In addition to identifying novel HSP90 inhibitors and anti-androgens, the biosensor will identify compounds that block the PPIs between AR and TIF2 and the subsequent recruitment of other AR coactivators, as well as the basal transcriptional machinery. By targeting the disruption of AR-TIF2 interactions we hope to identify novel PPI disruptors with the therapeutic potential to block the development and recurrence of CR CaP. The prescribed treatment for CaP is androgen ablation therapy (AAT) which targets the production or action of the testicular androgens. AAT methods include orchiectomy or medical castration by chronic administration of gonadotrophin-releasing hormone agonists, estrogens, and AR antiandrogens including bicalutamide (Casodex™), flutamide (Eulexin™) and nilutamide (Nilandron™). Aromatase inhibitors like Abiraterone™ block testosterone synthesis. However, despite good initial clinical responses to these frontline CaP therapies tumors inevitably recur despite continued treatment. To date no chemotherapy regimen has proven effective for CR CaP and current therapies are limited by toxicities including muscle atrophy, osteoporosis, anemia, and cognitive dysfunction. Additionally marked and rapid reductions in circulating testosterone levels produce significant effects on bone metabolism and cancer treatment-induced bone loss (CTIBL). The accelerated bone loss and increased number of bone fractures from CTIBL is a significant concern because most CaP patients are over 65 and are already at risk for osteoporosis. Our novel approach offers significant promise for identifying molecules with modalities distal to androgen receptor binding and with the potential to modulate transcriptional activity in a cell-specific manner that is distinct from the traditional approaches targeting androgen receptor activation.

We have proposed testing the AR-TIF2 biosensor in the DU-145, PC-3, LNCaP, LAPC4 and C4-2 cell lines because they are extensively utilized models that have been molecularly characterized and karyotyped as authentic prostate carcinoma cell lines. These cell lines represent different stages of CaP and we believe that differences in their endogenous levels of AR coactivators and corepessors will affect the responses of AR-TIF2 PPI biosensor and provide us valuable information on the regulation of AR transactivation that we can exploit for therapeutic intervention. DU-145 and PC-3 cells were selected because they provide an AR negative cell background that is a model for CR CaP. However because 90% of prostate cancers express wild-type AR we selected LAPC4 cells that express wild-type AR to serve as a model of androgen dependent CaP. Around 10% of CaPs express mutated AR, and LNCaP cells were selected because they express a mutant AR (T877A) that responds to androgens and exhibits promiscuity to other steroids. The C4-2 cell line was derived from the LNCaP cell line and although it was developed to be a model for CR CaP it expresses the mutant AR (T877A) and still retains a degree of sensitivity to androgens. The CWR-R1 cell line was derived from the CWR22 xenograft model and represents a different CR model that expresses a different mutant AR (H874Y) which also retains sensitivity to androgens. Systematic differences in the expression levels of known AR coregulatory genes between cell lines and in normal tissues have been shown to contribute to the significant differences in their androgen responsiveness For example, LNCaP cells expressed low levels of several AR corepressors with higher levels of coactivators including TIF2 when compared to cells that express AR but do not respond to androgens 3. This makes LNCaP cells a particularly valuable model for our studies.

Specific Aim 1: Generate and characterize large scale TIF2-GFP and AR-RFP recombinant adenovirus (rAV) banks to complete the assay development and HCS assay adaptation studies described herein, and to provide sufficient AR and TIF2 PPIB rAV to conduct an HCS campaign against ≥ 300,000 compounds.

Specific Aim 2: Develop and optimize of a novel AR-TIF2 PPIB high content image-based screening (HCS) assay in prostate cancer cell lines (DU-145, PC-3, LNCaP, LAPC4, C4-2, & CWR-R1) and select the most suitable model to screen for small molecule probes that block the formation of and/or disrupt interactions between AR and TIF2.

Specific Aim 3: Adapt the AR-TIF2 PPIB assay for HCS and validate the performance of the assay by screening the 1280 compound LOPAC and 446 compound NIH Clinical collections.

