Publications by authors named "Daniel K Nomura"

136 Publications

CYP27A1-dependent anti-melanoma activity of limonoid natural products targets mitochondrial metabolism.

Cell Chem Biol 2021 Mar 30. Epub 2021 Mar 30.

Novartis Institutes for BioMedical Research, 181 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

Three limonoid natural products with selective anti-proliferative activity against BRAF(V600E) and NRAS(Q61K)-mutation-dependent melanoma cell lines were identified. Differential transcriptome analysis revealed dependency of compound activity on expression of the mitochondrial cytochrome P450 oxidase CYP27A1, a transcriptional target of melanogenesis-associated transcription factor (MITF). We determined that CYP27A1 activity is necessary for the generation of a reactive metabolite that proceeds to inhibit cellular proliferation. A genome-wide small interfering RNA screen in combination with chemical proteomics experiments revealed gene-drug functional epistasis, suggesting that these compounds target mitochondrial biogenesis and inhibit tumor bioenergetics through a covalent mechanism. Our work suggests a strategy for melanoma-specific targeting by exploiting the expression of MITF target gene CYP27A1 and inhibiting mitochondrial oxidative phosphorylation in BRAF mutant melanomas.
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http://dx.doi.org/10.1016/j.chembiol.2021.03.004DOI Listing
March 2021

Reimagining Druggability Using Chemoproteomic Platforms.

Acc Chem Res 2021 Apr 18;54(7):1801-1813. Epub 2021 Mar 18.

Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States.

ConspectusOne of the biggest bottlenecks in modern drug discovery efforts is in tackling the undruggable proteome. Currently, over 85% of the proteome is still considered undruggable because most proteins lack well-defined binding pockets that can be functionally targeted with small molecules. Tackling the undruggable proteome necessitates innovative approaches for ligand discovery against undruggable proteins as well as the development of new therapeutic modalities to functionally manipulate proteins of interest. Chemoproteomic platforms, in particular activity-based protein profiling (ABPP), have arisen to tackle the undruggable proteome by using reactivity-based chemical probes and advanced quantitative mass spectrometry-based proteomic approaches to enable the discovery of "ligandable hotspots" or proteome-wide sites that can be targeted with small-molecule ligands. These sites can subsequently be pharmacologically targeted with covalent ligands to rapidly discover functional or nonfunctional binders against therapeutic proteins of interest. Chemoproteomic approaches have also revealed unique insights into ligandability such as the discovery of unique allosteric sites or intrinsically disordered regions of proteins that can be pharmacologically and selectively targeted for biological modulation and therapeutic benefit. Chemoproteomic platforms have also expanded the scope of emerging therapeutic modalities for targeted protein degradation and proteolysis-targeting chimeras (PROTACs) through the discovery of several new covalent E3 ligase recruiters. Looking into the future, chemoproteomic approaches will unquestionably have a major impact in further expansion of existing efforts toward proteome-wide ligandability mapping, targeted ligand discovery efforts against high-value undruggable therapeutic targets, further expansion of the scope of targeted protein degradation platforms, the discovery of new molecular glue scaffolds that enable unique modulation of protein function, and perhaps most excitingly the development of next-generation small-molecule induced-proximity-based therapeutic modalities that go beyond degradation. Exciting days lie ahead in this field as chemical biology becomes an increasingly major driver in drug discovery, and chemoproteomic approaches are sure to be a mainstay in developing next-generation therapeutics.
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http://dx.doi.org/10.1021/acs.accounts.1c00065DOI Listing
April 2021

4β-Hydroxycholesterol is a prolipogenic factor that promotes SREBP1c expression and activity through the liver X receptor.

J Lipid Res 2021 Feb 23;62:100051. Epub 2021 Feb 23.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA, USA; The Paul F. Glenn Center for Aging Research, University of California, Berkeley, Berkeley, CA, USA. Electronic address:

Oxysterols are oxidized derivatives of cholesterol that play regulatory roles in lipid biosynthesis and homeostasis. How oxysterol signaling coordinates different lipid classes such as sterols and triglycerides remains incompletely understood. Here, we show that 4β-hydroxycholesterol (HC) (4β-HC), a liver and serum abundant oxysterol of poorly defined functions, is a potent and selective inducer of the master lipogenic transcription factor, SREBP1c, but not the related steroidogenic transcription factor SREBP2. By correlating tracing of lipid synthesis with lipogenic gene expression profiling, we found that 4β-HC acts as a putative agonist for the liver X receptor (LXR), a sterol sensor and transcriptional regulator previously linked to SREBP1c activation. Unique among the oxysterol agonists of the LXR, 4β-HC induced expression of the lipogenic program downstream of SREBP1c and triggered de novo lipogenesis both in primary hepatocytes and in the mouse liver. In addition, 4β-HC acted in parallel to insulin-PI3K-dependent signaling to stimulate triglyceride synthesis and lipid-droplet accumulation. Thus, 4β-HC is an endogenous regulator of de novo lipogenesis through the LXR-SREBP1c axis.
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http://dx.doi.org/10.1016/j.jlr.2021.100051DOI Listing
February 2021

Lipidome-based Targeting of STAT3-driven Breast Cancer Cells Using Poly-l-glutamic Acid-coated Layer-by-Layer Nanoparticles.

