Publications by authors named "David J Maloney"

116 Publications

Optimization of ether and aniline based inhibitors of lactate dehydrogenase.

Bioorg Med Chem Lett 2021 Jun 24;41:127974. Epub 2021 Mar 24.

Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, United States. Electronic address:

Lactate dehydrogenase (LDH) is a critical enzyme in the glycolytic metabolism pathway that is used by many tumor cells. Inhibitors of LDH may be expected to inhibit the metabolic processes in cancer cells and thus selectively delay or inhibit growth in transformed versus normal cells. We have previously disclosed a pyrazole-based series of potent LDH inhibitors with long residence times on the enzyme. Here, we report the elaboration of a new subseries of LDH inhibitors based on those leads. These new compounds potently inhibit both LDHA and LDHB enzymes, and inhibit lactate production in cancer cell lines.
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http://dx.doi.org/10.1016/j.bmcl.2021.127974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113097PMC
June 2021

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

Sci Rep 2021 Jan 22;11(1):2121. Epub 2021 Jan 22.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.
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http://dx.doi.org/10.1038/s41598-021-81486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822874PMC
January 2021

Pyrazole-Based Lactate Dehydrogenase Inhibitors with Optimized Cell Activity and Pharmacokinetic Properties.

J Med Chem 2020 10 27;63(19):10984-11011. Epub 2020 Sep 27.

Urologic Oncology Branch, Center for Cancer Research, National Cancer Institute, 9000 Rockville Pike, Bethesda, Maryland 20892, United States.

Lactate dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate, with concomitant oxidation of reduced nicotinamide adenine dinucleotide as the final step in the glycolytic pathway. Glycolysis plays an important role in the metabolic plasticity of cancer cells and has long been recognized as a potential therapeutic target. Thus, potent, selective inhibitors of LDH represent an attractive therapeutic approach. However, to date, pharmacological agents have failed to achieve significant target engagement , possibly because the protein is present in cells at very high concentrations. We report herein a lead optimization campaign focused on a pyrazole-based series of compounds, using structure-based design concepts, coupled with optimization of cellular potency, drug-target residence times, and PK properties, to identify first-in-class inhibitors that demonstrate LDH inhibition . The lead compounds, named () and (), possess desirable attributes for further studying the effect of LDH inhibition.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830743PMC
October 2020

Dynamic Imaging of LDH Inhibition in Tumors Reveals Rapid In Vivo Metabolic Rewiring and Vulnerability to Combination Therapy.

Cell Rep 2020 02;30(6):1798-1810.e4

Leidos Biomedical, Frederick National Laboratory for Cancer Research, Frederick, MD 24060, USA.

The reliance of many cancers on aerobic glycolysis has stimulated efforts to develop lactate dehydrogenase (LDH) inhibitors. However, despite significant efforts, LDH inhibitors (LDHi) with sufficient specificity and in vivo activity to determine whether LDH is a feasible drug target are lacking. We describe an LDHi with potent, on-target, in vivo activity. Using hyperpolarized magnetic resonance spectroscopic imaging (HP-MRSI), we demonstrate in vivo LDH inhibition in two glycolytic cancer models, MIA PaCa-2 and HT29, and we correlate depth and duration of LDH inhibition with direct anti-tumor activity. HP-MRSI also reveals a metabolic rewiring that occurs in vivo within 30 min of LDH inhibition, wherein pyruvate in a tumor is redirected toward mitochondrial metabolism. Using HP-MRSI, we show that inhibition of mitochondrial complex 1 rapidly redirects tumor pyruvate toward lactate. Inhibition of both mitochondrial complex 1 and LDH suppresses metabolic plasticity, causing metabolic quiescence in vitro and tumor growth inhibition in vivo.
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http://dx.doi.org/10.1016/j.celrep.2020.01.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039685PMC
February 2020

Room-Temperature, Copper-Free Sonogashira Reactions Facilitated by Air-Stable, Monoligated Precatalyst [DTBNpP] Pd(crotyl)Cl.

ACS Omega 2018 Oct 10;3(10):12985-12998. Epub 2018 Oct 10.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland 20850, United States.