Specific Aim 4: Integrate panels of established counter screens and secondary or tertiary hit characterization assays to distinguish compounds that can block agonist induced AR translocation from the cytoplasm to the nucleus from those that can either block the formation of AR–TIF2 PPIs or that can disrupt established AR-TIF2 PPIs. Hits confirmed in these assays will also be filtered for drug–like properties, classified and clustered and the most chemically tractable probe molecules will be selected for further evaluation.

The goals of specific aim 4 are to characterize the confirmed hits from an AR-TIF2 PPIB HCS campaign, perform mechanism of action studies to identify the molecular targets of the hits, and select chemically tractable hits for lead optimization/SAR efforts.

We have an ongoing collaboration with Dr. Wang to characterize the inhibitors from an AR translocation HCS campaign. Dr. Wang’s group has established assays to examine the role of the AR in the development and progression of CR CaP and we will collaborate with Dr Wang to profile the confirmed hits from the AR-TIF2 PPIB screen in these assays. We will determine the ability of the AR-TIF2 PPI disruptor hits to inhibit AR activity in AR positive cell lines; C4-2, LNCaP, and LAPC4. We will measure the effects of AR-TIF2 PPI disruptor hits on the activity of AR-responsive PSA promoter driven reporters (GFP/luciferase) under normal conditions, and where TIF2 levels have been either over-expressed through transfection, or knocked down with siRNA’s. The activity of AR-TIF2 PPI hits in the PSA-reporter assays will be confirmed by Northern blots, real-time RT-PCR, and Western blots to explore their effects on the expression of endogenous AR-target genes. We will determine the cytotoxicity (LD50s) of the candidate AR-TIF2 PPI disruptor hits in the LNCaP, LAPC4, and C4-2 cell lines, and in AR-negative PC3 and COS-1 cells. After probe optimization, the most promising AR-TIF2 PPI disruptor leads would be tested in established prostate cancer xenograft animal models.

To identify AR DHT-binding antagonists, we will test confirmed hits in an AR fluorescence polarization binding assay kit (Invitrogen). Non-AR antagonists will be tested in an established microtubule (MT) morphology imaging assay to quantify the α-tubulin antibody staining intensity and MT organization of treated CaP cells. In collaboration with Dr. Day’s group we will profile the AR translocation hits in in vitro biochemical assays to identify compounds that inhibit components of the cytoplasmic dynein mediated cargo transport process along MTs. Inhibitors that produce significant alterations in the microtubule organization phenotype, cell shape (e.g., rounding) and/or the α-tubulin antibody fluorescent intensity measurements will be further examined for tubulin assembly inhibitory or MT-stabilizing actions in in vitro MT assembly assays. Compounds that do not significantly affect either MTs or inhibit AR ligand binding will be tested for the ability to inhibit both the basal and the MT-stimulated ATPase activity of the recombinant dynein (DYNH1C1) motor domain, and also against the ATPase activities of the hsp90 and hsp70 chaperones. Additional ATPase selectivity assays that would also be available in Dr. Day’s laboratory include myosin and kinesin 1.