Mol Cancer Ther 2021 Apr 3;20(4):726-738. Epub 2021 Feb 3.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, Massachusetts.

The oncogenic transcription factor STAT3 is aberrantly activated in 70% of breast cancers, including nearly all triple-negative breast cancers (TNBCs). Because STAT3 is difficult to target directly, we considered whether metabolic changes driven by activated STAT3 could provide a therapeutic opportunity. We found that STAT3 prominently modulated several lipid classes, with most profound effects on N-acyl taurine and arachidonic acid, both of which are involved in plasma membrane remodeling. To exploit these metabolic changes therapeutically, we screened a library of layer-by-layer (LbL) nanoparticles (NPs) differing in the surface layer that modulates interactivity with the cell membrane. We found that poly-l-glutamic acid (PLE)-coated NPs bind to STAT3-transformed breast cancer cells with 50% greater efficiency than to nontransformed cells, and the heightened PLE-NP binding to TNBC cells was attenuated by STAT3 inhibition. This effect was also observed in densely packed three-dimensional breast cancer organoids. As STAT3-transformed cells show greater resistance to cytotoxic agents, we evaluated whether enhanced targeted delivery via PLE-NPs would provide a therapeutic advantage. We found that cisplatin-loaded PLE-NPs induced apoptosis of STAT3-driven cells at lower doses compared with both unencapsulated cisplatin and cisplatin-loaded nontargeted NPs. In addition, because radiation is commonly used in breast cancer treatment, and may alter cellular lipid distribution, we analyzed its effect on PLE-NP-cell binding. Irradiation of cells enhanced the STAT3-targeting properties of PLE-NPs in a dose-dependent manner, suggesting potential synergies between these therapeutic modalities. These findings suggest that cellular lipid changes driven by activated STAT3 may be exploited therapeutically using unique LbL NPs.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026705PMC
April 2021

Chemoproteomics-enabled discovery of covalent RNF114-based degraders that mimic natural product function.

Cell Chem Biol 2021 Jan 22. Epub 2021 Jan 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Cambridge, MA 02139, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, Univerity of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, Berkeley, CA 94720, USA. Electronic address:

The translation of functionally active natural products into fully synthetic small-molecule mimetics has remained an important process in medicinal chemistry. We recently discovered that the terpene natural product nimbolide can be utilized as a covalent recruiter of the E3 ubiquitin ligase RNF114 for use in targeted protein degradation-a powerful therapeutic modality within modern-day drug discovery. Using activity-based protein profiling-enabled covalent ligand-screening approaches, here we report the discovery of fully synthetic RNF114-based recruiter molecules that can also be exploited for PROTAC applications, and demonstrate their utility in degrading therapeutically relevant targets, such as BRD4 and BCR-ABL, in cells. The identification of simple and easily manipulated drug-like scaffolds that can mimic the function of a complex natural product is beneficial in further expanding the toolbox of E3 ligase recruiters, an area of great importance in drug discovery and chemical biology.
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http://dx.doi.org/10.1016/j.chembiol.2021.01.005DOI Listing
January 2021

Bardoxolone conjugation enables targeted protein degradation of BRD4.

Sci Rep 2020 09 23;10(1):15543. Epub 2020 Sep 23.

Department of Chemistry, University of California, Berkeley, CA, 94720, USA.

Targeted protein degradation (TPD) has emerged as a powerful tool in drug discovery for the perturbation of protein levels using heterobifunctional small molecules. E3 ligase recruiters remain central to this process yet relatively few have been identified relative to the ~ 600 predicted human E3 ligases. While, initial recruiters have utilized non-covalent chemistry for protein binding, very recently covalent engagement to novel E3's has proven fruitful in TPD application. Herein we demonstrate efficient proteasome-mediated degradation of BRD4 by a bifunctional small molecule linking the KEAP1-Nrf2 activator bardoxolone to a BRD4 inhibitor JQ1.
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http://dx.doi.org/10.1038/s41598-020-72491-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511954PMC
September 2020

Discovery of a Functional Covalent Ligand Targeting an Intrinsically Disordered Cysteine within MYC.

Cell Chem Biol 2021 Jan 22;28(1):4-13.e17. Epub 2020 Sep 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Novartis-Berkeley Center for Proteomics and Chemistry Technologies, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA; Innovative Genomics Institute, Berkeley, CA 94720, USA. Electronic address:

MYC is a major oncogenic transcriptional driver of most human cancers that has remained intractable to direct targeting because much of MYC is intrinsically disordered. Here, we have performed a cysteine-reactive covalent ligand screen to identify compounds that could disrupt the binding of MYC to its DNA consensus sequence in vitro and also impair MYC transcriptional activity in situ in cells. We have identified a covalent ligand, EN4, that targets cysteine 171 of MYC within a predicted intrinsically disordered region of the protein. We show that EN4 directly targets MYC in cells, reduces MYC and MAX thermal stability, inhibits MYC transcriptional activity, downregulates multiple MYC transcriptional targets, and impairs tumorigenesis. We also show initial structure-activity relationships of EN4 and identify compounds that show improved potency. Overall, we identify a unique ligandable site within an intrinsically disordered region of MYC that leads to inhibition of MYC transcriptional activity.
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http://dx.doi.org/10.1016/j.chembiol.2020.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7854864PMC
January 2021

A Cellular Mechanism to Detect and Alleviate Reductive Stress.

