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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2015)

The human body is made up of over 35 trillion cells, each of which contains an identical copy or two copies of DNA. These cells, however, diverge functionally and morphologically from one another as they occupy specialized niches in various tissues and organs. For example, some cells produce antibodies to ward off invading microbes as part of the immune system, others contract in response to electrical stimuli as part of the cardiac ventricular walls, and yet others pump ions against a concentration gradient as part of the renal tubules. Such diversity in organization and function arising from a common genome requires differential regulation of gene expression in different cell types. This is achieved through epigenetic control of chromatin, which consists of chemical modifications that do not change the DNA code itself.

Each modification must be carefully placed at the right times and at the right locations in the genome in order to achieve a proper transcriptional outcome. Sets of proteins that enzymatically ‘write’ and ‘erase’ epigenetic modifications are tightly regulated to ensure the proper temporal and spatial distribution of each mark. When this system is perturbed, often by mutation in reader or writer proteins or by modulation of their expression, diseases such as cancer may occur.

We are particularly interested in a set of enzymes, the KDM4 family, which removes transcriptionally repressive methylation of histone H3 lysine 9. This family of demethylases is known to promote oncogenesis, likely through inappropriate activation of oncogenic transcription in the setting of KDM4 amplification and overexpression. In order to study the mechanism by which this family promotes oncogenesis and also to validate it as a therapeutic target in cancer we have developed a novel series of demethylase inhibitors using a combination of computational docking, synthetic chemistry, in vitro biochemistry and structural biology. These inhibitors display favorable potency and selectivity towards a subset of histone lysine demethylases, and we believe they will serve as the starting point for the development of chemical probes of the KDM4 family.

    Cover page: Inhibition of the Oncogenic KDM4 Family of Histone Lysine Demethylases
    • 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 (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 (2014)
      Organic cation transporter 1, OCT1 (SLC22A1), is the major hepatic uptake transporter for metformin, the most prescribed antidiabetic drug. However, its endogenous role is poorly understood. Here we show that similar to metformin treatment, loss of Oct1 caused an increase in the ratio of AMP to ATP, activated the energy sensor AMP-activated kinase (AMPK), and substantially reduced triglyceride (TG) levels in livers from healthy and leptin-deficient mice. Conversely, livers of human OCT1 transgenic mice fed high-fat diets were enlarged with high TG levels. Metabolomic and isotopic uptake methods identified thiamine as a principal endogenous substrate of OCT1. Thiamine deficiency enhanced the phosphorylation of AMPK and its downstream target, acetyl-CoA carboxylase. Metformin and the biguanide analog, phenformin, competitively inhibited OCT1-mediated thiamine uptake. Acute administration of metformin to wild-type mice reduced intestinal accumulation of thiamine. These findings suggest that OCT1 plays a role in hepatic steatosis through modulation of energy status. The studies implicate OCT1 as well as metformin in thiamine disposition, suggesting an intriguing and parallel mechanism for metformin and its major hepatic transporter in metabolic function.
        Cover page: OCT1 is a high-capacity thiamine transporter that regulates hepatic steatosis and is a target of metformin
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2023)
        Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen signaling. Androgen deprivation therapy and second-generation androgen receptor (AR)-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by AR and neuroendocrine (NE) markers. Molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterized treatment-emergent mCRPC by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing from 210 tumors. AR-/NE- tumors were clinically and molecularly distinct from other mCRPC subtypes, with the shortest survival, amplification of the chromatin remodeler CHD7, and PTEN loss. Methylation changes in CHD7 candidate enhancers were linked to elevated CHD7 expression in AR-/NE+ tumors. Genome-wide methylation analysis nominated Krüppel-like factor 5 (KLF5) as a driver of the AR-/NE- phenotype, and KLF5 activity was linked to RB1 loss. These observations reveal the aggressiveness of AR-/NE- mCRPC and could facilitate the identification of therapeutic targets in this highly aggressive disease.

        Significance

        Comprehensive characterization of the five subtypes of metastatic castration-resistant prostate cancer identified transcription factors that drive each subtype and showed that the double-negative subtype has the worst prognosis.
          Cover page: The genomic and epigenomic landscape of double-negative metastatic prostate cancerCreative Commons 'BY' version 4.0 license
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