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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC Santa Cruz Electronic Theses and Dissertations (2021)

Millions of people world-wide are affected by diseases and conditions related to microbial pathogens and the immune system. The demand for new therapies against antibiotic resistant bacteria and anti-inflammatories is urgent. My dissertation encompasses two distinct avenues of modern drug discovery: 1) A chemical biology approach to improve the physical properties and potency of novel cyclic peptide inhibitors to combat the virulence of aggressive gram negative bacteria by inhibiting the Type 3 Secretion System (T3SS) and 2) The development of a high-content image-based screening assay for the detection of anti-inflammatories. Chapter 1 describes the structural activity relationship studies of a novel cyclic peptide T3SS inhibitor and its potency against various gram negative bacteria T3SS. Chapter 2 describes the development of a high content image-based screen using macrophages and pro-inflammatory endotoxin lipopolysaccharide (LPS). We compare this screening platform to classic HeLa cytological profiling (CP) and observe the limitations of image based feature extraction techniques to cluster by MOA based on similarity profiles. Furthermore, we use supervised deep learning approaches to identify compounds that reverse the LPS-stimulated phenotype and confirm the selected deep learning hits exhibit an immunomodulatory response to LPS. Lastly we briefly describe our efforts in developing a costume-built online database for viewing the images generated from the HCS screen. This database brings side-by-side comparisons of macrophages treated with compounds +/- LPS stimuli and both stain sets. In addition, it's searchable and includes compound metadata associated with every image, allowing for quick inspection of the images.

    Cover page: Drug Discovery Efforts for Antibiotic Resistant Bacteria and Anti-InflammatoriesCreative Commons 'BY' version 4.0 license
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2014)
    Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca(2+)-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation.
      Cover page: Protein Kinase Cδ Promotes Transitional B Cell-Negative Selection and Limits Proximal B Cell Receptor Signaling To Enforce Tolerance
      • Article
      • Peer Reviewed
      UC Santa Cruz Previously Published Works (2024)
      We developed a high-content image-based screen that utilizes the pro-inflammatory stimulus lipopolysaccharide (LPS) and murine macrophages (RAW264.7) with the goal of enabling the identification of novel anti-inflammatory lead compounds. We screened 2,259 bioactive compounds with annotated mechanisms of action (MOA) to identify compounds that block the LPS-induced phenotype in macrophages. We utilized a set of seven fluorescence microscopy probes to generate images that were used to train and optimize a deep neural network classifier to distinguish between unstimulated and LPS-stimulated macrophages. The top hits from the deep learning classifier were validated using a linear classifier trained on individual cells and subsequently investigated in a multiplexed cytokine secretion assay. All 12 hits significantly modulated the expression of at least one cytokine upon LPS stimulation. Seven of these were allosteric inhibitors of the mitogen-activated protein kinase kinase (MEK1/2) and showed similar effects on cytokine expression. This deep learning morphological assay identified compounds that modulate the innate immune response to LPS and may aid in identifying new anti-inflammatory drug leads.
        Cover page: High‐Content Image‐Based Screening and Deep Learning for the Detection of Anti‐Inflammatory Drug Leads
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2014)
        Protein kinase Cδ (PKCδ) deficiency causes autoimmune pathology in humans and mice and is crucial for the maintenance of B cell homeostasis. However, the mechanisms underlying autoimmune disease in PKCδ deficiency remain poorly defined. Here, we address the antigen-dependent and -independent roles of PKCδ in B cell development, repertoire selection, and antigen responsiveness. We demonstrate that PKCδ is rapidly phosphorylated downstream of both the B cell receptor (BCR) and the B cell-activating factor (BAFF) receptor. We found that PKCδ is essential for antigen-dependent negative selection of splenic transitional B cells and is required for activation of the proapoptotic Ca(2+)-Erk pathway that is selectively activated during B cell-negative selection. Unexpectedly, we also identified a previously unrecognized role for PKCδ as a proximal negative regulator of BCR signaling that substantially impacts survival and proliferation of mature follicular B cells. As a consequence of these distinct roles, PKCδ deficiency leads to the survival and development of a B cell repertoire that is not only aberrantly autoreactive but also hyperresponsive to antigen stimulation.
          • Article
          • Peer Reviewed
          UC Santa Cruz Previously Published Works (2017)
          Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-κB activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-κB enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3'N, EpD-1,2N, EpD-1,3'N, EpD-1,2,3'N, and EpD-1,2,4'N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (>40% translocation inhibition at 7.5 μM) and showed no toxicity to mammalian cells at 240 μM. In addition, EpD-3'N and EpD-1,2,4'N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.
            Cover page: Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2021)
            Antibiotic-resistant bacteria are an emerging global health threat. New antimicrobials are urgently needed. The injectisome type III secretion system (T3SS), required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, is an attractive antimicrobial target. We previously identified synthetic cyclic peptomers, inspired by the natural product phepropeptin D, that inhibit protein secretion through the Yersinia Ysc and Pseudomonas aeruginosa Psc T3SSs but do not inhibit bacterial growth. Here, we describe the identification of an isomer, 4EpDN, that is 2-fold more potent (50% inhibitory concentration [IC50] of 4 μM) than its parental compound. Furthermore, 4EpDN inhibited the Yersinia Ysa and the Salmonella SPI-1 T3SSs, suggesting that this cyclic peptomer has broad efficacy against evolutionarily distant injectisome T3SSs. Indeed, 4EpDN strongly inhibited intracellular growth of Chlamydia trachomatis in HeLa cells, which requires the T3SS. 4EpDN did not inhibit the unrelated twin arginine translocation (Tat) system, nor did it impact T3SS gene transcription. Moreover, although the injectisome and flagellar T3SSs are evolutionarily and structurally related, the 4EpDN cyclic peptomer did not inhibit secretion of substrates through the Salmonella flagellar T3SS, indicating that cyclic peptomers broadly but specifically target the injectisome T3SS. 4EpDN reduced the number of T3SS needles detected on the surface of Yersinia pseudotuberculosis as detected by microscopy. Collectively, these data suggest that cyclic peptomers specifically inhibit the injectisome T3SS from a variety of Gram-negative bacteria, possibly by preventing complete T3SS assembly.
              Cover page: Developing Cyclic Peptomers as Broad-Spectrum Type III Secretion System Inhibitors in Gram-Negative Bacteria.
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2017)
              CD4+ T cells differentiate into T helper cell subsets in feedforward manners with synergistic signals from the T cell receptor (TCR), cytokines, and lineage-specific transcription factors. Naive CD4+ T cells avoid spontaneous engagement of feedforward mechanisms but retain a prepared state. T cells lacking the adaptor molecule LAT demonstrate impaired TCR-induced signals yet cause a spontaneous lymphoproliferative T helper 2 (TH2) cell syndrome in mice. Thus, LAT constitutes an unexplained maintenance cue. Here, we demonstrate that tonic signals through LAT constitutively export the repressor HDAC7 from the nucleus of CD4+ T cells. Without such tonic signals, HDAC7 target genes Nur77 and Irf4 are repressed. We reveal that Nur77 suppresses CD4+ T cell proliferation and uncover a suppressive role for Irf4 in TH2 polarization; halving Irf4 gene-dosage leads to increases in GATA3+ and IL-4+ cells. Our studies reveal that naive CD4+ T cells are dynamically tuned by tonic LAT-HDAC7 signals.
                Cover page: Tonic LAT-HDAC7 Signals Sustain Nur77 and Irf4 Expression to Tune Naive CD4 T Cells
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