Publications by authors named "William P Janzen"

49 Publications

Prosocial effects of an oxytocin metabolite, but not synthetic oxytocin receptor agonists, in a mouse model of autism.

Neuropharmacology 2019 01 3;144:301-311. Epub 2018 Nov 3.

Carolina Institute for Developmental Disabilities, University of North Carolina School of Medicine, Chapel Hill, NC, 27599, USA; Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, 27599, USA. Electronic address:

Currently, there are no established pharmaceutical strategies that effectively treat social deficits in autism spectrum disorder (ASD). Oxytocin, a neurohormone that plays a role in multiple types of social behaviors, has been proposed as a possible therapeutic against social impairment and other symptoms in ASD. However, from the standpoint of pharmacotherapy, oxytocin has several liabilities as a standard clinical treatment, including rapid metabolism, low brain penetrance, and activity at the vasopressin (antidiuretic hormone) receptors. The present studies describe findings from a preclinical screening program to evaluate oxytocin receptor (OXTR) agonists and oxytocin metabolites for potential clinical use as more optimal treatments. We first investigated two synthetic oxytocin analogs, TC-OT-39 and carbetocin, using in vitro cell-based assays for pharmacological characterization and behavioral tests in the BALB/cByJ mouse model of ASD-like social deficits. Although both TC-OT-39 and carbetocin selectively activate the OXTR, neither synthetic agonist had prosocial efficacy in the BALB/cByJ model. We next evaluated two oxytocin metabolites: OT(4-9) and OT(5-9). While OT(5-9) failed to affect social deficits, the metabolite OT(4-9) led to significant social preference in the BALB/cByJ model, in a dose-dependent manner. The increased sociability was observed at both 24 h and 12 days following the end of a subchronic regimen with OT(4-9) (2.0 mg/kg). Overall, these results suggest that the prosocial effects of oxytocin could be mediated by downstream activity of oxytocin metabolites, raising the possibility of new pathways to target for drug discovery relevant to ASD.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384330PMC
January 2019

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

PLoS One 2018 1;13(6):e0197372. Epub 2018 Jun 1.

Epizyme, Inc., Cambridge, Massachusetts, United States of America.

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197372PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983452PMC
December 2018

Identification of a peptide inhibitor for the histone methyltransferase WHSC1.

PLoS One 2018 9;13(5):e0197082. Epub 2018 May 9.

Epizyme Inc., Cambridge, Massachusetts, United States of America.

WHSC1 is a histone methyltransferase that is responsible for mono- and dimethylation of lysine 36 on histone H3 and has been implicated as a driver in a variety of hematological and solid tumors. Currently, there is a complete lack of validated chemical matter for this important drug discovery target. Herein we report on the first fully validated WHSC1 inhibitor, PTD2, a norleucine-containing peptide derived from the histone H4 sequence. This peptide exhibits micromolar affinity towards WHSC1 in biochemical and biophysical assays. Furthermore, a crystal structure was solved with the peptide in complex with SAM and the SET domain of WHSC1L1. This inhibitor is an important first step in creating potent, selective WHSC1 tool compounds for the purposes of understanding the complex biology in relation to human disease.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197082PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5942779PMC
August 2018

Quantitative high-throughput phenotypic screening of pediatric cancer cell lines identifies multiple opportunities for drug repurposing.

Oncotarget 2018 Jan 19;9(4):4758-4772. Epub 2017 Dec 19.