Cell 2020 Oct 16;183(1):46-61.e21. Epub 2020 Sep 16.

Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley CA 94720, USA; Howard Hughes Medical Institute, University of California at Berkeley, Berkeley CA 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, CA 94720, USA. Electronic address:

Metazoan organisms rely on conserved stress response pathways to alleviate adverse conditions and preserve cellular integrity. Stress responses are particularly important in stem cells that provide lifetime support for tissue formation and repair, but how these protective systems are integrated into developmental programs is poorly understood. Here we used myoblast differentiation to identify the E3 ligase CUL2 and its substrate FNIP1 as core components of the reductive stress response. Reductive stress, as caused by prolonged antioxidant signaling or mitochondrial inactivity, reverts the oxidation of invariant Cys residues in FNIP1 and allows CUL2 to recognize its target. The ensuing proteasomal degradation of FNIP1 restores mitochondrial activity to preserve redox homeostasis and stem cell integrity. The reductive stress response is therefore built around a ubiquitin-dependent rheostat that tunes mitochondrial activity to redox needs and implicates metabolic control in coordination of stress and developmental signaling.
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http://dx.doi.org/10.1016/j.cell.2020.08.034DOI Listing
October 2020

The regulation of glucose and lipid homeostasis via PLTP as a mediator of BAT-liver communication.

EMBO Rep 2020 09 16;21(9):e49828. Epub 2020 Jul 16.

Diabetes Center, University of California, San Francisco, CA, USA.

While brown adipose tissue (BAT) is well-recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT-derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT-specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT-liver interorgan communication.
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http://dx.doi.org/10.15252/embr.201949828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7507062PMC
September 2020

Manumycin polyketides act as molecular glues between UBR7 and P53.

Nat Chem Biol 2020 11 22;16(11):1189-1198. Epub 2020 Jun 22.

Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.

Molecular glues are an intriguing therapeutic modality that harness small molecules to induce interactions between proteins that typically do not interact. However, such molecules are rare and have been discovered fortuitously, thus limiting their potential as a general strategy for therapeutic intervention. We postulated that natural products bearing one or more electrophilic sites may be an unexplored source of new molecular glues, potentially acting through multicovalent attachment. Using chemoproteomic platforms, we show that members of the manumycin family of polyketides, which bear multiple potentially reactive sites, target C374 of the putative E3 ligase UBR7 in breast cancer cells, and engage in molecular glue interactions with the neosubstrate tumor-suppressor TP53, leading to p53 transcriptional activation and cell death. Our results reveal an anticancer mechanism of this natural product family, and highlight the potential for combining chemoproteomics and multicovalent natural products for the discovery of new molecular glues.
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http://dx.doi.org/10.1038/s41589-020-0557-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7572527PMC
November 2020

A Nimbolide-Based Kinase Degrader Preferentially Degrades Oncogenic BCR-ABL.

ACS Chem Biol 2020 07 25;15(7):1788-1794. Epub 2020 Jun 25.

Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, United States.

Targeted protein degradation (TPD) and proteolysis-targeting chimeras (PROTACs) have arisen as powerful therapeutic modalities for degrading specific proteins in a proteasome-dependent manner. However, a major limitation of TPD is the lack of E3 ligase recruiters. Recently, we discovered the natural product nimbolide as a covalent recruiter for the E3 ligase RNF114. Here, we show the broader utility of nimbolide as an E3 ligase recruiter for TPD applications. We demonstrate that a PROTAC linking nimbolide to the kinase and BCR-ABL fusion oncogene inhibitor dasatinib, BT1, selectively degrades BCR-ABL over c-ABL in leukemia cancer cells, compared to previously reported cereblon or VHL-recruiting BCR-ABL degraders that show opposite selectivity or, in some cases, inactivity. Thus, we further establish nimbolide as an additional general E3 ligase recruiter for PROTACs, and we demonstrate the importance of expanding upon the arsenal of E3 ligase recruiters, as such molecules confer differing selectivity for the degradation of neo-substrate proteins.
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http://dx.doi.org/10.1021/acschembio.0c00348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7891886PMC
July 2020

Unbiased Proteomic Profiling Uncovers a Targetable GNAS/PKA/PP2A Axis in Small Cell Lung Cancer Stem Cells.

Cancer Cell 2020 07 11;38(1):129-143.e7. Epub 2020 Jun 11.