A novel application of [DTBNpP] Pd(crotyl)Cl (DTBNpP = di--butylneopentylphosphine) (), an air-stable, commercially available palladium precatalyst that allows rapid access to a monoligated state, has been identified for room-temperature, copper-free Sonogashira couplings of challenging aryl bromides and alkynes. The mild reaction conditions with TMP in dimethyl sulfoxide afford up to 97% yields, excellent functional group tolerability, and broad reaction compatibility with access to one-pot indole formation.
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http://dx.doi.org/10.1021/acsomega.8b01868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6644404PMC
October 2018

Lead optimization and efficacy evaluation of quinazoline-based BET family inhibitors for potential treatment of cancer and inflammatory diseases.

Bioorg Med Chem Lett 2019 05 12;29(10):1220-1226. Epub 2019 Mar 12.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States. Electronic address:

Extensive optimization of quinazoline-based lead 8 is described. The structure-activity relationship studies indicate the S-configuration is preferred for the phenylmorpholine substitution. Together with incorporation of a (2-hydroxyl-2-methylpropyl)pyrazole moiety at the 2-position leads to analogs with comparable potency and marked improvement in the pharmacokinetic profile over our previously reported lead compounds. Further in vivo efficacy studies in Kasumi-1 xenograft mouse model demonstrates that the selected inhibitors are well tolerated and highly efficacious in the inhibition of tumor growth. Additionally, the representative analog 19 also demonstrated significant improvement of arthritis severity in a collagen-induced arthritis (CIA) mouse model. These results indicate potential use of these quinazoline-based BET inhibitors for treatment of cancer and inflammatory diseases. A brief discussion of the co-crystallized structure of 19 with BRD4 (BD1) is also highlighted.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405260PMC
May 2019

A high-throughput screen to identify novel small molecule inhibitors of the Werner Syndrome Helicase-Nuclease (WRN).

PLoS One 2019 9;14(1):e0210525. Epub 2019 Jan 9.

Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, United States of America.

Werner syndrome (WS), an autosomal recessive genetic disorder, displays accelerated clinical symptoms of aging leading to a mean lifespan less than 50 years. The WS helicase-nuclease (WRN) is involved in many important pathways including DNA replication, recombination and repair. Replicating cells are dependent on helicase activity, leading to the pursuit of human helicases as potential therapeutic targets for cancer treatment. Small molecule inhibitors of DNA helicases can be used to induce synthetic lethality, which attempts to target helicase-dependent compensatory DNA repair pathways in tumor cells that are already genetically deficient in a specific pathway of DNA repair. Alternatively, helicase inhibitors may be useful as tools to study the specialized roles of helicases in replication and DNA repair. In this study, approximately 350,000 small molecules were screened based on their ability to inhibit duplex DNA unwinding by a catalytically active WRN helicase domain fragment in a high-throughput fluorometric assay to discover new non-covalent small molecule inhibitors of the WRN helicase. Select compounds were screened to exclude ones that inhibited DNA unwinding by other helicases in the screen, bound non-specifically to DNA, acted as irreversible inhibitors, or possessed unfavorable chemical properties. Several compounds were tested for their ability to impair proliferation of cultured tumor cells. We observed that two of the newly identified WRN helicase inhibitors inhibited proliferation of cancer cells in a lineage-dependent manner. These studies represent the first high-throughput screen for WRN helicase inhibitors and the results have implications for anti-cancer strategies targeting WRN in different cancer cells and genetic backgrounds.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210525PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326523PMC
October 2019

Molecular basis for activation of lecithin:cholesterol acyltransferase by a compound that increases HDL cholesterol.

Elife 2018 11 27;7. Epub 2018 Nov 27.

Department of Biological Sciences, Purdue University, Indiana, United States.