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

Drug repurposing approaches have the potential advantage of facilitating rapid and cost-effective development of new therapies. Particularly, the repurposing of drugs with known safety profiles in children could bypass or streamline toxicity studies. We employed a phenotypic screening paradigm on a panel of well-characterized cell lines derived from pediatric solid tumors against a collection of ∼3,800 compounds spanning approved drugs and investigational agents. Specifically, we employed titration-based screening where compounds were tested at multiple concentrations for their effect on cell viability. Molecular and cellular target enrichment analysis indicated that numerous agents across different therapeutic categories and modes of action had an antiproliferative effect, notably antiparasitic/protozoal drugs with non-classic antineoplastic activity. Focusing on active compounds with dosing and safety information in children according to the Children's Pharmacy Collaborative database, we identified compounds with therapeutic potential through further validation using 3D tumor spheroid models. Moreover, we show that antiparasitic agents induce cell death apoptosis induction. This study demonstrates that our screening platform enables the identification of chemical agents with cytotoxic activity in pediatric cancer cell lines of which many have known safety/toxicity profiles in children. These agents constitute attractive candidates for efficacy studies in pre-clinical models of pediatric solid tumors.
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http://dx.doi.org/10.18632/oncotarget.23462DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5797010PMC
January 2018

High-throughput small molecule screen identifies inhibitors of aberrant chromatin accessibility.

Proc Natl Acad Sci U S A 2016 Mar 29;113(11):3018-23. Epub 2016 Feb 29.

Department of Genetics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Carolina Center for Genome Sciences, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

Mutations in chromatin-modifying proteins and transcription factors are commonly associated with a wide variety of cancers. Through gain- or loss-of-function, these mutations may result in characteristic alterations of accessible chromatin, indicative of shifts in the landscape of regulatory elements genome-wide. The identification of compounds that reverse a specific chromatin signature could lead to chemical probes or potential therapies. To explore whether chromatin accessibility could serve as a platform for small molecule screening, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE), a chemical method to enrich for nucleosome-depleted genomic regions, as a high-throughput, automated assay. After demonstrating the validity and robustness of this approach, we applied this method to screen an epigenetically targeted small molecule library by evaluating regions of aberrant nucleosome depletion mediated by EWSR1-FLI1, the chimeric transcription factor critical for the bone and soft tissue tumor Ewing sarcoma. As a class, histone deacetylase inhibitors were greatly overrepresented among active compounds. These compounds resulted in diminished accessibility at targeted sites by disrupting transcription of EWSR1-FLI1. Capitalizing on precise differences in chromatin accessibility for drug discovery efforts offers significant advantages because it does not depend on the a priori selection of a single molecular target and may detect novel biologically relevant pathways.
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http://dx.doi.org/10.1073/pnas.1521827113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801272PMC
March 2016

Towards a hit for every target.

Nat Rev Drug Discov 2016 01 20;15(1):1-2. Epub 2015 Nov 20.

AstraZeneca, Cambridge Science Park, 310 Milton Road, Cambridge CB4 0FZ, UK.

Technological advances coupled with novel collaborative strategies for compound sourcing and management are poised to transform the utility of high-throughput screening.
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http://dx.doi.org/10.1038/nrd.2015.19DOI Listing
January 2016

Cavitation Enhancing Nanodroplets Mediate Efficient DNA Fragmentation in a Bench Top Ultrasonic Water Bath.

PLoS One 2015 17;10(7):e0133014. Epub 2015 Jul 17.

Joint Department of Biomedical Engineering, University of North Carolina, Chapel Hill, North Carolina, and North Carolina State University, Raleigh, North Carolina, United States of America.

A perfluorocarbon nanodroplet formulation is shown to be an effective cavitation enhancement agent, enabling rapid and consistent fragmentation of genomic DNA in a standard ultrasonic water bath. This nanodroplet-enhanced method produces genomic DNA libraries and next-generation sequencing results indistinguishable from DNA samples fragmented in dedicated commercial acoustic sonication equipment, and with higher throughput. This technique thus enables widespread access to fast bench-top genomic DNA fragmentation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0133014PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505845PMC
May 2016

Identification of a fragment-like small molecule ligand for the methyl-lysine binding protein, 53BP1.

ACS Chem Biol 2015 Apr 28;10(4):1072-81. Epub 2015 Jan 28.

†Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

Improving our understanding of the role of chromatin regulators in the initiation, development, and suppression of cancer and other devastating diseases is critical, as they are integral players in regulating DNA integrity and gene expression. Developing small molecule inhibitors for this target class with cellular activity is a crucial step toward elucidating their specific functions. We specifically targeted the DNA damage response protein, 53BP1, which uses its tandem tudor domain to recognize histone H4 dimethylated on lysine 20 (H4K20me2), a modification related to double-strand DNA breaks. Through a cross-screening approach, we identified UNC2170 (1) as a micromolar ligand of 53BP1, which demonstrates at least 17-fold selectivity for 53BP1 as compared to other methyl-lysine (Kme) binding proteins tested. Structural studies revealed that the tert-butyl amine of UNC2170 anchors the compound in the methyl-lysine (Kme) binding pocket of 53BP1, making it competitive with endogenous Kme substrates. X-ray crystallography also demonstrated that UNC2170 binds at the interface of two tudor domains of a 53BP1 dimer. Importantly, this compound functions as a 53BP1 antagonist in cellular lysates and shows cellular activity by suppressing class switch recombination, a process which requires a functional 53BP1 tudor domain. These results demonstrate that UNC2170 is a functionally active, fragment-like ligand for 53BP1.
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http://dx.doi.org/10.1021/cb500956gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4402254PMC
April 2015

Expression of Ror2 mediates invasive phenotypes in renal cell carcinoma.

PLoS One 2014 26;9(12):e116101. Epub 2014 Dec 26.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America; Departments of Medicine and Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599, United States of America.

Ror2 is a Wnt ligand receptor that is overexpressed in a variety of tumors including clear cell renal cell carcinoma (ccRCC). Here we demonstrate that expression of wild type Ror2 results in increased tumorigenic properties in in vitro cell culture and in vivo xenograft models. In addition, Ror2 expression produced positive changes in both cell migration and invasion, which were dependent on matrix metalloprotease 2 (MMP2) activity. Mutations in key regions of the kinase domain of Ror2 resulted in the abrogation of increased tumor growth, cell migration, and cell invasion observed with expression of wild-type Ror2. Finally, we examined Ror2 expression as a prognostic biomarker for ccRCC utilizing the TCGA ccRCC dataset. High expression of Ror2 showed a significant correlation with higher clinical stage, nuclear grade, and tumor stage. Furthermore, high expression of Ror2 in ccRCC patients correlated with significant lower overall survival, cancer specific survival, and recurrence free survival. Together, these findings suggest that Ror2 plays a central role in influencing the ccRCC phenotype, and can be considered as a negative prognostic biomarker and potential therapeutic target in this cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116101PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277431PMC
November 2015

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C.

J Biomol Screen 2015 Jun 22;20(5):655-62. Epub 2014 Dec 22.

Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Center for Integrative Chemical Biology and Drug Discovery, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z' values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.
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http://dx.doi.org/10.1177/1087057114564057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610143PMC
June 2015

Screening technologies for small molecule discovery: the state of the art.

Authors:
William P Janzen

Chem Biol 2014 Sep;21(9):1162-70

Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Screening, high-throughput screening, and ultra-high-throughput screening are all really just points on a spectrum that represent differing applications of the same process: the creation of biologically relevant assays that are relevant, reproducible, reliable, and robust. Whether the discovery program is developing a pharmaceutical, an academic probe, cosmetics, pesticides, or a toxicity monitoring assay, the development of a screen focuses on generating a method that will reliably deliver reproducible results over a period of weeks, months, or years and that will generate consistent results for every test along the way. This review provides both historical perspective on how this unique scientific discipline evolved and commentary on the current state of the art technologies and techniques.
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http://dx.doi.org/10.1016/j.chembiol.2014.07.015DOI Listing
September 2014

Expanding the scope of drug repurposing in pediatrics: the Children's Pharmacy Collaborative.

Drug Discov Today 2014 Nov 19;19(11):1696-1698. Epub 2014 Aug 19.

Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA; Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.

Drug repurposing is the use of 'old' drugs for new indications, avoiding the need for time- and cost-intensive toxicity studies. This approach should be particularly attractive for pediatrics, but its use in this population has been limited. One obstacle has been the lack of a comprehensive database of drugs for which there already is at least one indication in children. We describe the development of The Children's Pharmacy Collaborative, which should grow over time, serve as a resource for professionals and families, and stimulate drug-repurposing efforts for a range of pediatric disorders.
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http://dx.doi.org/10.1016/j.drudis.2014.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4578872PMC
November 2014

UNC2025, a potent and orally bioavailable MER/FLT3 dual inhibitor.