Department of Pediatrics, Stanford University, 265 Campus Drive, Stanford, CA 94305-5457, USA; Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address:

Using unbiased kinase profiling, we identified protein kinase A (PKA) as an active kinase in small cell lung cancer (SCLC). Inhibition of PKA activity genetically, or pharmacologically by activation of the PP2A phosphatase, suppresses SCLC expansion in culture and in vivo. Conversely, GNAS (G-protein α subunit), a PKA activator that is genetically activated in a small subset of human SCLC, promotes SCLC development. Phosphoproteomic analyses identified many PKA substrates and mechanisms of action. In particular, PKA activity is required for the propagation of SCLC stem cells in transplantation studies. Broad proteomic analysis of recalcitrant cancers has the potential to uncover targetable signaling networks, such as the GNAS/PKA/PP2A axis in SCLC.
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http://dx.doi.org/10.1016/j.ccell.2020.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363571PMC
July 2020

Neuronal modulation of hepatic lipid accumulation induced by bingelike drinking.

Am J Physiol Endocrinol Metab 2020 05 11;318(5):E655-E666. Epub 2020 Feb 11.

Diabetes Center, Department of Anatomy, University of California, San Francisco, California.

Excessive alcohol consumption, including binge drinking, is a common cause of fatty liver disease. Binge drinking rapidly induces hepatic steatosis, an early step in the pathogenesis of chronic liver injury. Despite its prevalence, the process by which excessive alcohol consumption promotes hepatic lipid accumulation remains unclear. Alcohol exerts potent effects on the brain, including hypothalamic neurons crucial for metabolic regulation. However, whether or not the brain plays a role in alcohol-induced hepatic steatosis is unknown. In the brain, alcohol increases extracellular levels of adenosine, a potent neuromodulator, and previous work implicates adenosine signaling as being important for the development of alcoholic fatty liver disease. Acute alcohol exposure also increases both the activity of agouti-related protein (AgRP)-expressing neurons and AgRP immunoreactivity. Here, we show that adenosine receptor A signaling in the brain modulates the extent of alcohol-induced fatty liver in mice and that both the AgRP neuropeptide and the sympathetic nervous system are indispensable for hepatic steatosis induced by bingelike alcohol consumption. Together, these results indicate that the brain plays an integral role in alcohol-induced hepatic lipid accumulation and that central adenosine signaling, hypothalamic AgRP, and the sympathetic nervous system are crucial mediators of this process.
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http://dx.doi.org/10.1152/ajpendo.00218.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272724PMC
May 2020

A Metabolic Dependency for Host Isoprenoids in the Obligate Intracellular Pathogen Rickettsia parkeri Underlies a Sensitivity to the Statin Class of Host-Targeted Therapeutics.

mSphere 2019 11 13;4(6). Epub 2019 Nov 13.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

Gram-negative bacteria in the order have an obligate intracellular growth requirement, and some species cause human diseases such as typhus and spotted fever. The bacteria have evolved a dependence on essential nutrients and metabolites from the host cell as a consequence of extensive genome reduction. However, it remains largely unknown which nutrients they acquire and whether their metabolic dependency can be exploited therapeutically. Here, we describe a genetic rewiring of bacterial isoprenoid biosynthetic pathways in the that has resulted from reductive genome evolution. Furthermore, we investigated whether the spotted fever group species scavenges isoprenoid precursors directly from the host. Using targeted mass spectrometry, we found that infection caused decreases in host isoprenoid products and concomitant increases in bacterial isoprenoid metabolites. Additionally, we report that treatment of infected cells with statins, which inhibit host isoprenoid synthesis, prohibited bacterial growth. We show that growth inhibition correlates with changes in bacterial size and shape that mimic those caused by antibiotics that inhibit peptidoglycan biosynthesis, suggesting that statins lead to an inhibition of cell wall synthesis. Altogether, our results describe a potential Achilles' heel of obligate intracellular pathogens that can potentially be exploited with host-targeted therapeutics that interfere with metabolic pathways required for bacterial growth. Obligate intracellular pathogens, which include viruses as well as certain bacteria and eukaryotes, are a subset of infectious microbes that are metabolically dependent on and unable to grow outside an infected host cell because they have lost or lack essential biosynthetic pathways. In this study, we describe a metabolic dependency of the bacterial pathogen on host isoprenoid molecules that are used in the biosynthesis of downstream products, including cholesterol, steroid hormones, and heme. Bacteria make products from isoprenoids, such as an essential lipid carrier for making the bacterial cell wall. We show that bacterial metabolic dependency can represent a potential Achilles' heel and that inhibiting host isoprenoid biosynthesis with the FDA-approved statin class of drugs inhibits bacterial growth by interfering with the integrity of the cell wall. This work supports the potential to treat infections by obligate intracellular pathogens through inhibition of host biosynthetic pathways that are susceptible to parasitism.
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http://dx.doi.org/10.1128/mSphere.00536-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6854040PMC
November 2019

The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis.

Nature 2019 11 21;575(7784):688-692. Epub 2019 Oct 21.

Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA, USA.

