Publications by authors named "Ajit Jadhav"

167 Publications

FEN1 Blockade for Platinum Chemo-Sensitization and Synthetic Lethality in Epithelial Ovarian Cancers.

Cancers (Basel) 2021 Apr 14;13(8). Epub 2021 Apr 14.

Translational Oncology, Division of Cancer and Stem Cells, School of Medicine, University of Nottingham Biodiscovery Institute, Nottingham NG7 2RD, UK.

FEN1 plays critical roles in long patch base excision repair (LP-BER), Okazaki fragment maturation, and rescue of stalled replication forks. In a clinical cohort, FEN1 overexpression is associated with aggressive phenotype and poor progression-free survival after platinum chemotherapy. Pre-clinically, FEN1 is induced upon cisplatin treatment, and nuclear translocation of FEN1 is dependent on physical interaction with importin β. FEN1 depletion, gene inactivation, or inhibition re-sensitizes platinum-resistant ovarian cancer cells to cisplatin. BRCA2 deficient cells exhibited synthetic lethality upon treatment with a FEN1 inhibitor. FEN1 inhibitor-resistant PEO1R cells were generated, and these reactivated BRCA2 and overexpressed the key repair proteins, POLβ and XRCC1. FEN1i treatment was selectively toxic to POLβ deficient but not XRCC1 deficient ovarian cancer cells. High throughput screening of 391,275 compounds identified several FEN1 inhibitor hits that are suitable for further drug development. We conclude that FEN1 is a valid target for ovarian cancer therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13081866DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070745PMC
April 2021

A target-agnostic screen identifies approved drugs to stabilize the endoplasmic reticulum-resident proteome.

Cell Rep 2021 Apr;35(4):109040

National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA. Electronic address:

Endoplasmic reticulum (ER) dysregulation is associated with pathologies including neurodegenerative, muscular, and diabetic conditions. Depletion of ER calcium can lead to the loss of resident proteins in a process termed exodosis. To identify compounds that attenuate the redistribution of ER proteins under pathological conditions, we performed a quantitative high-throughput screen using the Gaussia luciferase (GLuc)-secreted ER calcium modulated protein (SERCaMP) assay, which monitors secretion of ER-resident proteins triggered by calcium depletion. We identify several clinically used drugs, including bromocriptine, and further characterize them using assays to measure effects on ER calcium, ER stress, and ER exodosis. Bromocriptine elicits protective effects in cell-based models of exodosis as well as in vivo models of stroke and diabetes. Bromocriptine analogs with reduced dopamine receptor activity retain similar efficacy in stabilizing the ER proteome, indicating a non-canonical mechanism of action. This study describes a strategic approach to identify small-molecule drugs capable of improving ER proteostasis in human disease conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2021.109040DOI Listing
April 2021

Discovery and Optimization of 2-1λ-Pyridin-2-one Inhibitors of Mutant Isocitrate Dehydrogenase 1 for the Treatment of Cancer.

J Med Chem 2021 Apr 6;64(8):4913-4946. Epub 2021 Apr 6.

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

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are oncogenic for a number of malignancies, primarily low-grade gliomas and acute myeloid leukemia. We report a medicinal chemistry campaign around a 7,7-dimethyl-7,8-dihydro-2-1λ-quinoline-2,5(6)-dione screening hit against the R132H and R132C mutant forms of isocitrate dehydrogenase (IDH1). Systematic SAR efforts produced a series of potent pyrid-2-one mIDH1 inhibitors, including the atropisomer (-, ). In an engineered mIDH1-U87-xenograft mouse model, after a single oral dose of 30 mg/kg, 16 h post dose, between 16 and 48 h, showed higher tumoral concentrations that corresponded to lower 2-HG concentrations, when compared with the approved drug AG-120 (ivosidenib).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.1c00019DOI Listing
April 2021

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2021.127974DOI Listing
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-81486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822874PMC
January 2021

Therapeutic candidates for the Zika virus identified by a high-throughput screen for Zika protease inhibitors.

Proc Natl Acad Sci U S A 2020 12 23;117(49):31365-31375. Epub 2020 Nov 23.

National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892;

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.2005463117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733812PMC
December 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830743PMC
October 2020

Optimization of High-Throughput Methyltransferase Assays for the Discovery of Small Molecule Inhibitors.

ACS Comb Sci 2020 08 27;22(8):422-432. Epub 2020 Jun 27.

Department of Medicinal Chemistry and Molecular Pharmacology, Center for Cancer Research, Institute for Drug Discovery, Purdue University, West Lafayette, Indiana 47907, United States.