Future Johnston Independent Research Projects

I believe that the protein-protein interaction (PPIs) positional biosensor assay technology that we have utilized to investigate p53-hDM2 and AR-TIF2 PPIs should be directly applicable to measuring PPIs between other nuclear hormone receptors (NRs) and their coactivators or corepressors. Ligand-induced coactivator recruitment determines the outcome of NR activation because they serve as critical amplifiers of gene expression and transcription complex assembly in a context-specific fashion. Although NRs selectively recruit specific coactivator complexes, distinct ligands may ellicit preferential recruitment of different coregulator cohorts. The chemical structure of an NR ligand directs the specificity of receptor binding and can be translated into agonist, partial agonist, or antagonist outputs in terms of target gene responses. Some NR ligands only activate a subset of the NRs target genes, and it is believed that such gene selectivity is cell/tissue specific and reflects the ratio of coactivators and corepressors which codetermine whether an on or off signal is processed. Coregulators may also exhibit different functions depending upon the specific promoter context. NR coactivator recruitment profiles therefore influence the tissue specific spatiotemporal gene expression responses to NR ligands. For example, increased NR coactivator levels produce a more rapid transcriptional response and shift the ligand induction response to the left. Selective estrogen and androgen receptor modulators (SERMs & SARMs) take advantage of the different repertoires and concentrations of coregulatory proteins in various cells to exert their tissue selective behaviors. After we have established the utility of this approach with the AR-TIF2 biosensor it is my intention to expand the application of the assay technology to other NRs including, but not limited to, other steroid family members such as GR, ERα, and ERβ. There are very few HTS compatible assay formats available to investigate NR coactivator and corepressor interactions, and my plan would be to develop reagents similar to the TIF2 biosensor for a panel of other NR coregulators. Once we have developed this panel of NR and coregulator reagents this will provide us very powerful tools to investigate how different NR agonists, antagonists, or small molecules affect the PPIs between specific NRs and their coregulators in a systematic and combinatorial fashion. I believe that these studies will provide valuable new approaches to identify selective molecules with therapeutic value in cancer and metabolic diseases.

Collaborative Research Projects

My current research interests are focused on the application of my high throughput screening (HTS), high content screening (HCS), and lead optimization experience to academic drug discovery and research. My expertise in pharmaceutical drug discovery shaped the design of the UPDDI academic screening center with respect to the selection of assay technologies, robotic platforms, detection instruments, compound libraries, HTS informatics systems, and the overall assay development process and screening philosophy of the facility. For example, we selected a workstation based HTS approach rather than a fully integrated robotic approach because it provided greater flexibility with respect to the assay formats and target classes that we could address, cost less to purchase and maintain in service, and required fewer people to operate. Once we had established the necessary HTS infrastructure we had to assemble an IT network, servers, databases and informatics tools to handle the large amounts of data transfer and analysis, and to provide secure storage for these data. We selected the ActivityBase™ LIMS package for compound management, inventory, and sample tracking to associate compound data with the bioassay data, HTS data processing, and data visualizations for quality control review. Based on prior experience we selected the Spotfire data visualization package to compliment our LIMS package and the LeadScope™ cheminformatics software for compound classification, clustering, similarity searching, structure activity relationship and R-group analysis.