Ferroptosis is a form of regulated cell death that is caused by the iron-dependent peroxidation of lipids. The glutathione-dependent lipid hydroperoxidase glutathione peroxidase 4 (GPX4) prevents ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols. Ferroptosis has previously been implicated in the cell death that underlies several degenerative conditions, and induction of ferroptosis by the inhibition of GPX4 has emerged as a therapeutic strategy to trigger cancer cell death. However, sensitivity to GPX4 inhibitors varies greatly across cancer cell lines, which suggests that additional factors govern resistance to ferroptosis. Here, using a synthetic lethal CRISPR-Cas9 screen, we identify ferroptosis suppressor protein 1 (FSP1) (previously known as apoptosis-inducing factor mitochondrial 2 (AIFM2)) as a potent ferroptosis-resistance factor. Our data indicate that myristoylation recruits FSP1 to the plasma membrane where it functions as an oxidoreductase that reduces coenzyme Q (CoQ) (also known as ubiquinone-10), which acts as a lipophilic radical-trapping antioxidant that halts the propagation of lipid peroxides. We further find that FSP1 expression positively correlates with ferroptosis resistance across hundreds of cancer cell lines, and that FSP1 mediates resistance to ferroptosis in lung cancer cells in culture and in mouse tumour xenografts. Thus, our data identify FSP1 as a key component of a non-mitochondrial CoQ antioxidant system that acts in parallel to the canonical glutathione-based GPX4 pathway. These findings define a ferroptosis suppression pathway and indicate that pharmacological inhibition of FSP1 may provide an effective strategy to sensitize cancer cells to ferroptosis-inducing chemotherapeutic agents.
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http://dx.doi.org/10.1038/s41586-019-1705-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883167PMC
November 2019

ER-lysosome contacts enable cholesterol sensing by mTORC1 and drive aberrant growth signalling in Niemann-Pick type C.

Nat Cell Biol 2019 10 23;21(10):1206-1218. Epub 2019 Sep 23.

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.

Cholesterol activates the master growth regulator, mTORC1 kinase, by promoting its recruitment to the surface of lysosomes by the Rag guanosine triphosphatases (GTPases). The mechanisms that regulate lysosomal cholesterol content to enable mTORC1 signalling are unknown. Here, we show that oxysterol binding protein (OSBP) and its anchors at the endoplasmic reticulum (ER), VAPA and VAPB, deliver cholesterol across ER-lysosome contacts to activate mTORC1. In cells lacking OSBP, but not other VAP-interacting cholesterol carriers, the recruitment of mTORC1 by the Rag GTPases is inhibited owing to impaired transport of cholesterol to lysosomes. By contrast, OSBP-mediated cholesterol trafficking drives constitutive mTORC1 activation in a disease model caused by the loss of the lysosomal cholesterol transporter, Niemann-Pick C1 (NPC1). Chemical and genetic inactivation of OSBP suppresses aberrant mTORC1 signalling and restores autophagic function in cellular models of Niemann-Pick type C (NPC). Thus, ER-lysosome contacts are signalling hubs that enable cholesterol sensing by mTORC1, and targeting the sterol-transfer activity of these signalling hubs could be beneficial in patients with NPC.
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http://dx.doi.org/10.1038/s41556-019-0391-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936960PMC
October 2019

Covalent targeting of the vacuolar H-ATPase activates autophagy via mTORC1 inhibition.

Nat Chem Biol 2019 08 8;15(8):776-785. Epub 2019 Jul 8.

Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.

Autophagy is a lysosomal degradation pathway that eliminates aggregated proteins and damaged organelles to maintain cellular homeostasis. A major route for activating autophagy involves inhibition of the mTORC1 kinase, but current mTORC1-targeting compounds do not allow complete and selective mTORC1 blockade. Here, we have coupled screening of a covalent ligand library with activity-based protein profiling to discover EN6, a small-molecule in vivo activator of autophagy that covalently targets cysteine 277 in the ATP6V1A subunit of the lysosomal v-ATPase, which activates mTORC1 via the Rag guanosine triphosphatases. EN6-mediated ATP6V1A modification decouples the v-ATPase from the Rags, leading to inhibition of mTORC1 signaling, increased lysosomal acidification and activation of autophagy. Consistently, EN6 clears TDP-43 aggregates, a causative agent in frontotemporal dementia, in a lysosome-dependent manner. Our results provide insight into how the v-ATPase regulates mTORC1, and reveal a unique approach for enhancing cellular clearance based on covalent inhibition of lysosomal mTORC1 signaling.
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http://dx.doi.org/10.1038/s41589-019-0308-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6641988PMC
August 2019

Harnessing the anti-cancer natural product nimbolide for targeted protein degradation.

Nat Chem Biol 2019 07 17;15(7):747-755. Epub 2019 Jun 17.

Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA.

Nimbolide, a terpenoid natural product derived from the Neem tree, impairs cancer pathogenicity; however, the direct targets and mechanisms by which nimbolide exerts its effects are poorly understood. Here, we used activity-based protein profiling (ABPP) chemoproteomic platforms to discover that nimbolide reacts with a novel functional cysteine crucial for substrate recognition in the E3 ubiquitin ligase RNF114. Nimbolide impairs breast cancer cell proliferation in-part by disrupting RNF114-substrate recognition, leading to inhibition of ubiquitination and degradation of tumor suppressors such as p21, resulting in their rapid stabilization. We further demonstrate that nimbolide can be harnessed to recruit RNF114 as an E3 ligase in targeted protein degradation applications and show that synthetically simpler scaffolds are also capable of accessing this unique reactive site. Our study highlights the use of ABPP platforms in uncovering unique druggable modalities accessed by natural products for cancer therapy and targeted protein degradation applications.
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http://dx.doi.org/10.1038/s41589-019-0304-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6592714PMC
July 2019

Parthenolide Covalently Targets and Inhibits Focal Adhesion Kinase in Breast Cancer Cells.