Methyltransferases (MTases) play diverse roles in cellular processes. Aberrant methylation levels have been implicated in many diseases, indicating the need for the identification and development of small molecule inhibitors for each MTase. Specific inhibitors can serve as probes to investigate the function and validate therapeutic potential for the respective MTase. High-throughput screening (HTS) is a powerful method to identify initial hits for further optimization. Here, we report the development of a fluorescence-based MTase assay and compare this format with the recently developed MTase-Glo luminescence assay for application in HTS. Using protein N-terminal methyltransferase 1 (NTMT1) as a model system, we miniaturized to 1536-well quantitative HTS format. Through a pilot screen of 1428 pharmacologically active compounds and subsequent validation, we discovered that MTase-Glo produced lower false positive rates than the fluorescence-based MTase assay. Nevertheless, both assays displayed robust performance along with low reagent requirements and can potentially be employed as general HTS formats for the discovery of inhibitors for any MTase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscombsci.0c00077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7429283PMC
August 2020

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma.

Sci Rep 2020 03 30;10(1):5688. Epub 2020 Mar 30.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAF mutant melanoma cell line, SB-3123 which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123 melanoma cells by Pmel-1 CD8 T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-62369-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105471PMC
March 2020

Endonuclease FEN1 Coregulates ERα Activity and Provides a Novel Drug Interface in Tamoxifen-Resistant Breast Cancer.

Cancer Res 2020 05 19;80(10):1914-1926. Epub 2020 Mar 19.

Division of Oncogenomics, The Netherlands Cancer Institute, Amsterdam, the Netherlands.

Estrogen receptor α (ERα) is a key transcriptional regulator in the majority of breast cancers. ERα-positive patients are frequently treated with tamoxifen, but resistance is common. In this study, we refined a previously identified 111-gene outcome prediction-classifier, revealing FEN1 as the strongest determining factor in ERα-positive patient prognostication. FEN1 levels were predictive of outcome in tamoxifen-treated patients, and FEN1 played a causal role in ERα-driven cell growth. FEN1 impacted the transcriptional activity of ERα by facilitating coactivator recruitment to the ERα transcriptional complex. FEN1 blockade induced proteasome-mediated degradation of activated ERα, resulting in loss of ERα-driven gene expression and eradicated tumor cell proliferation. Finally, a high-throughput 465,195 compound screen identified a novel FEN1 inhibitor, which effectively blocked ERα function and inhibited proliferation of tamoxifen-resistant cell lines as well as -cultured ERα-positive breast tumors. Collectively, these results provide therapeutic proof of principle for FEN1 blockade in tamoxifen-resistant breast cancer. SIGNIFICANCE: These findings show that pharmacologic inhibition of FEN1, which is predictive of outcome in tamoxifen-treated patients, effectively blocks ERα function and inhibits proliferation of tamoxifen-resistant tumor cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-19-2207DOI Listing
May 2020

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells.

Sci Rep 2020 03 6;10(1):4203. Epub 2020 Mar 6.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-61082-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060218PMC
March 2020

Anxiolytic Drug FGIN-1-27 Ameliorates Autoimmunity by Metabolic Reprogramming of Pathogenic Th17 Cells.

Sci Rep 2020 02 28;10(1):3766. Epub 2020 Feb 28.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, MD, USA.

Th17 cells are critical drivers of autoimmune diseases and immunopathology. There is an unmet need to develop therapies targeting pathogenic Th17 cells for the treatment of autoimmune disorders. Here, we report that anxiolytic FGIN-1-27 inhibits differentiation and pathogenicity of Th17 cells in vitro and in vivo using the experimental autoimmune encephalomyelitis (EAE) model of Th17 cell-driven pathology. Remarkably, we found that the effects of FGIN-1-27 were independent of translocator protein (TSPO), the reported target for this small molecule, and instead were driven by a metabolic switch in Th17 cells that led to the induction of the amino acid starvation response and altered cellular fatty acid composition. Our findings suggest that the small molecule FGIN-1-27 can be re-purposed to relieve autoimmunity by metabolic reprogramming of pathogenic Th17 cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-60610-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048748PMC
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.01.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7039685PMC
February 2020

Characterization of Lead Compounds Targeting the Selenoprotein Thioredoxin Glutathione Reductase for Treatment of Schistosomiasis.

ACS Infect Dis 2020 03 24;6(3):393-405. Epub 2020 Jan 24.

Department of Microbial Pathogens and Immunity, Rush University Medical Center, Chicago, Illinois 60612, United States.