Lecithin:cholesterol acyltransferase (LCAT) and LCAT-activating compounds are being investigated as treatments for coronary heart disease (CHD) and familial LCAT deficiency (FLD). Herein we report the crystal structure of human LCAT in complex with a potent piperidinylpyrazolopyridine activator and an acyl intermediate-like inhibitor, revealing LCAT in an active conformation. Unlike other LCAT activators, the piperidinylpyrazolopyridine activator binds exclusively to the membrane-binding domain (MBD). Functional studies indicate that the compound does not modulate the affinity of LCAT for HDL, but instead stabilizes residues in the MBD and facilitates channeling of substrates into the active site. By demonstrating that these activators increase the activity of an FLD variant, we show that compounds targeting the MBD have therapeutic potential. Our data better define the substrate binding site of LCAT and pave the way for rational design of LCAT agonists and improved biotherapeutics for augmenting or restoring reverse cholesterol transport in CHD and FLD patients.
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http://dx.doi.org/10.7554/eLife.41604DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277198PMC
November 2018

KDM5 Histone Demethylase Activity Links Cellular Transcriptomic Heterogeneity to Therapeutic Resistance.

Cancer Cell 2018 12 21;34(6):939-953.e9. Epub 2018 Nov 21.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Center for Cancer Evolution, Dana-Farber Cancer Institute, Boston, MA 02215, USA; The Eli and Edythe L Broad Institute of MIT and Harvard, Cambridge, MA 02139, USA; Center for Functional Cancer Epigenetics, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Ludwig Center at Harvard, Boston, MA 02215, USA. Electronic address:

Members of the KDM5 histone H3 lysine 4 demethylase family are associated with therapeutic resistance, including endocrine resistance in breast cancer, but the underlying mechanism is poorly defined. Here we show that genetic deletion of KDM5A/B or inhibition of KDM5 activity increases sensitivity to anti-estrogens by modulating estrogen receptor (ER) signaling and by decreasing cellular transcriptomic heterogeneity. Higher KDM5B expression levels are associated with higher transcriptomic heterogeneity and poor prognosis in ER breast tumors. Single-cell RNA sequencing, cellular barcoding, and mathematical modeling demonstrate that endocrine resistance is due to selection for pre-existing genetically distinct cells, while KDM5 inhibitor resistance is acquired. Our findings highlight the importance of cellular phenotypic heterogeneity in therapeutic resistance and identify KDM5A/B as key regulators of this process.
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http://dx.doi.org/10.1016/j.ccell.2018.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310147PMC
December 2018

Predicting Novel Therapies and Targets: Regulation of Notch3 by the Bromodomain Protein BRD4.

Mol Cancer Ther 2019 02 12;18(2):421-436. Epub 2018 Nov 12.

Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, Texas.

Systematic approaches for accurate repurposing of targeted therapies are needed. We developed and aimed to biologically validate our therapy predicting tool (TPT) for the repurposing of targeted therapies for specific tumor types by testing the role of Bromodomain and Extra-Terminal motif inhibitors (BETi) in inhibiting BRD4 function and downregulating Notch3 signaling in ovarian cancer.Utilizing established ovarian cancer preclinical models, we carried out and studies with clinically relevant BETis to determine their therapeutic effect and impact on Notch3 signaling.Treatment with BETis or siRNA-mediated BRD4 knockdown resulted in decreased cell viability, reduced cell proliferation, and increased cell apoptosis studies with orthotopic mouse models demonstrated that treatment with BETi decreased tumor growth. In addition, knockdown of BRD4 with doxycycline-inducible shRNA increased survival up to 50% ( < 0.001). Treatment with either BETis or BRD4 siRNA decreased Notch3 expression both and BRD4 inhibition also decreased the expression of targets, including Chromatin immunoprecipitation revealed that BRD4 was present at the promoter.Our findings provide biological validation for the TPT by demonstrating that BETis can be an effective therapeutic agent for ovarian cancer by downregulating Notch3 expression.The TPT could rapidly identify candidate drugs for ovarian or other cancers along with novel companion biomarkers.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363833PMC
February 2019

Structure-Based Engineering of Irreversible Inhibitors against Histone Lysine Demethylase KDM5A.

J Med Chem 2018 12 15;61(23):10588-10601. Epub 2018 Nov 15.

Department of Molecular and Cellular Oncology , The University of Texas MD Anderson Cancer Center , Houston , Texas 77030 , United States.

