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Open Access Publications from the University of California

Scholarly Works (7 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2013)

Epigenetic regulation of gene expression is critical to development, cellular homeostasis, and disease pathology. One important factor of the epigenetic signaling system is chromatin methylation. In particular, histone protein methylation of lysine and arginine residues is widespread and influences myriad cellular processes. To aid in this research, a facile method was developed to install methylarginine analogs on recombinant protein for use in biochemical, biophysical and structural studies. Through chemical conjugation of novel alpha,beta-unsaturated amidine precursors with proteins, methylarginine mimics can be displayed with control of methylation site, extent and regiospecificity. Moreover, a discussion of the enzymes responsible for installing chromatin methylation further highlights the importance of protein and nucleic acid methylation in cellular physiology and disease. Complementary to the activity of methylating enzymes, the removal of methylation marks by demethylase proteins is essential for dynamic gene expression. Of particular interest is the demethylation of methyllysine residues in histone proteins, which has been shown to strongly influence transcription of associated genes. In certain cancer cells overexpressing such lysine demethylase enzymes, epigenetic signaling is dysregulated, resulting in aberrant gene expression conducive to cancer phenotypes. Therefore, chemical probes were developed against a cancer-associated demethylase, JMJD2C, to study the cellular role of said enzyme during normal transcription as well as in cancer pathology. This work has produced several novel and potent inhibitors, which will be used in cellular assays. With the acquisition of structural data from crystallography efforts, structure-based design is feasible for compound optimization in regard to potency and paralog selectivity. Ultimately, these chemical probes are intended to explore the cellular functions of JMJD2C and its role in cancer. Taken together, this body of work represents a broad investigation of chromatin methylation and its influence on epigenetics from the perspective of chemical biology.

    Cover page: Development of chemical tools to interrogate the roles of lysine and arginine methylation in epigenetic regulation
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2013)
    Arginine methylation has emerged as a widespread post-translational modification with influence over myriad cellular processes. However, the molecular mechanisms underlying such methylarginine-dependent phenomena remain unclear. To aid in this research, a facile method was developed to install methylarginine analogues on recombinant protein for use in biochemical, biophysical, and structural studies. Through chemical conjugation of novel α,β-unsaturated amidine precursors with proteins, methylarginine mimics can be displayed with control of methylation site, extent, and regiospecificity. Analogue installation into histones using this strategy produced modified proteins that were recognized by antibodies specific to endogenous methylarginine, and these histones retained the capacity to form mononucleosomes. Moreover, a native methylarginine-specific binding domain was shown to interact with methylarginine analogue-modified substrates. This chemical conjugation method for installing methylarginine analogues provides an efficient route to produce homogeneous modified proteins for subsequent investigations of methylarginine-dependent processes.
      Cover page: Site-Specific and Regiospecific Installation of Methylarginine Analogues into Recombinant Histones and Insights into Effector Protein Binding
      • Multimedia
      • Peer Reviewed
      UC San Francisco Previously Published Works (2014)
      The JmjC domain-containing proteins are hydroxylases that confer posttranslational modifications on histone tails, by removing methylation marks on methylated lysine residues. This serves to either promote or repress gene transcription. The JMJD2A-D family members include the enzyme Jumonji domain 2C (JMJD2C), which specifically demethylates di- and trimethylated histone H3 at Lys 9 or Lys 36.[1] Dysregulation of JMJD2C has been implicated in prostate, colonic, and breast cancer as the demethylase can modify the expression levels of oncogenes.[2] The goal of the present study was to identify potent and selective small-molecule inhibitors of JMJD2C, to be used as chemical biology tools to further investigate the role of JMJD2C in cell proliferation and survival. Using high-resolution crystal structures of the JMJD2 subfamily members as templates, we have performed a small molecule virtual docking screen. From the ~3 million molecules that were docked, this experiment identified 21 compounds as possible leads. These compounds were tested against JMJD2C in enzymatic assays and here we report an overall hit rate of 76%, with 8 compounds demonstrating an IC50 of 176μM to 1.18μM. A molecule containing a salicylate core was selected as a candidate for optimization and thus far we have completed several rounds of iterative target-specific compound docking, hybrid molecule design, compound synthesis and in vitro characterization. Notably, our method demonstrated a substantial increase in potency when we linked two docked fragments together and further derivatized this new scaffold, through which we have successfully derived a 65nM inhibitor of JMJD2C. A compound representing the inhibitor scaffold has been co-crystallized with JMJD2A to a resolution of 2.4 Å. In the crystal structure each asymmetric unit contains two JMJD2A monomers, each bound to a single inhibitor molecule. This complex-structure superposes well with the docked pose for the hybrid series of compounds. We are now focusing our efforts on identifying an inhibitor that is selective for the JMJD2 family over other JmjC domain-containing proteins.
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      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.
        Cover page: Chloride channels regulate differentiation and barrier functions of the mammalian airway
        • Article
        UC San Francisco Previously Published Works (2019)
        SUMMARYThe conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. To better understand how airway developmental programs are established to support air breathing and barrier functions, we constructed a single-cell atlas of the human and mouse developing trachea. In this study, we uncover hitherto unrecognized heterogeneity of cell states with distinct differentiation programs and immune features of the developing airway. In addition, we find ubiquitous expression ofCFTRandANO1/TMEM16Achloride channels in the embryonic airway epithelium. We show that genetic inactivation of TMEM16A leads to airway defects commonly seen in cystic fibrosis patients with deficient CFTR, alters the differentiation trajectory of airway basal progenitors, and results in mucus cell hyperplasia and aberrant epithelial antimicrobial expression. Together, our study illuminates conserved developmental features of the mammalian airway and implicates chloride homeostasis as a key player in regulating mucosal barrier formation and function relevant to early onset airway diseases.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2016)
          Development of tool molecules that inhibit Jumonji demethylases allows for the investigation of cancer-associated transcription. While scaffolds such as 2,4-pyridinedicarboxylic acid (2,4-PDCA) are potent inhibitors, they exhibit limited selectivity. To discover new inhibitors for the KDM4 demethylases, enzymes overexpressed in several cancers, we docked a library of 600,000 fragments into the high-resolution structure of KDM4A. Among the most interesting chemotypes were the 5-aminosalicylates, which docked in two distinct but overlapping orientations. Docking poses informed the design of covalently linked fragment compounds, which were further derivatized. This combined approach improved affinity by ∼ 3 log-orders to yield compound 35 (Ki = 43 nM). Several hybrid inhibitors were selective for KDM4C over the related enzymes FIH, KDM2A, and KDM6B while lacking selectivity against the KDM3 and KDM5 subfamilies. Cocrystal structures corroborated the docking predictions. This study extends the use of structure-based docking from fragment discovery to fragment linking optimization, yielding novel KDM4 inhibitors.
            Cover page: Docking and Linking of Fragments To Discover Jumonji Histone Demethylase Inhibitors
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2010)
            Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.
              Cover page: The p110δ structure: mechanisms for selectivity and potency of new PI(3)K inhibitors
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