Schistosomiasis is a widespread human parasitic disease currently affecting over 200 million people. Chemotherapy for schistosomiasis relies exclusively on praziquantel. Although significant advances have been made in recent years to reduce the incidence and intensity of schistosome infections, these gains will be at risk should drug-resistant parasites evolve. Thioredoxin glutathione reductase (TGR) is a selenoprotein of the parasite essential for the survival of schistosomes in the mammalian host. Several high-throughput screening campaigns have identified inhibitors of TGR. Follow up analyses of select active compounds form the basis of the present study. We identified eight compounds effective against worms. Compounds - are active against all major species and development stages. The ability of these compounds to target immature worms is especially critical because praziquantel is poorly active against this stage. Compounds -, , and displayed schistosomicidal activity even after only 1 h incubation with the worms. Compounds - meet or exceed standards set by the World Health Organization for leads for schistosomiasis therapy activity. The mechanism of TGR inhibition was studied further with wild-type and mutant TGR proteins. Compounds - were found to induce an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in TGR, leading to the production of superoxide and hydrogen peroxide. Collectively, this effort has identified several active compound series that may serve as the basis for the development of new schistosomicidal compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsinfecdis.9b00354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072008PMC
March 2020

A Comparative Study of Target Engagement Assays for HDAC1 Inhibitor Profiling.

SLAS Discov 2020 03 29;25(3):253-264. Epub 2019 Oct 29.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.

Histone deacetylases (HDACs) are epigenetic modulators linked to diseases including cancer and neurodegeneration. Given their therapeutic potential, highly sensitive biochemical and cell-based profiling technologies have been developed to discover small-molecule HDAC inhibitors. Ultimately, the therapeutic action of these inhibitors is dependent on a physical engagement with their intended targets in cellular and tissue environments. Confirming target engagement in the cellular environment is particularly relevant for HDACs since they function as part of cell type-specific multiprotein complexes. Here we implemented two recently developed high-throughput target engagement technologies, NanoBRET and SplitLuc CETSA, to profile 349 compounds in the Epigenetic-Focused collection for HDAC1 binding. We found that the two HDAC1 target engagement assays correlated well with each other and with orthogonal activity-based assays, in particular those carried out in cellular environments rather than with isolated HDAC proteins. The assays detected a majority of the previously described HDAC1 inhibitors in the collection and, importantly, triaged HDAC inhibitors known to target other HDACs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2472555219883625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848789PMC
March 2020

A novel P300 inhibitor reverses DUX4-mediated global histone H3 hyperacetylation, target gene expression, and cell death.

Sci Adv 2019 09 11;5(9):eaaw7781. Epub 2019 Sep 11.

Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.

Facioscapulohumeral muscular dystrophy (FSHD) results from mutations causing overexpression of the transcription factor, DUX4, which interacts with the histone acetyltransferases, EP300 and CBP. We describe the activity of a new spirocyclic EP300/CBP inhibitor, iP300w, which inhibits the cytotoxicity of the DUX4 protein and reverses the overexpression of most DUX4 target genes, in engineered cell lines and FSHD myoblasts, as well as in an FSHD animal model. In evaluating the effect of iP300w on global histone H3 acetylation, we discovered that DUX4 overexpression leads to a dramatic global increase in the total amount of acetylated histone H3. This unexpected effect requires the C-terminus of DUX4, is conserved with mouse Dux, and may facilitate zygotic genome activation. This global increase in histone H3 acetylation is reversed by iP300w, highlighting the central role of EP300 and CBP in the transcriptional mechanism underlying DUX4 cytotoxicity and the translational potential of blocking this interaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aaw7781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739093PMC
September 2019

Scaffold-Based Analytics: Enabling Hit-to-Lead Decisions by Visualizing Chemical Series Linked across Large Datasets.

J Chem Inf Model 2019 11 29;59(11):4880-4892. Epub 2019 Oct 29.

National Center for Advancing Translational Science , 9800 Medical Center Drive , Rockville , Maryland 20850 , United States.

We present a method for visualizing and navigating large screening datasets while also taking into account their activities and properties. Our approach is to annotate the data with all possible scaffolds contained within each molecule. We have developed a Spotfire visualization, coupled to a fuzzy clustering approach based on the scaffold decomposition of the screening deck, used to drive the hit triage process. Progression decisions can be made using aggregate scaffold parameters and data from multiple datasets merged at the scaffold level. This visualization reveals overlaps that help prioritize hits, highlight tractable series, and posit ways to combine aspects of multiple hits. The structure-activity relationship of a large and complex hit is automatically mapped onto all constituent scaffolds making it possible to navigate, via any shared scaffold, to all related hits. This scaffold "walking" helps address bias toward a handful of potent and ligand-efficient molecules at the expense of coverage of chemical space. We consider two scaffold generation methods and explored their similarities and differences both qualitatively and quantitatively. The workflow of a Spotfire visualization used in combination with fuzzy clustering and structure annotation provides an intuitive view of large and diverse screening datasets. This allows teams to effortlessly navigate between structurally related molecules and enriches the population of leads considered and progressed in a manner complementary to established approaches.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jcim.9b00243DOI Listing
November 2019

Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening.