The active sites of hundreds of human α-ketoglutarate (αKG) and Fe(II)-dependent dioxygenases are exceedingly well preserved, which challenges the design of selective inhibitors. We identified a noncatalytic cysteine (Cys481 in KDM5A) near the active sites of KDM5 histone H3 lysine 4 demethylases, which is absent in other histone demethylase families, that could be explored for interaction with the cysteine-reactive electrophile acrylamide. We synthesized analogs of a thienopyridine-based inhibitor chemotype, namely, 2-((3-aminophenyl)(2-(piperidin-1-yl)ethoxy)methyl)thieno[3,2- b]pyridine-7-carboxylic acid (N70) and a derivative containing a (dimethylamino)but-2-enamido)phenyl moiety (N71) designed to form a covalent interaction with Cys481. We characterized the inhibitory and binding activities against KDM5A and determined the cocrystal structures of the catalytic domain of KDM5A in complex with N70 and N71. Whereas the noncovalent inhibitor N70 displayed αKG-competitive inhibition that could be reversed after dialysis, inhibition by N71 was dependent on enzyme concentration and persisted even after dialysis, consistent with covalent modification.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467790PMC
December 2018

Discovery and lead identification of quinazoline-based BRD4 inhibitors.

Bioorg Med Chem Lett 2018 11 31;28(21):3483-3488. Epub 2018 Aug 31.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States. Electronic address:

A new series of quinazoline-based analogs as potent bromodomain-containing protein 4 (BRD4) inhibitors is described. The structure-activity relationships on 2- and 4-position of quinazoline ring, and the substitution at 6-position that mimic the acetylated lysine are discussed. A co-crystallized structure of 48 (CN750) with BRD4 (BD1) including key inhibitor-protein interactions is also highlighted. Together with preliminary rodent pharmacokinetic results, a new lead (65, CN427) is identified which is suitable for further lead optimization.
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http://dx.doi.org/10.1016/j.bmcl.2018.08.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7392372PMC
November 2018

Inhibition of spinal 15-LOX-1 attenuates TLR4-dependent, nonsteroidal anti-inflammatory drug-unresponsive hyperalgesia in male rats.

Pain 2018 Dec;159(12):2620-2629

Department of Anesthesiology, University of California, San Diego, La Jolla, CA, United States.

Although nonsteroidal anti-inflammatory drugs are the first line of therapeutics for the treatment of mild to moderate somatic pain, they are not generally considered to be effective for neuropathic pain. In the current study, direct activation of spinal Toll-like 4 receptors (TLR4) by the intrathecal (IT) administration of KDO2 lipid A (KLA), the active component of lipopolysaccharide, elicits a robust tactile allodynia that is unresponsive to cyclooxygenase inhibition, despite elevated expression of cyclooxygenase metabolites in the spinal cord. Intrathecal KLA increases 12-lipoxygenase-mediated hepoxilin production in the lumbar spinal cord, concurrent with expression of the tactile allodynia. The TLR4-induced hepoxilin production was also observed in primary spinal microglia, but not in astrocytes, and was accompanied by increased microglial expression of the 12/15-lipoxygenase enzyme 15-LOX-1. Intrathecal KLA-induced tactile allodynia was completely prevented by spinal pretreatment with the 12/15-lipoxygenase inhibitor CDC or a selective antibody targeting rat 15-LOX-1. Similarly, pretreatment with the selective inhibitors ML127 or ML351 both reduced activity of the rat homolog of 15-LOX-1 heterologously expressed in HEK-293T cells and completely abrogated nonsteroidal anti-inflammatory drug-unresponsive allodynia in vivo after IT KLA. Finally, spinal 12/15-lipoxygenase inhibition by nordihydroguaiaretic acid (NDGA) both prevents phase II formalin flinching and reverses formalin-induced persistent tactile allodynia. Taken together, these findings suggest that spinal TLR4-mediated hyperpathic states are mediated at least in part through activation of microglial 15-LOX-1.
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http://dx.doi.org/10.1097/j.pain.0000000000001373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237621PMC
December 2018

KDM5 histone demethylases repress immune response via suppression of STING.

PLoS Biol 2018 08 6;16(8):e2006134. Epub 2018 Aug 6.

Department of Pathology, Yale School of Medicine, New Haven, Connecticut, United States of America.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy.
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http://dx.doi.org/10.1371/journal.pbio.2006134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095604PMC
August 2018

Discovery of Orally Bioavailable, Quinoline-Based Aldehyde Dehydrogenase 1A1 (ALDH1A1) Inhibitors with Potent Cellular Activity.