J Med Chem 2014 Aug 6;57(16):7031-41. Epub 2014 Aug 6.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, ‡Department of Pharmacology, School of Medicine, and §Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

We previously reported a potent small molecule Mer tyrosine kinase inhibitor UNC1062. However, its poor PK properties prevented further assessment in vivo. We report here the sequential modification of UNC1062 to address DMPK properties and yield a new potent and highly orally bioavailable Mer inhibitor, 11, capable of inhibiting Mer phosphorylation in vivo, following oral dosing as demonstrated by pharmaco-dynamic (PD) studies examining phospho-Mer in leukemic blasts from mouse bone marrow. Kinome profiling versus more than 300 kinases in vitro and cellular selectivity assessments demonstrate that 11 has similar subnanomolar activity against Flt3, an additional important target in acute myelogenous leukemia (AML), with pharmacologically useful selectivity versus other kinases examined.
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http://dx.doi.org/10.1021/jm500749dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4148167PMC
August 2014

Discovery of a selective, substrate-competitive inhibitor of the lysine methyltransferase SETD8.

J Med Chem 2014 Aug 25;57(15):6822-33. Epub 2014 Jul 25.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡Department of Pharmacology, School of Medicine, §Lineberger Comprehensive Cancer Center, and ∥Department of Biochemistry and Biophysics, UNC Macromolecular Interactions Facility, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

The lysine methyltransferase SETD8 is the only known methyltransferase that catalyzes monomethylation of histone H4 lysine 20 (H4K20). Monomethylation of H4K20 has been implicated in regulating diverse biological processes including the DNA damage response. In addition to H4K20, SETD8 monomethylates non-histone substrates including proliferating cell nuclear antigen (PCNA) and promotes carcinogenesis by deregulating PCNA expression. However, selective inhibitors of SETD8 are scarce. The only known selective inhibitor of SETD8 to date is nahuoic acid A, a marine natural product, which is competitive with the cofactor. Here, we report the discovery of the first substrate-competitive inhibitor of SETD8, UNC0379 (1). This small-molecule inhibitor is active in multiple biochemical assays. Its affinity to SETD8 was confirmed by ITC (isothermal titration calorimetry) and SPR (surface plasmon resonance) studies. Importantly, compound 1 is selective for SETD8 over 15 other methyltransferases. We also describe structure-activity relationships (SAR) of this series.
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http://dx.doi.org/10.1021/jm500871sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136711PMC
August 2014

The lipid kinase PIP5K1C regulates pain signaling and sensitization.

Neuron 2014 May;82(4):836-47

Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Cell Biology and Physiology, UNC Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. Electronic address:

Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c(+/-) mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2-dependent nociceptive signaling and suggest that PIP5K1C is a therapeutic target for chronic pain.
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http://dx.doi.org/10.1016/j.neuron.2014.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4074510PMC
May 2014

Filtration improves the performance of a high-throughput screen for anti-mycobacterial compounds.

PLoS One 2014 2;9(5):e96348. Epub 2014 May 2.

Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, United States of America; Cancer Genetics Program, Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.

The tendency for mycobacteria to aggregate poses a challenge for their use in microplate based assays. Good dispersions have been difficult to achieve in high-throughput screening (HTS) assays used in the search for novel antibacterial drugs to treat tuberculosis and other related diseases. Here we describe a method using filtration to overcome the problem of variability resulting from aggregation of mycobacteria. This method consistently yielded higher reproducibility and lower variability than conventional methods, such as settling under gravity and vortexing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096348PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008622PMC
June 2015

The structure-activity relationships of L3MBTL3 inhibitors: flexibility of the dimer interface.

Medchemcomm 2013 Nov;4(11):1501-1507

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Eshelman School of Pharmacy University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. ; Tel: (919) 843-5486.