SLAS Discov 2020 01 14;25(1):43-56. Epub 2019 Sep 14.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA.

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a "tough target" to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2472555219873559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340117PMC
January 2020

The NCATS BioPlanet - An Integrated Platform for Exploring the Universe of Cellular Signaling Pathways for Toxicology, Systems Biology, and Chemical Genomics.

Front Pharmacol 2019 26;10:445. Epub 2019 Apr 26.

Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, United States.

Chemical genomics aims to comprehensively define, and ultimately predict, the effects of small molecule compounds on biological systems. Chemical activity profiling approaches must consider chemical effects on all pathways operative in mammalian cells. To enable a strategic and maximally efficient chemical profiling of pathway space, we have created the NCATS BioPlanet, a comprehensive integrated pathway resource that incorporates the universe of 1,658 human pathways sourced from publicly available, manually curated sources, which have been subjected to thorough redundancy and consistency cross-evaluation. BioPlanet supports interactive browsing, retrieval, and analysis of pathways, exploration of pathway connections, and pathway search by gene targets, category, and availability of corresponding bioactivity assay, as well as visualization of pathways on a 3-dimensional globe, in which the distance between any two pathways is proportional to their degree of gene component overlap. Using this resource, we propose a strategy to identify a minimal set of 362 biological assays that can interrogate the universe of human pathways. The NCATS BioPlanet is a public resource, which will be continually expanded and updated, for systems biology, toxicology, and chemical genomics, available at http://tripod.nih.gov/bioplanet/.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphar.2019.00445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6524730PMC
April 2019

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2019.03.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405260PMC
May 2019

Canvass: A Crowd-Sourced, Natural-Product Screening Library for Exploring Biological Space.

ACS Cent Sci 2018 Dec 5;4(12):1727-1741. Epub 2018 Dec 5.

Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington, Indiana 47405, United States.

Natural products and their derivatives continue to be wellsprings of nascent therapeutic potential. However, many laboratories have limited resources for biological evaluation, leaving their previously isolated or synthesized compounds largely or completely untested. To address this issue, the Canvass library of natural products was assembled, in collaboration with academic and industry researchers, for quantitative high-throughput screening (qHTS) across a diverse set of cell-based and biochemical assays. Characterization of the library in terms of physicochemical properties, structural diversity, and similarity to compounds in publicly available libraries indicates that the Canvass library contains many structural elements in common with approved drugs. The assay data generated were analyzed using a variety of quality control metrics, and the resultant assay profiles were explored using statistical methods, such as clustering and compound promiscuity analyses. Individual compounds were then sorted by structural class and activity profiles. Differential behavior based on these classifications, as well as noteworthy activities, are outlined herein. One such highlight is the activity of (-)-2()-cathafoline, which was found to stabilize calcium levels in the endoplasmic reticulum. The workflow described here illustrates a pilot effort to broadly survey the biological potential of natural products by utilizing the power of automation and high-throughput screening.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscentsci.8b00747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311695PMC
December 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2018.10.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310147PMC
December 2018

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/j.pain.0000000000001373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237621PMC
December 2018

Identifying hotspots in land use land cover change and the drivers in a semi-arid region of India.

Environ Monit Assess 2018 Aug 20;190(9):535. Epub 2018 Aug 20.

Centre for Resilience Studies, Watershed Organisation Trust, Pune, 411009, India.

The study examines long-term land use/land cover change (LUCC) at a finer scale in a semi-arid region of India. The objectives were to study and quantify the spatiotemporal LUCC and uncover the major drivers causing the change in the Mula Pravara river basin, which is located in a semi-arid region of Maharashtra state, India. Advanced very high-resolution radiometer (AVHRR)-Normalized Difference Vegetation Index (NDVI) 3g data for the years 1982-2015 were used to identify the 'hotspot' with significant positive and negative LUCC. Multi-temporal Landsat imagery was used to produce finer scale land use maps. From 1991 to 2016, the agricultural land area increased by approximately 98% due to the conversion of uncultivable and fallow lands to agriculture. The built-up area increased by 195%, and in recent years, an urban expansion has occurred in agricultural lands close to the urban fringe areas. There has been a shift from food crops to commercial crops, as observed from the steep increase in the amount of land under horticultural plantations, by 1601% from 2001 to 2016. In addition, the area under forest canopy was reduced in the protected regions. Institutional factors that improved access to water resources were the major drivers of change in hotspots, especially in the context of agriculture. Technological and economic factors were the other supporting factors that contributed to the change. This study demonstrates the advantages of using satellite remote sensing techniques to monitor the LUCC, which is useful for predicting future land changes and aids in planning adaptation strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10661-018-6919-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105204PMC
August 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2006134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095604PMC
August 2018