Cell Chem Biol 2019 07 9;26(7):1027-1035.e22. Epub 2019 May 9.

Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Novartis-Berkeley Center for Proteomics and Chemistry Technologies, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Parthenolide, a natural product from the feverfew plant and member of the large family of sesquiterpene lactones, exerts multiple biological and therapeutic activities including anti-inflammatory and anti-cancer effects. Here, we further study the parthenolide mechanism of action using activity-based protein profiling-based chemoproteomic platforms to map additional covalent targets engaged by parthenolide in human breast cancer cells. We find that parthenolide, as well as other related exocyclic methylene lactone-containing sesquiterpenes, covalently modify cysteine 427 of focal adhesion kinase 1 (FAK1), leading to impairment of FAK1-dependent signaling pathways and breast cancer cell proliferation, survival, and motility. These studies reveal a functional target exploited by members of a large family of anti-cancer natural products.
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http://dx.doi.org/10.1016/j.chembiol.2019.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756182PMC
July 2019

Covalent Ligand Screening Uncovers a RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications.

ACS Chem Biol 2019 11 13;14(11):2430-2440. Epub 2019 May 13.

Department of Molecular and Cell Biology , University of California, Berkeley , Berkeley , California 94720 , United States.

Targeted protein degradation has arisen as a powerful strategy for drug discovery allowing the targeting of undruggable proteins for proteasomal degradation. This approach most often employs heterobifunctional degraders consisting of a protein-targeting ligand linked to an E3 ligase recruiter to ubiquitinate and mark proteins of interest for proteasomal degradation. One challenge with this approach, however, is that only a few E3 ligase recruiters currently exist for targeted protein degradation applications, despite the hundreds of known E3 ligases in the human genome. Here, we utilized activity-based protein profiling (ABPP)-based covalent ligand screening approaches to identify cysteine-reactive small-molecules that react with the E3 ubiquitin ligase RNF4 and provide chemical starting points for the design of RNF4-based degraders. The hit covalent ligand from this screen reacted with either of two zinc-coordinating cysteines in the RING domain, C132 and C135, with no effect on RNF4 activity. We further optimized the potency of this hit and incorporated this potential RNF4 recruiter into a bifunctional degrader linked to JQ1, an inhibitor of the BET family of bromodomain proteins. We demonstrate that the resulting compound CCW 28-3 is capable of degrading BRD4 in a proteasome- and RNF4-dependent manner. In this study, we have shown the feasibility of using chemoproteomics-enabled covalent ligand screening platforms to expand the scope of E3 ligase recruiters that can be exploited for targeted protein degradation applications.
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http://dx.doi.org/10.1021/acschembio.8b01083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7422721PMC
November 2019

Effective breast cancer combination therapy targeting BACH1 and mitochondrial metabolism.

Nature 2019 04 6;568(7751):254-258. Epub 2019 Mar 6.

Ben May Department for Cancer Research, University of Chicago, Chicago, IL, USA.

Mitochondrial metabolism is an attractive target for cancer therapy. Reprogramming metabolic pathways could improve the ability of metabolic inhibitors to suppress cancers with limited treatment options, such as triple-negative breast cancer (TNBC). Here we show that BTB and CNC homology1 (BACH1), a haem-binding transcription factor that is increased in expression in tumours from patients with TNBC, targets mitochondrial metabolism. BACH1 decreases glucose utilization in the tricarboxylic acid cycle and negatively regulates transcription of electron transport chain (ETC) genes. BACH1 depletion by shRNA or degradation by hemin sensitizes cells to ETC inhibitors such as metformin, suppressing growth of both cell line and patient-derived tumour xenografts. Expression of a haem-resistant BACH1 mutant in cells that express a short hairpin RNA for BACH1 rescues the BACH1 phenotype and restores metformin resistance in hemin-treated cells and tumours. Finally, BACH1 gene expression inversely correlates with ETC gene expression in tumours from patients with breast cancer and in other tumour types, which highlights the clinical relevance of our findings. This study demonstrates that mitochondrial metabolism can be exploited by targeting BACH1 to sensitize breast cancer and potentially other tumour tissues to mitochondrial inhibitors.
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http://dx.doi.org/10.1038/s41586-019-1005-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6698916PMC
April 2019

Suppressing fatty acid uptake has therapeutic effects in preclinical models of prostate cancer.

Sci Transl Med 2019 02;11(478)

Monash Partners Comprehensive Cancer Consortium, Monash Biomedicine Discovery Institute Cancer Program, Prostate Cancer Research Group, Department of Physiology, Monash University, Clayton, VIC 3800, Australia.