After establishing the UPDDI HTS/HCS facility, my research has focused on initiating and directing HTS/HCS research projects through a blend of collaborations with other investigators and my own research. As the assay ambassador and point of contact for the UPDDI academic outreach effort, I have initiated collaborations leading to grant submissions and several ongoing research projects. In the intervening years, I have directed the development and optimization of HTS/HCS assays and implemented fifteen screening campaigns that have included the development and implementation of the primary screen, active confirmation, potency and efficacy determinations, hit characterization in secondary and tertiary assays, and in some cases lead optimization. More than 2.15 M compounds have been screened in these fifteen primary HTS/HCS campaigns and > 2.5 M data points were generated. Eight of the primary HTS/HCS campaigns were conducted in in vitro biochemical assay formats, six were cell based assays, and one was a whole organism screen. Nine of the HTS/HCS campaigns were against cancer therapeutic targets, three were against anti-viral targets, two were phenotypic screens, and one was to identify redox cycling compounds that are promiscuous artifacts in HTS data sets. With respect to target classes, two were kinase targets, two were dual specificity phosphatase targets, two were protease targets, two were protein-protein interaction targets, two were cell based necroptosis screens, two were cellular motor screens, two were transcriptional factor screens, and one screen was against HIV reverse transcriptase. A list of the fifteen HTS campaigns and my collaborators is presented: a 65K compound in vitro HTS campaign to identify Mitogen-Activated Protein Kinase Phosphatase 1 (MKP-1) inhibitors – a collaboration with John S. Lazo (Pitt); a 65K compound in vitro HTS campaign to identify Inhibitors of West Nile Virus NS2bNS3 Proteinase – a collaboration with Alex Strongin (Burnham Institute); a 65K compound in vitro HTS campaign to identify inhibitors of HIV-1 Reverse Transcriptase RNase H – a collaboration with Michael Parniak (Pitt); a 97K compound fluorescence polarization-based in vitro HTS campaign to screen for small molecule inhibitors of the Polo Box Domain of PLK-1 – a collaboration with Michael Yaffee (MIT); a 65K compound in vitro HTS campaign to identify inhibitors of the Cdc25B Catalytic Domain protein tyrosine phosphatase – collaboration with Marni Brisson (Paul Johnston Co-PI, Pitt); a 221,515 compound in vitro HTS campaign to identify inhibitors of proprotein convertases furin, PC5, SKI-1, and PCSK9 – a collaboration with Nabil Seidah (Clinical Research Institute of Montreal, Canada); two cell based 220K compound HTS campaigns to identify inhibitors of the Necrotic Cell Death Pathway in the Jurkat-FADD-/- TNFα and L929-zVAD cell models – a collaboration with Junying Yuan (Harvard); a 4.5K compound whole organism HTS campaign to identify small molecule inhibitors of Cilia using the Chlamydomonas Gravitaxis Assay – a collaboration with Wallace Marshall (UCSF); a 220K compound cell based HCS campaign to identify inhibitors of Dynein mediated cargo transport towards the minus ends of microtubules – a collaboration with Billy Day (Pitt); a 220K compound cell based HCS campaign using the p53-HDM2 Protein-Protein Interaction Biosensor to identify disruptors of p53-hDM2 interactions (Paul Johnston PI, Pitt); a 199K compound in vitro HTS campaign to profile the NIH small molecule repository for compounds that generate H2O2 by redox cycling in reducing environments (Paul Johnston PI, Pitt); a 220K compound in vitro HTS campaign to identify compounds that target HIV-1 Nef – a collaboration with Thomas Smithgall (Pitt); a 220K compound cell based HCS campaign to identify compounds that alter the predominant nuclear localization of AR-GFP in the C4-2 model of castration resistant prostate cancer – collaboration with Zhou Wang (Pitt); a 97K compound cell based HCS campaign to identify selective inhibitors of STAT3 activation in squamous cell carcinoma of the head and neck – a collaboration with Jennifer Grandis (Pitt).

Since building the UPDDI screening facility, I have managed the Pittsburgh Molecular Library Screening Center (PMLSC) which was federally funded by the NIH in the pilot phase of the NIH Roadmap Initiative Molecular Library Screening Center Network. I am also the co-Principal investigator and manger of the Pittsburgh Specialized Application Center (PSAC) which is a component of the NCI’s Chemical Biology Consortium (CBC). The CBC is part of the Experimental Therapeutics (NExT) Program, a partnership between the NCI’s Division of Cancer Treatment and Diagnosis and Center for Cancer Research tasked with streamlining the development and testing of promising new anticancer drugs to expedite their delivery to the bedside. I also manage the Chemical Biology Facility (ChBF) for the University of Pittsburgh Cancer Center (UPCI) and direct an ARRA funded project to screen for effective cancer drug combinations in the NCI 60 cell line panel. The UPDDI HTS/HCS facility is recognized as one of the nation’s top academic screening facilities and provides a service to investigators who are interested in screening large libraries of small molecules against targets of interest, using such advanced technologies as liquid-handling robots; multimode detection readers (absorbance, luminescence, fluorescence intensity, fluorescence polarization, and time-resolved fluorescence); automated HCS fluorescent microscopy platforms with live cell and kinetic screening options combined with image analysis algorithms and image database storage; and biosafety level 2 tissue culture hoods and incubators. I have collaborated with investigators to help them select an assay format that is compatible with HTS/HCS, to develop and optimize these assays for HTS, to adapt and validate these assays on the UPDDI automated robotic platforms, and to implement these assays in HTS campaigns. I have assisted investigators with grant applications, crafting and writing assay development and screening specific aims, and providing preliminary data for these applications. To date these efforts have contributed to ten HTS/HCS assay development/screening collaborations funded by the NIH, NCI or research foundations, and eighteen peer reviewed publications.