We recently reported the discovery of UNC1215, a potent and selective chemical probe for the L3MBTL3 methyllysine reader domain. In this article, we describe the development of structure-activity relationships (SAR) of a second series of potent L3MBTL3 antagonists which evolved from the structure of the chemical probe UNC1215. These compounds are selective for L3MBTL3 against a panel of methyllysine reader proteins, particularly the related MBT family proteins, L3MBTL1 and MBTD1. A co-crystal structure of L3MBTL3 and one of the most potent compounds suggests that the L3MBTL3 dimer rotates about the dimer interface to accommodate ligand binding.
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http://dx.doi.org/10.1039/C3MD00197KDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3897169PMC
November 2013

Development of a Novel Screening Strategy Designed to Discover a New Class of HIV Drugs.

J Lab Autom 2014 Jun 4;19(3):297-303. Epub 2013 Dec 4.

Center for Integrative Chemical Biology and Drug Discovery, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, NC, USA Cancer Genetics Program, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, NC, USA

Current antiretroviral treatments target multiple pathways important for human immunodeficiency virus (HIV) multiplication, including viral entry, synthesis and integration of the DNA provirus, and the processing of viral polyprotein precursors. However, HIV is becoming increasingly resistant to these "combination therapies." Recent findings show that inhibition of HIV Gag protein cleavage into its two structural proteins, matrix (MA) and capsid (CA), has a devastating effect on viral production, revealing a potential new target class for HIV treatment. Unlike the widely used HIV protease inhibitors, this new class of inhibitor would target the substrate, not the protease enzyme itself. This approach offers a distinct advantage in that inhibitors of MA/CA would only need to affect a subset of the Gag molecules to disable viral replication. To discover MA/CA-specific inhibitors, we constructed a modified MA/CA fusion peptide (MA/CAΔ) that contains the HIV protease (PR) cleavage site as well as a tetracysteine motif for fluorescent labeling. The HIV PR cleavage of MA/CAΔ can then be monitored via fluorescence polarization (FP). We have adapted this FP assay for high-throughput screening and validated it according to industry standards using a 384-well plate format. We have currently tested 24,000 compounds in this assay and here detail the screening methodology and the results of this screening campaign.
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http://dx.doi.org/10.1177/2211068213513453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4216240PMC
June 2014

Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis.

J Med Chem 2013 Dec 20;56(23):9693-700. Epub 2013 Nov 20.

Center for Integrative Chemical Biology and Drug Discovery ‡Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy §Department of Pharmacology ∥Lineberger Comprehensive Cancer Center, Department of Medicine, School of Medicine, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.
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http://dx.doi.org/10.1021/jm4013888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962266PMC
December 2013

Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new Mer kinase inhibitors.

J Med Chem 2013 Dec 20;56(23):9683-92. Epub 2013 Nov 20.

Center for Integrative Chemical Biology and Drug Discovery and ‡Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.
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http://dx.doi.org/10.1021/jm401387jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980660PMC
December 2013

Discovery of an in vivo chemical probe of the lysine methyltransferases G9a and GLP.

J Med Chem 2013 Nov 31;56(21):8931-42. Epub 2013 Oct 31.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, ‡National Institute of Mental Health Psychoactive Drug Screening Program, §Department of Pharmacology, School of Medicine, and ∥Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

Among epigenetic "writers", "readers", and "erasers", the lysine methyltransferases G9a and GLP, which catalyze mono- and dimethylation of histone H3 lysine 9 (H3K9me2) and nonhistone proteins, have been implicated in a variety of human diseases. A "toolkit" of well-characterized chemical probes will allow biological and disease hypotheses concerning these proteins to be tested in cell-based and animal models with high confidence. We previously discovered potent and selective G9a/GLP inhibitors including the cellular chemical probe UNC0638, which displays an excellent separation of functional potency and cell toxicity. However, this inhibitor is not suitable for animal studies due to its poor pharmacokinetic (PK) properties. Here, we report the discovery of the first G9a and GLP in vivo chemical probe UNC0642, which not only maintains high in vitro and cellular potency, low cell toxicity, and excellent selectivity, but also displays improved in vivo PK properties, making it suitable for animal studies.
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http://dx.doi.org/10.1021/jm401480rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3880643PMC
November 2013

Small-molecule ligands of methyl-lysine binding proteins: optimization of selectivity for L3MBTL3.