J Med Chem 2018 06 31;61(11):4883-4903. Epub 2018 May 31.

National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive , Rockville , Maryland 20850 , United States.

Aldehyde dehydrogenases (ALDHs) are responsible for the metabolism of aldehydes (exogenous and endogenous) and possess vital physiological and toxicological functions in areas such as CNS, inflammation, metabolic disorders, and cancers. Overexpression of certain ALDHs (e.g., ALDH1A1) is an important biomarker in cancers and cancer stem cells (CSCs) indicating the potential need for the identification and development of small molecule ALDH inhibitors. Herein, a newly designed series of quinoline-based analogs of ALDH1A1 inhibitors is described. Extensive medicinal chemistry optimization and biological characterization led to the identification of analogs with significantly improved enzymatic and cellular ALDH inhibition. Selected analogs, e.g., 86 (NCT-505) and 91 (NCT-506), demonstrated target engagement in a cellular thermal shift assay (CETSA), inhibited the formation of 3D spheroid cultures of OV-90 cancer cells, and potentiated the cytotoxicity of paclitaxel in SKOV-3-TR, a paclitaxel resistant ovarian cancer cell line. Lead compounds also exhibit high specificity over other ALDH isozymes and unrelated dehydrogenases. The in vitro ADME profiles and pharmacokinetic evaluation of selected analogs are also highlighted.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004562PMC
June 2018

DDB2 represses ovarian cancer cell dedifferentiation by suppressing ALDH1A1.

Cell Death Dis 2018 05 1;9(5):561. Epub 2018 May 1.

Department of Radiology, College of Medicine, The Ohio State University, Columbus, OH, 43210, USA.

Cancer stem cells (CSCs), representing the root of many solid tumors including ovarian cancer, have been implicated in disease recurrence, metastasis, and therapeutic resistance. Our previous study has demonstrated that the CSC subpopulation in ovarian cancer can be limited by DNA damage-binding protein 2 (DDB2). Here, we demonstrated that the ovarian CSC subpopulation can be maintained via cancer cell dedifferentiation, and DDB2 is able to suppress this non-CSC-to-CSC conversion by repression of ALDH1A1 transcription. Mechanistically, DDB2 binds to the ALDH1A1 gene promoter, facilitating the enrichment of histone H3K27me3, and competing with the transcription factor C/EBPβ for binding to this region, eventually inhibiting the promoter activity of the ALDH1A1 gene. The de-repression of ALDH1A1 expression contributes to DDB2 silencing-augmented non-CSC-to-CSC conversion and expansion of the CSC subpopulation. We further showed that treatment with a selective ALDH1A1 inhibitor blocked DDB2 silencing-induced expansion of CSCs, and halted orthotopic xenograft tumor growth. Together, our data demonstrate that DDB2, functioning as a transcription repressor, can abrogate ovarian CSC properties by downregulating ALDH1A1 expression.
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http://dx.doi.org/10.1038/s41419-018-0585-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5948213PMC
May 2018

Insights into the Action of Inhibitor Enantiomers against Histone Lysine Demethylase 5A.

J Med Chem 2018 04 23;61(7):3193-3208. Epub 2018 Mar 23.

Department of Molecular and Cellular Oncology , The University of Texas MD Anderson Cancer Center , Houston , Texas 77030 , United States.

Isomers of chiral drugs can exhibit marked differences in biological activities. We studied the binding and inhibitory activities of 12 compounds against KDM5A. Among them are two pairs of enantiomers representing two distinct inhibitor chemotypes, namely, ( R)- and ( S)-2-((2-chlorophenyl)(2-(piperidin-1-yl)ethoxy)methyl)-1 H-pyrrolo[3,2- b]pyridine-7-carboxylic acid (compounds N51 and N52) and ( R) - and ( S) -N-(1-(3-isopropyl-1 H-pyrazole-5-carbonyl)pyrrolidin-3-yl)cyclopropanecarboxamide (compounds N54 and N55). In vitro, the S enantiomer of the N51/N52 pair (N52) and the R enantiomer of the N54/N55 pair (N54) exhibited about 4- to 5-fold greater binding affinity. The more potent enzyme inhibition of KDM5A by the R-isoform for the cell-permeable N54/N55 pair translated to differences in growth inhibitory activity. We determined structures of the KDM5A catalytic domain in complex with all 12 inhibitors, which revealed the interactions (or lack thereof) responsible for the differences in binding affinity. These results provide insights to guide improvements in binding potency and avenues for development of cell permeable inhibitors of the KDM5 family.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322411PMC
April 2018