Future Collaborative Research Projects

In the future it would be my intention to continue to use my expertise in screening and drug discovery to collaborate with other investigators on grant submissions and research projects. However, in anticipation of the increased demands of my own independent research effort I will have to be more selective in my collaborative projects, and in the future I will focus mainly on projects that utilize HCS technology and/or address cancer targets.

REFEREED ARTICLES:

1. Johnston, P.A. and A. Coddington (1982). Multiple Drug Resistance in the Fission Yeast Schizosaccharomyces pombe: Evidence for the Existence of Pleiotropic Mutations Affecting Energy Dependent Transport systems. Mol. Gen. Genet. 185: 311-314.
2. Johnston, P.A. and A. Coddington (1983). Multiple Drug Resistance in the Fission Yeast Schizosaccharomyces pombe: Correlation between Drug and Amino Acid Uptake and Membrane ATPase Activities. Current Genetics 7: 299-307.
3. Johnston, P.A. and A. Coddington (1984). Drug Resistance in the Fission Yeast Schizosaccharomyces pombe: Pleiotropic Mutations Affecting the Oleic Acid and Sterol Composition of Cell Membranes. Current Genetics 8: 37-43.
4. Johnston, P.A., D.O. Adams, and T.A. Hamilton (1984). Fc-receptor-Mediated Protein Phosphorylation in Murine Peritoneal Macrophages. Biochem. Biophys. Res. Commun. 124: 197-202.
5. Johnston, P.A., D.O. Adams, and T.A. Hamilton (1985). Regulation of the Fc-receptor-mediated Respiratory Burst: Treatment of Primed Murine Peritoneal macrophages with Lipopolysaccharide Selectively Inhibits H202 Secretion Stimulated by Immune Complexes. J. Immunol.135: 513-518.
6. Johnston, P.A., D.O. Adams and T.A. Hamilton (1986). Regulation of Respiratory Burst in Murine Peritoneal macrophages: Differential Sensitivity to Phorbol Diesters by Macrophages in Different Stages of Functional Activation. Cell. Immunol. 100: 400-410.
7. Johnston, P.A., M.M Jansen, S.D. Somers, D.O. Adams and T.A. Hamilton (1987). Ligands for the Scavenger Receptor on Murine Peritoneal Macrophages Induce Expression of a Set of Early Proteins. J. Immunol. 138: 1551-1558.
8. Johnston, P.A. S.D. Somers and T.A. Hamilton (1987). Expression of a 120 Kilodalton Protein During Tumoricidal Activation in Murine Peritoneal Macrophages. J. Immunol. 138: 2739-2744.
9. Introna, M., R.C. Bast, Jr., P.A. Johnston, D.O. Adams and T.A. Hamilton (1987). Homologous and Heterologous Desensitization of Proto-Oncogene cFos Expression in Murine Peritoneal Macrophages. J. Cell. Physiol. 131: 36-42.
10. Johnston, P.A., T.J. Koerner, M.M. Jansen and T.A. Hamilton (1989). Expression of Macrophage p120 Depends upon Early Protein Synthesis. J. Immunol. 142: 2728-2735.
11. Johnston, P.A., G.A. Reynolds, S. Wasserman and T.C. Sudhof (1990). Two Novel Annexins from Drosophila Melanogaster. Cloning, Characterization and Differential Expression in Development. J. Biol. Chem 265: 11382-11388.
12. Johnston, P.A., F. Yu, G.A. Reynolds, H.L. Yin, C.R. Moomaw, C.A. Slaughter C.A. and T.A. Sudhof (1990). Purification and Expression of gCap39. An Intracellular and Secreted Ca2+-Dependent Actin-Binding Protein Enriched in Mononuclear Phagocytes. J. Biol. Chem. 265: 17946-17952.