J Med Chem 2013 Sep 16;56(18):7358-71. Epub 2013 Sep 16.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.

Lysine methylation is a key epigenetic mark, the dysregulation of which is linked to many diseases. Small-molecule antagonism of methyl-lysine (Kme) binding proteins that recognize such epigenetic marks can improve our understanding of these regulatory mechanisms and potentially validate Kme binding proteins as drug-discovery targets. We previously reported the discovery of 1 (UNC1215), the first potent and selective small-molecule chemical probe of a methyl-lysine reader protein, L3MBTL3, which antagonizes the mono- and dimethyl-lysine reading function of L3MBTL3. The design, synthesis, and structure-activity relationship studies that led to the discovery of 1 are described herein. These efforts established the requirements for potent L3MBTL3 binding and enabled the design of novel antagonists, such as compound 2 (UNC1679), that maintain in vitro and cellular potency with improved selectivity against other MBT-containing proteins. The antagonists described were also found to effectively interact with unlabeled endogenous L3MBTL3 in cells.
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http://dx.doi.org/10.1021/jm400919pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846386PMC
September 2013

UNC569, a novel small-molecule mer inhibitor with efficacy against acute lymphoblastic leukemia in vitro and in vivo.

Mol Cancer Ther 2013 Nov 30;12(11):2367-77. Epub 2013 Aug 30.

Corresponding Author: Douglas K. Graham, University of Colorado Anschutz Medical Campus, Mail Stop 8302, Building RC1-N, Room P18-4400, 12800 E. 19th Ave, Aurora, CO 80045.

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Although survival rates have improved, patients with certain biologic subtypes still have suboptimal outcomes. Current chemotherapeutic regimens are associated with short- and long-term toxicities and novel, less toxic therapeutic strategies are needed. Mer receptor tyrosine kinase is ectopically expressed in ALL patient samples and cell lines. Inhibition of Mer expression reduces prosurvival signaling, increases chemosensitivity, and delays development of leukemia in vivo, suggesting that Mer tyrosine kinase inhibitors are excellent candidates for targeted therapies. Brain and spinal tumors are the second most common malignancies in childhood. Multiple chemotherapy approaches and radiotherapies have been attempted, yet overall survival remains dismal. Mer is also abnormally expressed in atypical teratoid/rhabdoid tumors (AT/RT), providing a rationale for targeting Mer as a therapeutic strategy. We have previously described UNC569, the first small-molecule Mer inhibitor. This article describes the biochemical and biologic effects of UNC569 in ALL and AT/RT. UNC569 inhibited Mer activation and downstream signaling through ERK1/2 and AKT, determined by Western blot analysis. Treatment with UNC569 reduced proliferation/survival in liquid culture, decreased colony formation in methylcellulose/soft agar, and increased sensitivity to cytotoxic chemotherapies. MYC transgenic zebrafish with T-ALL were treated with UNC569 (4 μmol/L for two weeks). Fluorescence was quantified as indicator of the distribution of lymphoblasts, which express Mer and enhanced GFP. UNC569 induced more than 50% reduction in tumor burden compared with vehicle- and mock-treated fish. These data support further development of Mer inhibitors as effective therapies in ALL and AT/RT.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823742PMC
November 2013

UNC1062, a new and potent Mer inhibitor.