Metabolism alterations are hallmarks of cancer, but the involvement of lipid metabolism in disease progression is unclear. We investigated the role of lipid metabolism in prostate cancer using tissue from patients with prostate cancer and patient-derived xenograft mouse models. We showed that fatty acid uptake was increased in human prostate cancer and that these fatty acids were directed toward biomass production. These changes were mediated, at least partly, by the fatty acid transporter CD36, which was associated with aggressive disease. Deleting in the prostate of cancer-susceptible mice reduced fatty acid uptake and the abundance of oncogenic signaling lipids and slowed cancer progression. Moreover, CD36 antibody therapy reduced cancer severity in patient-derived xenografts. We further demonstrated cross-talk between fatty acid uptake and de novo lipogenesis and found that dual targeting of these pathways more potently inhibited proliferation of human cancer-derived organoids compared to the single treatments. These findings identify a critical role for CD36-mediated fatty acid uptake in prostate cancer and suggest that targeting fatty acid uptake might be an effective strategy for treating prostate cancer.
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http://dx.doi.org/10.1126/scitranslmed.aau5758DOI Listing
February 2019

Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State.

Cell Chem Biol 2019 03 24;26(3):420-432.e9. Epub 2019 Jan 24.

Department of Biology, Stanford University, Stanford, CA 94305, USA. Electronic address:

The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in an ACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.
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http://dx.doi.org/10.1016/j.chembiol.2018.11.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6430697PMC
March 2019

Estrogen signaling in arcuate Kiss1 neurons suppresses a sex-dependent female circuit promoting dense strong bones.

Nat Commun 2019 01 11;10(1):163. Epub 2019 Jan 11.

Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA, 94158, USA.

Central estrogen signaling coordinates energy expenditure, reproduction, and in concert with peripheral estrogen impacts skeletal homeostasis in females. Here, we ablate estrogen receptor alpha (ERα) in the medial basal hypothalamus and find a robust bone phenotype only in female mice that results in exceptionally strong trabecular and cortical bones, whose density surpasses other reported mouse models. Stereotaxic guided deletion of ERα in the arcuate nucleus increases bone mass in intact and ovariectomized females, confirming the central role of estrogen signaling in this sex-dependent bone phenotype. Loss of ERα in kisspeptin (Kiss1)-expressing cells is sufficient to recapitulate the bone phenotype, identifying Kiss1 neurons as a critical node in this powerful neuroskeletal circuit. We propose that this newly-identified female brain-to-bone pathway exists as a homeostatic regulator diverting calcium and energy stores from bone building when energetic demands are high. Our work reveals a previously unknown target for treatment of age-related bone disease.
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http://dx.doi.org/10.1038/s41467-018-08046-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329772PMC
January 2019

The ER membrane protein complex promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis.

J Cell Sci 2019 01 16;132(2). Epub 2019 Jan 16.

Ludwig Institute for Cancer Research, University of Oxford, ORCRB, Headington, Oxford, OX3 7DQ, UK

The eukaryotic endoplasmic reticulum (ER) membrane contains essential complexes that oversee protein biogenesis and lipid metabolism, impacting nearly all aspects of cell physiology. The ER membrane protein complex (EMC) is a newly described transmembrane domain (TMD) insertase linked with various phenotypes, but whose clients and cellular responsibilities remain incompletely understood. We report that EMC deficiency limits the cellular boundaries defining cholesterol tolerance, reflected by diminished viability with limiting or excessive extracellular cholesterol. Lipidomic and proteomic analyses revealed defective biogenesis and concomitant loss of the TMD-containing ER-resident enzymes sterol-O-acyltransferase 1 (SOAT1) and squalene synthase (SQS, also known as FDFT1), which serve strategic roles in the adaptation of cells to changes in cholesterol availability. Insertion of the weakly hydrophobic tail-anchor (TA) of SQS into the ER membrane by the EMC ensures sufficient flux through the sterol biosynthetic pathway while biogenesis of polytopic SOAT1 promoted by the EMC provides cells with the ability to store free cholesterol as inert cholesteryl esters. By facilitating insertion of TMDs that permit essential mammalian sterol-regulating enzymes to mature accurately, the EMC is an important biogenic determinant of cellular robustness to fluctuations in cholesterol availability.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.223453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6362398PMC
January 2019

Development of alkyl glycerone phosphate synthase inhibitors: Structure-activity relationship and effects on ether lipids and epithelial-mesenchymal transition in cancer cells.

Eur J Med Chem 2019 Feb 28;163:722-735. Epub 2018 Nov 28.

Department of Chemistry and Technologies of Drugs, Sapienza University of Rome, P. le A. Moro 5, 00185, Rome, Italy. Electronic address:

In aggressive tumors, alkylglyceronephosphate synthase (AGPS) controls cellular ether phospholipid utilization and metabolism to promote cancer cell proliferation and motility. SAR studies on the first-in-class AGPS inhibitor 1, discovered by our group, led to the 2,6-difluoro analog 2i which showed higher binding affinity than 1in vitro. In 231MFP cancer cells, 2i reduced ether lipids levels and cell migration rate. When tested in PC-3 and MDA-MB-231 cancer cells, 2i specifically impaired epithelial to mesenchymal transition (EMT) by modulating E-cadherin, Snail and MMP2 expression levels. Moreover, the combination of siRNAs against AGPS and 2i provided no additive effect, confirming that the modulation of 2i on EMT specifically relies on AGPS inhibition. Finally, this compound also affected cancer cell proliferation especially in MDA-MB-231 cells expressing higher AGPS level, whereas it provided negligible effects on MeT5A, a non-tumorigenic cell line, thus showing cancer specificity.
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http://dx.doi.org/10.1016/j.ejmech.2018.11.050DOI Listing
February 2019

Metabolomic Markers of Phthalate Exposure in Plasma and Urine of Pregnant Women.