13. Yu, Fu-Xin, P.A. Johnston, T.C. Sudhof and H.L. Yin (1990). gCap39, a Calcium Ion and Polyphosphoinositide-Regulated Actin Capping Protein. Science 250: 1413-1415.
14. Johnston, P.A. and T.C. Sudhof (1990). Evolutionary Conservation and Structure-Function Relationships in the Calelectrins (Annnexins). Biochem. Soc. Trans. 18: 1097-1098.
15. Perrin, M., P.A. Johnston, T. Ozcelik, R. Jahn, U. Franke and T.C. Sudhof (1991). Structural and Functional Conservation of Synaptotagnin p65) in Drosophila and Humans. J. Biol. Chem. 266: 615-622.
16. Mignery, G.A., P.A. Johnston and T.C. Sudhof (1992). Mechanism of Ca2+ Inhibition of Inositol 1,4,5-Trisphosphate (InsP3) Binding to the Cerebellar InsP3 Receptor. J. Biol. Chem. 267: 7450-7455.
17. Daley, M.J., G. Furda, R. Dougherty, P.A. Coyle, T.J. Williams and P.A. Johnston (1992). Potentiation of Antibiotic Therapy for Bovine Mastitis by Recombinant Bovine Interleukin-2. J. Dairy Science 75: 3330-3338.
18. Tao, W.T., R. Dougherty, P.A. Johnston and W. Pickett (1993). Recombinant Bovine GM-CSF Primes Platlet Activating Factor, rHuIL-8 but not rBoIL-1 Induced Bovine Neutrophil Degranulation. J. Leukocyte Biology 53: 679-684.
19. Bolnet, C., P.A. Johnston, A. Kemper, C. Ricks, & J. Petite (1995). Influence of Avian Con A Induced Splenocyte Conditioned Media on Cells of the Hematopoietic Lineage. J. Poultry Sci. 74: 1102-1116.
20. Johnston, P. A. and P. A. Johnston (2002). Cellular Platforms for HTS: three case studies. Drug Discovery Today 7: 353-363.
21. Pratt S, Shepard RL, Kandasamy RA, Johnston PA, Perry W 3rd, and Dantzig AH. (2005). The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites. Mol. Cancer Ther. 4:855-63.
22. Razonable RR, Henault M, Lee LN, Laethem C, Johnston PA, Watson HL, Paya CV. (2005) Secretion of proinflammatory cytokines and chemokines during amphotericin B exposure is mediated by coactivation of toll-like receptors 1 and 2 Antimicrob Agents Chemother. 49(4):1617-21.
23. Arnold DM, Foster C, Huryn DM, Lazo JS, Johnston PA, Wipf P. (2007) Synthesis and Biological Activity of a Focused Library of Mitogen-activated Protein Kinase Phosphatase Inhibitors. Chem. Biol. Drug Des. 69:23-30.
24. Brisson-Tierno M*, Johnston PA*, Foster C, Skoko JJ, Shun TY, Lazo JS. (2007) Development and Optimization of high-throughput in vitro protein phosphatase screening assays for drug discovery. * Authors contributed equally to this work. Nature Protocols. 2:1134-44.
25. Johnston PA. An interview with Paul A. Johnston, Ph.D., by Vicki Glaser. (2007) Assay and Drug Development Technologies. 5: 289-97.
26. Johnston PA, Foster CA, Shun TY, Skoko JJ, Shinde S, Wipf P, and Lazo JS. (2007) Development and Implementation of a 384-well Homogeneous Fluorescence Intensity HTS Assay to Identify Mitogen-Activated Protein Kinase Phosphatase-1 Dual Specificity Protein Phosphatase Inhibitors. Assays and Drug. Discovery Technologies. 5: 319-332.
27. Johnston PA, Phillips J, Shun TY, Shinde SN, Lazo JS, Huryn DM, Myers MC, Ratnikov BI, Smith JW, Su Y, Dahl R, Cosford NDP, Shiryaev SA, and Strongin AY. (2007) HTS Identifies Novel and Specific Uncompetitive Inhibitors of the Two-Component NS2B-NS3 Proteinase of West Nile Virus. Assay and Drug Development Technologies. Assays and Drug. Discovery Technologies. 5: 737-750.