Irreversible inhibition of cytosolic thioredoxin reductase 1 as a mechanistic basis for anticancer therapy.

Sci Transl Med 2018 02;10(428)

Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE 171 77 Stockholm, Sweden.

Cancer cells adapt to their inherently increased oxidative stress through activation of the glutathione (GSH) and thioredoxin (TXN) systems. Inhibition of both of these systems effectively kills cancer cells, but such broad inhibition of antioxidant activity also kills normal cells, which is highly unwanted in a clinical setting. We therefore evaluated targeting of the TXN pathway alone and, more specifically, selective inhibition of the cytosolic selenocysteine-containing enzyme TXN reductase 1 (TXNRD1). TXNRD1 inhibitors were discovered in a large screening effort and displayed increased specificity compared to pan-TXNRD inhibitors, such as auranofin, that also inhibit the mitochondrial enzyme TXNRD2 and additional targets. For our lead compounds, TXNRD1 inhibition correlated with cancer cell cytotoxicity, and inhibitor-triggered conversion of TXNRD1 from an antioxidant to a pro-oxidant enzyme correlated with corresponding increases in cellular production of HO In mice, the most specific TXNRD1 inhibitor, here described as TXNRD1 inhibitor 1 (TRi-1), impaired growth and viability of human tumor xenografts and syngeneic mouse tumors while having little mitochondrial toxicity and being better tolerated than auranofin. These results display the therapeutic anticancer potential of irreversibly targeting cytosolic TXNRD1 using small molecules and present potent and selective TXNRD1 inhibitors. Given the pronounced up-regulation of TXNRD1 in several metastatic malignancies, it seems worthwhile to further explore the potential benefit of specific irreversible TXNRD1 inhibitors for anticancer therapy.
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http://dx.doi.org/10.1126/scitranslmed.aaf7444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7059553PMC
February 2018

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH).

J Med Chem 2017 11 9;60(22):9184-9204. Epub 2017 Nov 9.

National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, Rockville, Maryland 20850, United States.

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as 63 exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug-target residence time was determined via SPR. Analysis of these data suggests that drug-target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5894102PMC
November 2017

Inhibition of 12/15-Lipoxygenase Protects Against β-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes.

Diabetes 2017 11 25;66(11):2875-2887. Epub 2017 Aug 25.

Department of Pediatrics and the Center for Diabetes and Metabolic Diseases, Indiana University School of Medicine, Indianapolis, IN

Islet β-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in β-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate β-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced β-cell oxidative stress, and preservation of β-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced β-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of β-cell dysfunction and glycemic deterioration in models of T1D.
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http://dx.doi.org/10.2337/db17-0215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5652601PMC
November 2017

Parallel Chemistry Approach to Identify Novel Nuclear Receptor Ligands Based on the GW0742 Scaffold.

ACS Comb Sci 2017 10 5;19(10):646-656. Epub 2017 Sep 5.

Department of Chemistry and Biochemistry, Milwaukee Institute for Drug Discovery, University of Wisconsin , Milwaukee, Wisconsin 53211, United States.

We describe the parallel synthesis of novel analogs of GW0742, a peroxisome proliferator-activated receptor δ (PPARδ) agonist. For that purpose, modified reaction conditions were applied, such as a solid-phase palladium-catalyzed Suzuki coupling. In addition, tetrazole-based compounds were generated as a bioisostere for carboxylic acid-containing ligand GW0742. The new compounds were investigated for their ability to activate PPARδ mediated transcription and their cross-reactivity with the vitamin D receptor (VDR), another member of the nuclear receptor superfamily. We identified many potent PPARδ agonists that were less toxic than GW0742, where ∼65 of the compounds synthesized exhibited partial PPARδ activity (23-98%) with EC values ranging from 0.007-18.2 μM. Some ligands, such as compound 32, were more potent inhibitors of VDR-mediated transcription with significantly reduced PPARδ activity than GW0742, however, none of the ligands were completely selective for VDR inhibition over PPARδ activation of transcription.
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http://dx.doi.org/10.1021/acscombsci.7b00066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643073PMC
October 2017

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis.