Eur J Med Chem 2013 Jul 2;65:83-93. Epub 2013 Apr 2.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Abnormal activation of Mer kinase has been implicated in the oncogenesis of many human cancers including acute lymphoblastic and myeloid leukemia, non-small cell lung cancer, and glioblastoma. We have discovered a new family of small molecule Mer inhibitors, pyrazolopyrimidine sulfonamides, that potently inhibit the kinase activity of Mer. Importantly, these compounds do not demonstrate significant hERG activity in the PatchXpress assay. Through structure-activity relationship studies, 35 (UNC1062) was identified as a potent (IC50 = 1.1 nM) and selective Mer inhibitor. When applied to live tumor cells, UNC1062 inhibited Mer phosphorylation and colony formation in soft agar. Given the potential of Mer as a therapeutic target, UNC1062 is a promising candidate for further drug development.
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http://dx.doi.org/10.1016/j.ejmech.2013.03.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3720808PMC
July 2013

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain.

Nat Chem Biol 2013 Mar 6;9(3):184-91. Epub 2013 Jan 6.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, University of North Carolina Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyllysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin-interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a K(d) of 120 nM, competitively displacing mono- or dimethyllysine-containing peptides, and is greater than 50-fold more potent toward L3MBTL3 than other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a unique 2:2 polyvalent mode of interaction between UNC1215 and L3MBTL3. In cells, UNC1215 is nontoxic and directly binds L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins, and point mutants that disrupt the Kme-binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215 on localization. Finally, UNC1215 was used to reveal a new Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis.
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http://dx.doi.org/10.1038/nchembio.1157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3577944PMC
March 2013

Inhibitors of Streptococcus pneumoniae surface endonuclease EndA discovered by high-throughput screening using a PicoGreen fluorescence assay.

J Biomol Screen 2013 Mar 26;18(3):247-57. Epub 2012 Sep 26.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

The human commensal pathogen Streptococcus pneumoniae expresses a number of virulence factors that promote serious pneumococcal diseases, resulting in significant morbidity and mortality worldwide. These virulence factors may give S. pneumoniae the capacity to escape immune defenses, resist antimicrobial agents, or a combination of both. Virulence factors also present possible points of therapeutic intervention. The activities of the surface endonuclease, EndA, allow S. pneumoniae to establish invasive pneumococcal infection. EndA's role in DNA uptake during transformation contributes to gene transfer and genetic diversification. Moreover, EndA's nuclease activity degrades the DNA backbone of neutrophil extracellular traps (NETs), allowing pneumococcus to escape host immune responses. Given its potential impact on pneumococcal pathogenicity, EndA is an attractive target for novel antimicrobial therapy. Herein, we describe the development of a high-throughput screening assay for the discovery of nuclease inhibitors. Nuclease-mediated digestion of double-stranded DNA was assessed using fluorescence changes of the DNA dye ligand, PicoGreen. Under optimized conditions, the assay provided robust and reproducible activity data (Z'= 0.87) and was used to screen 4727 small molecules against an imidazole-rescued variant of EndA. In total, six small molecules were confirmed as novel EndA inhibitors, some of which may have utility as research tools for understanding pneumococcal pathogenesis and for drug discovery.
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http://dx.doi.org/10.1177/1087057112461153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773034PMC
March 2013

Design and synthesis of diarylamines and diarylethers as cytotoxic antitumor agents.

Bioorg Med Chem Lett 2012 Oct 9;22(19):6224-8. Epub 2012 Aug 9.

Beijing Institute of Pharmacology & Toxicology, 27 Tai-Ping Road, Beijing 100850, China.

Based on a shared structural core of diarylamine in several known anticancer drugs as well as a new cytotoxic hit 6-chloro-2-(4-cyanophenyl)amino-3-nitropyridine (7), 30 diarylamines and diarylethers were designed, synthesized, and evaluated for cytotoxic activity against A549, KB, KB-vin, and DU145 human tumor cell lines (HTCL). Four new leads 11e, 12, 13a, and 13b were discovered with GI(50) values ranging from 0.33 to 3.45μM. Preliminary SAR results revealed that a diarylamine or diarylether could serve as an active structural core, meta-chloro and ortho-nitro groups on the A-ring (either pyridine or phenyl ring) were necessary and crucial for cytotoxic activity, and the para-substituents on the other phenyl ring (B-ring) were related to inhibitory selectivity for different tumor cells. In an investigation of potential biological targets of the new leads, high thoughput kinase screening discovered that new leads 11e, 12 and 13b especially inhibit Mer tyrosine kinase, a proto-oncogene associated with munerous tumor types, with IC(50) values of 2.2-3.0μM. Therefore, these findings provide a good starting point to optimize a new class of compounds as potential anticancer agents, particularly targeting Mer tyrosine kinase.
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http://dx.doi.org/10.1016/j.bmcl.2012.08.014DOI Listing
October 2012

Development of a high-throughput assay for identifying inhibitors of TBK1 and IKKε.