28. Keinan S, Paquette WD, Skoko JJ, Beratan DN, Yang W, Shinde S, Johnston PA, Lazo JS, and Wipf P. (2008) Computational design, synthesis and biological evaluation of para-quinone-based inhibitors for redox regulation of the dual-specificity phosphatase Cdc25B. Org. Biomol. Chem. 6: 3256-3263.
29. Johnston PA, Soares KM, Shinde SN, Foster CA, Shun TY, Takyi HK, Wipf P, and Lazo JS. (2008) Development of a 384-well Colorimetric Assay to Quantify Hydrogen Peroxide Generated by the Redox Cycling of Compounds in the Presence of Reducing Agents. Assay and Drug Development Technologies. 6: 505-518.
30. Johnston PA, Foster CA, Tierno MB, Shun TY, Shinde SN, Paquette WD, Brummond KM, Wipf P, and Lazo JS. (2009). Cdc25B Dual Specificity Phosphatase Inhibitors Identified in a High Throughput Screen of the NIH Compound Library. Assay and Drug Development Technologies. 7: 250-265.
31. Kay M. Brummond, Shuli Mao, Sunita N. Shinde, Paul A. Johnston, and Billy W. Day. (2009). Design and Synthesis of a Library of Tetracyclic Hydroazulenoisoindoles. J. Comb. Chem., 11: 486-494.
32. Wipf P, Arnold D, Carter K, Dong S, Johnston PA, Sharlow E, Lazo JS, Huryn D. (2009). A case study from the chemistry core of the Pittsburgh Molecular Library Screening Center: the Polo-like kinase polo-box domain (Plk1-PBD). Curr Top Med Chem., 9:1194-205.
33. Soares KM, Blackmon N, Shun TY, Shinde SN, Takyi HK, Wipf P, Lazo JS, Johnston PA. (2010). Profiling the NIH Small Molecule Repository for Compounds That Generate H(2)O(2) by Redox Cycling in Reducing Environments. Assay Drug Dev Technol. 8: 152-171.
34. Dudgeon D, Shinde, SN, Shun, TY, Lazo, JS, Strock, CJ, Giuliano, KA, Taylor, DL, Johnston, PA, and Johnston, PA. (2010). Characterization and Optimization of a Novel Protein-Protein Interaction Biosensor HCS Assay to Identify Disruptors of the Interactions between p53 and hDM2. Assay Drug Dev Technol. 8: 437-58.
35. Dudgeon D, Shinde, SN, Hua, Y, Shun, TY, Lazo, JS, Strock, CJ, Giuliano, KA, Taylor, DL, Johnston, PA, and Johnston, PA. (2010). Implementation of a 220,000 Compound HCS Campaign to Identify Disruptors of the Interaction between p53 and hDM2, and Characterization of the Confirmed Hits. J Biomol Screen. 15: 766-782.
36. Gosai SJ, Kwak JH, Luke CJ, Long OS, King DE, Kovatch KJ, Johnston PA, Shun TY, Lazo JS, Perlmutter DH, Silverman GA and Pak SC. (2010) Automated high –content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z. PLoS One. 2010 Nov 12;5(11):e15460.
37. Shun TY, Lazo JS, Sharlow ER, Johnston PA. (2011) Identifying Actives from HTS Data Sets, Practical Approaches for the Selection of an Appropriate HTS Data Processing Method and Quality Control Review. J Biomol Screen. 16: 1-14.
38. Daghestani HN, Zhu G, Johnston PA, Shinde SN, Brodsky JL, Vallee RB, and Day BW. (2011) "Characterization of Inhibitors of Glucocorticoid Receptor Nuclear Translocation: A Model of Cytoplasmic Dynein-Mediated Cargo Transport" Assay Drug Dev Technol. Sep 15. [Epub ahead of print]