Arterioscler Thromb Vasc Biol 2017 10 3;37(10):1828-1839. Epub 2017 Aug 3.

From the Department of Pharmacology (R.A., B.E.T., K.M., J.Y., M.H.) and Department of Internal Medicine, Division of Cardiovascular Medicine (M.H.), University of Michigan, Ann Arbor; Chemistry and Biochemistry, University of California Santa Cruz (J.C.F., A.G., T.R.H.); and National Institutes of Health Chemical Genomics Center, National Center for Advancing Translational Sciences, Rockville, MD (D.K.L., A.J., A.S., D.J.M.).

Objective: Adequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis.

Approach And Results: ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model.

Conclusions: Our data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.
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http://dx.doi.org/10.1161/ATVBAHA.117.309868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620123PMC
October 2017

12-Lipoxygenase Inhibitor Improves Functions of Cytokine-Treated Human Islets and Type 2 Diabetic Islets.

J Clin Endocrinol Metab 2017 08;102(8):2789-2797

Department of Internal Medicine, Eastern Virginia Medical School, Norfolk, Virginia 23507.

Context: The 12-lipoxygenase (12-LO) pathway produces proinflammatory metabolites, and its activation is implicated in islet inflammation associated with type 1 and type 2 diabetes (T2D).

Objectives: We aimed to test the efficacy of ML355, a highly selective, small molecule inhibitor of 12-LO, for the preservation of islet function.

Design: Human islets from nondiabetic donors were incubated with a mixture of tumor necrosis factor α , interluekin-1β, and interferon-γ to model islet inflammation. Cytokine-treated islets and human islets from T2D donors were incubated in the presence and absence of ML355.

Setting: In vitro study.

Participants: Human islets from organ donors aged >20 years of both sexes and any race were used. T2D status was defined from either medical history or most recent hemoglobin A1c value >6.5%.

Intervention: Glucose stimulation.

Main Outcome Measures: Static and dynamic insulin secretion and oxygen consumption rate (OCR).

Results: ML355 prevented the reduction of insulin secretion and OCR in cytokine-treated human islets and improved both parameters in human islets from T2D donors.

Conclusions: ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.
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http://dx.doi.org/10.1210/jc.2017-00267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546865PMC
August 2017

A high-throughput small molecule screen identifies synergism between DNA methylation and Aurora kinase pathways for X reactivation.

Proc Natl Acad Sci U S A 2016 12 23;113(50):14366-14371. Epub 2016 Nov 23.

Howard Hughes Medical Institute, Massachusetts General Hospital, Boston, MA 02114;

X-chromosome inactivation is a mechanism of dosage compensation in which one of the two X chromosomes in female mammals is transcriptionally silenced. Once established, silencing of the inactive X (Xi) is robust and difficult to reverse pharmacologically. However, the Xi is a reservoir of >1,000 functional genes that could be potentially tapped to treat X-linked disease. To identify compounds that could reactivate the Xi, here we screened ∼367,000 small molecules in an automated high-content screen using an Xi-linked GFP reporter in mouse fibroblasts. Given the robust nature of silencing, we sensitized the screen by "priming" cells with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5azadC). Compounds that elicited GFP activity include VX680, MLN8237, and 5azadC, which are known to target the Aurora kinase and DNA methylation pathways. We demonstrate that the combinations of VX680 and 5azadC, as well as MLN8237 and 5azadC, synergistically up-regulate genes on the Xi. Thus, our work identifies a synergism between the DNA methylation and Aurora kinase pathways as being one of interest for possible pharmacological reactivation of the Xi.
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http://dx.doi.org/10.1073/pnas.1617597113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5167172PMC
December 2016

A High-Content Assay Enables the Automated Screening and Identification of Small Molecules with Specific ALDH1A1-Inhibitory Activity.