PLoS One 2012 30;7(7):e41494. Epub 2012 Jul 30.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, North Carolina, United States of America.

IKKε and TBK1 are noncanonical IKK family members which regulate inflammatory signaling pathways and also play important roles in oncogenesis. However, few inhibitors of these kinases have been identified. While the substrate specificity of IKKε has recently been described, the substrate specificity of TBK1 is unknown, hindering the development of high-throughput screening technologies for inhibitor identification. Here, we describe the optimal substrate phosphorylation motif for TBK1, and show that it is identical to the phosphorylation motif previously described for IKKε. This information enabled the design of an optimal TBK1/IKKε substrate peptide amenable to high-throughput screening and we assayed a 6,006 compound library that included 4,727 kinase-focused compounds to discover in vitro inhibitors of TBK1 and IKKε. 227 compounds in this library inhibited TBK1 at a concentration of 10 µM, while 57 compounds inhibited IKKε. Together, these data describe a new high-throughput screening assay which will facilitate the discovery of small molecule TBK1/IKKε inhibitors possessing therapeutic potential for both inflammatory diseases and cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041494PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3408500PMC
April 2013

Orally active adenosine A(1) receptor agonists with antinociceptive effects in mice.

J Med Chem 2012 Jul 16;55(14):6467-77. Epub 2012 Jul 16.

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Adenosine A(1) receptor (A(1)AR) agonists have antinociceptive effects in multiple preclinical models of acute and chronic pain. Although numerous A(1)AR agonists have been developed, clinical applications of these agents have been hampered by their cardiovascular side effects. Herein we report a series of novel A(1)AR agonists, some of which are structurally related to adenosine 5'-monophosphate (5'-AMP), a naturally occurring nucleotide that itself activates A(1)AR. These novel compounds potently activate A(1)AR in several orthogonal in vitro assays and are subtype selective for A(1)AR over A(2A)AR, A(2B)AR, and A(3)AR. Among them, UNC32A (3a) is orally active and has dose-dependent antinociceptive effects in wild-type mice. The antinociceptive effects of 3a were completely abolished in A(1)AR knockout mice, revealing a strict dependence on A(1)AR for activity. The apparent lack of cardiovascular side effects when administered orally and high affinity (K(i) of 36 nM for the human A(1)AR) make this compound potentially suitable as a therapeutic.
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http://dx.doi.org/10.1021/jm3004834DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3501123PMC
July 2012

Discovery of Novel Small Molecule Mer Kinase Inhibitors for the Treatment of Pediatric Acute Lymphoblastic Leukemia.

ACS Med Chem Lett 2012 Feb;3(2):129-134

Center for Integrative Chemical Biology and Drug Discovery, Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Ectopic Mer expression promotes pro-survival signaling and contributes to leukemogenesis and chemoresistance in childhood acute lymphoblastic leukemia (ALL). Consequently, Mer kinase inhibitors may promote leukemic cell death and further act as chemosensitizers increasing efficacy and reducing toxicities of current ALL regimens. We have applied a structure-based design approach to discover novel small molecule Mer kinase inhibitors. Several pyrazolopyrimidine derivatives effectively inhibit Mer kinase activity at sub-nanomolar concentrations. Furthermore, the lead compound shows a promising selectivity profile against a panel of 72 kinases and has excellent pharmacokinetic properties. We also describe the crystal structure of the complex between the lead compound and Mer, opening new opportunities for further optimization and new template design.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3365829PMC
http://dx.doi.org/10.1021/ml200239kDOI Listing
February 2012