Front Public Health 2018 22;6:298. Epub 2018 Oct 22.

School of Public Health, Center for Environmental Research and Children's Health, University of California, Berkeley, Berkeley, CA, United States.

Phthalates are known endocrine disruptors and found in almost all people with several associated adverse health outcomes reported in humans and animal models. Limited data are available on the relationship between exposure to endocrine disrupting chemicals and the human metabolome. We examined the relationship of metabolomic profiles in plasma and urine of 115 pregnant women with eleven urine phthalate metabolites measured at 26 weeks of gestation to identify potential biomarkers and relevant pathways. Targeted metabolomics was performed by selected reaction monitoring liquid chromatography and triple quadrupole mass spectrometry to measure 415 metabolites in plasma and 151 metabolites in urine samples. We have chosen metabolites with the best defined peaks for more detailed analysis (138 in plasma and 40 in urine). Relationship between urine phthalate metabolites and concurrent metabolomic markers in plasma and urine suggested potential involvement of diverse pathways including lipid, steroid, and nucleic acid metabolism and enhanced inflammatory response. Most of the correlations were positive for both urine and plasma, and further confirmed by regression and PCA analysis. However, after the FDR adjustment for multiple comparisons, only 9 urine associations remained statistically significant (-values 0.0001-0.0451), including Nicotinamide mononucleotide, Cysteine T2, Cystine, and L-Aspartic acid. Additionally, we found negative associations of maternal pre-pregnancy body mass index (BMI) with more than 20 metabolomic markers related to lipid and amino-acid metabolism and inflammation pathways in plasma ( = 0.01-0.0004), while Mevalonic acid was positively associated ( = 0.009). Nicotinic acid, the only significant metabolite in urine, had a positive association with maternal BMI ( = 0.002). In summary, when evaluated in the context of metabolic pathways, the findings suggest enhanced lipid biogenesis, inflammation and altered nucleic acid metabolism in association with higher phthalate levels. These results provide new insights into the relationship between phthalates, common in most human populations, and metabolomics, a novel approach to exposure and health biomonitoring.
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http://dx.doi.org/10.3389/fpubh.2018.00298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204535PMC
October 2018

Acyl-CoA synthetase 6 enriches the neuroprotective omega-3 fatty acid DHA in the brain.

Proc Natl Acad Sci U S A 2018 12 6;115(49):12525-12530. Epub 2018 Nov 6.

Department of Physiology, East Carolina University, Greenville, NC 25878;

Docosahexaenoic acid (DHA) is an omega-3 fatty acid that is highly abundant in the brain and confers protection against numerous neurological diseases, yet the fundamental mechanisms regulating the enrichment of DHA in the brain remain unknown. Here, we have discovered that a member of the long-chain acyl-CoA synthetase family, Acsl6, is required for the enrichment of DHA in the brain by generating an Acsl6-deficient mouse (Acsl6). Acsl6 is highly enriched in the brain and lipid profiling of Acsl6 tissues reveals consistent reductions in DHA-containing lipids in tissues highly abundant with Acsl6. Acsl6 mice demonstrate motor impairments, altered glutamate metabolism, and increased astrogliosis and microglia activation. In response to a neuroinflammatory lipopolysaccharide injection, Acsl6 brains show similar increases in molecular and pathological indices of astrogliosis compared with controls. These data demonstrate that Acsl6 is a key mediator of neuroprotective DHA enrichment in the brain.
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http://dx.doi.org/10.1073/pnas.1807958115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298081PMC
December 2018

Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues.

Nat Chem Biol 2018 11 16;14(11):1021-1031. Epub 2018 Oct 16.

Department of Bioengineering, University of California, San Diego, La Jolla, CA, USA.

Fatty acid synthase (FASN) predominantly generates straight-chain fatty acids using acetyl-CoA as the initiating substrate. However, monomethyl branched-chain fatty acids (mmBCFAs) are also present in mammals but are thought to be primarily diet derived. Here we demonstrate that mmBCFAs are de novo synthesized via mitochondrial BCAA catabolism, exported to the cytosol by adipose-specific expression of carnitine acetyltransferase (CrAT), and elongated by FASN. Brown fat exhibits the highest BCAA catabolic and mmBCFA synthesis fluxes, whereas these lipids are largely absent from liver and brain. mmBCFA synthesis is also sustained in the absence of microbiota. We identify hypoxia as a potent suppressor of BCAA catabolism that decreases mmBCFA synthesis in obese adipose tissue, such that mmBCFAs are significantly decreased in obese animals. These results identify adipose tissue mmBCFA synthesis as a novel link between BCAA metabolism and lipogenesis, highlighting roles for CrAT and FASN promiscuity influencing acyl-chain diversity in the lipidome.
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http://dx.doi.org/10.1038/s41589-018-0132-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245668PMC
November 2018