PLoS One 2017 27;12(1):e0170937. Epub 2017 Jan 27.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, United States of America.

Aldehyde dehydrogenase enzymes (ALDHs) have a broad spectrum of biological activities through the oxidation of both endogenous and exogenous aldehydes. Increased expression of ALDH1A1 has been identified in a wide-range of human cancer stem cells and is associated with cancer relapse and poor prognosis, raising the potential of ALDH1A1 as a therapeutic target. To facilitate quantitative high-throughput screening (qHTS) campaigns for the discovery, characterization and structure-activity-relationship (SAR) studies of small molecule ALDH1A1 inhibitors with cellular activity, we show herein the miniaturization to 1536-well format and automation of a high-content cell-based ALDEFLUOR assay. We demonstrate the utility of this assay by generating dose-response curves on a comprehensive set of prior art inhibitors as well as hundreds of ALDH1A1 inhibitors synthesized in house. Finally, we established a screening paradigm using a pair of cell lines with low and high ALDH1A1 expression, respectively, to uncover novel cell-active ALDH1A1-specific inhibitors from a collection of over 1,000 small molecules.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0170937PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5271370PMC
August 2017

A High-Throughput Screen Identifies 2,9-Diazaspiro[5.5]Undecanes as Inducers of the Endoplasmic Reticulum Stress Response with Cytotoxic Activity in 3D Glioma Cell Models.

PLoS One 2016 29;11(8):e0161486. Epub 2016 Aug 29.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, 20850, United States of America.

The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein folding. ER Ca2+ depletion and accumulation of unfolded proteins activate the molecular chaperone GRP78 (glucose-regulated protein 78) which in turn triggers the ER stress response (ERSR) pathway aimed to restore ER homeostasis. Failure to adapt to stress, however, results in apoptosis. We and others have shown that malignant cells are more susceptible to ERSR-induced apoptosis than their normal counterparts, implicating the ERSR as a potential target for cancer therapeutics. Predicated on these findings, we developed an assay that uses a GRP78 biosensor to identify small molecule activators of ERSR in glioma cells. We performed a quantitative high-throughput screen (qHTS) against a collection of ~425,000 compounds and a comprehensive panel of orthogonal secondary assays was formulated for stringent compound validation. We identified novel activators of ERSR, including a compound with a 2,9-diazaspiro[5.5]undecane core, which depletes intracellular Ca2+ stores and induces apoptosis-mediated cell death in several cancer cell lines, including patient-derived and 3D cultures of glioma cells. This study demonstrates that our screening platform enables the identification and profiling of ERSR inducers with cytotoxic activity and advocates for characterization of these compound in in vivo models.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161486PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5003374PMC
August 2017

Structural Basis for KDM5A Histone Lysine Demethylase Inhibition by Diverse Compounds.

Cell Chem Biol 2016 07 14;23(7):769-781. Epub 2016 Jul 14.

Department of Biochemistry, Emory University, Atlanta, GA 30322, USA; The Winship Cancer Institute, Emory University, Atlanta, GA 30322, USA. Electronic address:

The KDM5/JARID1 family of Fe(II)- and α-ketoglutarate-dependent demethylases removes methyl groups from methylated lysine 4 of histone H3. Accumulating evidence supports a role for KDM5 family members as oncogenic drivers. We compare the in vitro inhibitory properties and binding affinity of ten diverse compounds with all four family members, and present the crystal structures of the KDM5A-linked Jumonji domain in complex with eight of these inhibitors in the presence of Mn(II). All eight inhibitors structurally examined occupy the binding site of α-ketoglutarate, but differ in their specific binding interactions, including the number of ligands involved in metal coordination. We also observed inhibitor-induced conformational changes in KDM5A, particularly those residues involved in the binding of α-ketoglutarate, the anticipated peptide substrate, and intramolecular interactions. We discuss how particular chemical moieties contribute to inhibitor potency and suggest strategies that might be utilized in the successful design of selective and potent epigenetic inhibitors.
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http://dx.doi.org/10.1016/j.chembiol.2016.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4958579PMC
July 2016