REVIEWS, PROCEEDINGS OF CONFERENCE & SYMPOSIA, & BOOK CHAPTERS:

1. P. A. Johnston, H. Liu, T. O'Connell, P. Phelps, M. Bland, J. Tyczkowski, A. Kemper, T. Harding, A. Avakian, E. Haddad, C. Whitfill, R. Gildersleeve, and C. Ricks (1995). Proceedings of the Symposium - Current Advances in Avian Embryology and Incubation, Session V: Interventions in Embryo Development. Applications of In ovo Technology". J. Poultry Sci. 76: (1) 165-178.
2. Carpenter, J; C. Laethem, F. R. Hubbard, T. K. Eckols, M. Baez, D. McClure, D. L. G. Nelson, and P. A. Johnston. (2002). Configuring Radioligand receptor Binding Assays for HTS using Scintillation Proximity Assay Technology. High Throughput Screening: Methods and Protocols, in Methods in Molecular Biology, 190: 31-49. The Humana Press, Inc. Edited by W. P. Janzen.
3. A. H. Gough and P. A. Johnston. (2006) Requirements, Features and Performance of High Content Screening Platforms, in High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Discovery. Methods in Molecular Biology 356: 41-61. Humana Press, Totowa, NJ. Editors: D. L. Taylor, J. R. Haskins and K.A. Giuliano
4. R.G.Williams, R.Kandasamy, D.Nickischer, O.J. Trask, C.Laethem, P.A Johnston, and P.A. Johnston. (2006). “Generation and Characterization of a Stable MK2-EGFP Cell Line and Subsequent Development of a High Content Imaging Assay on the Cellomics ArrayScan® Platform to Screen for p38 MAPK Inhibitors” in Methods in Enzymology 414: 364-89. “Automated Microscopy Screening” Elsevier/Academic Press Edited by Jim Inglese.
5. O.J. Trask, A.Baker, R.G.Williams, D.Nickischer, R. Kandasamy, C. Laethem, P. A. Johnston, and P. A. Johnston. (2006). “Assay Development and Case History of a 32K Biased Library High Content MK2-EGFP Translocation Screen to Identify p38 MAPK Inhibitors on the ArrayScan® 3.1 Imaging Platform” in Methods in Enzymology 414: 419-39. “Automated Microscopy Screening” Elsevier/Academic Press Edited by Jim Inglese.
6. D. Nickischer, C. L. Laethem, O. J. Trask, Jr., R. G. Williams, R. Kandasamy, P. A. Johnston, and P. A. Johnston. (2006). “Development and implementation of three MAPK signaling pathway imaging assays to provide MAPK module selectivity profiling for kinase inhibitors; MK2-EGFP translocation, c-Jun & ERK activation” in Methods in Enzymology 414: 389-418. “Automated Microscopy Screening” Elsevier/Academic Press Edited by Jim Inglese.
7. P. A. Johnston. (2007) “Automated High Content Screening Microscopy” Chapter 2, p25-42, in High Content Screening: Science, Technology and Applications. John Wiley & Sons, Inc; Edited by: Stephen A. Haney.
8. Trask OJ, Nickischer D, Burton A, Williams RG, Kandasamy RA, Johnston PA, Johnston PA. (2009). High-throughput automated confocal microscopy imaging screen of a kinase-focused library to identify p38 mitogen-activated protein kinase inhibitors using the GE InCell 3000 analyzer. Methods Mol Biol. 565:159-186.
9. P. A. Johnston. (2011). Redox cycling compounds generate H(2)O(2) in HTS buffers containing strong reducing reagents - real hits or promiscuous artifacts? Curr Opin Chem Biol. 15(1):174-82.
10. P. A. Johnston and J. R. Grandis. (2011). STAT3 signaling: anticancer strategies and challenges. Mol Interv. 2011 Feb;11(1):18-26.
11. P. A. Johnston. (2012) High content analysis and screening: basics, instrumentation and applications. Chemical Genomics. Cambridge University Press, Edited by: Haian Fu.