Publications by authors named "Violeta Yu"

29 Publications

  • Page 1 of 1

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of Na1.7 Inhibitors.

SLAS Discov 2020 06 15;25(5):434-446. Epub 2020 Apr 15.

Q-State Biosciences, Cambridge, MA, USA.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.
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http://dx.doi.org/10.1177/2472555220914532DOI Listing
June 2020

Pharmacological characterization of potent and selective NaV1.7 inhibitors engineered from Chilobrachys jingzhao tarantula venom peptide JzTx-V.

PLoS One 2018 3;13(5):e0196791. Epub 2018 May 3.

Therapeutic Discovery, Amgen Discovery Research, Thousand Oaks, California, United States of America.

Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0196791PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933747PMC
August 2018

1,2,4-Triazolsulfone: A novel isosteric replacement of acylsulfonamides in the context of Na1.7 inhibition.

Bioorg Med Chem Lett 2018 06 17;28(11):2103-2108. Epub 2018 Apr 17.

Amgen, Inc., 360 Binney Street, Cambridge, MA 02142, United States.

Recently, the identification of several classes of aryl sulfonamides and acyl sulfonamides that potently inhibit Na1.7 and demonstrate high levels of selectivity over other Na isoforms have been reported. The fully ionizable nature of these inhibitors has been shown to be an important part of the pharmacophore for the observed potency and isoform selectivity. The requirement of this functionality, however, has presented challenges associated with optimization toward inhibitors with drug-like properties and minimal off-target activity. In an effort to obviate these challenges, we set out to develop an orally bioavailable, selective Na1.7 inhibitor, lacking these acidic functional groups. Herein, we report the discovery of a novel series of inhibitors wherein a triazolesulfone has been designed to serve as a bioisostere for the acyl sulfonamide. This work culminated in the delivery of a potent series of inhibitors which demonstrated good levels of selectivity over Na1.5 and favorable pharmacokinetics in rodents.
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http://dx.doi.org/10.1016/j.bmcl.2018.04.035DOI Listing
June 2018

Discovery of a biarylamide series of potent, state-dependent Na1.7 inhibitors.

Bioorg Med Chem Lett 2017 08 26;27(16):3817-3824. Epub 2017 Jun 26.

Department of Therapeutic Discovery, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, United States.

The Na1.7 ion channel has garnered considerable attention as a target for the treatment of pain. Herein we detail the discovery and structure-activity relationships of a novel series of biaryl amides. Optimization led to the identification of several state-dependent, potent and metabolically stable inhibitors which demonstrated promising levels of selectivity over Na1.5 and good rat pharmacokinetics. Compound 18, which demonstrated preferential inhibition of a slow inactivated state of Na1.7, was advanced into a rat formalin study where upon reaching unbound drug levels several fold over the rat Na1.7 IC it failed to demonstrate a robust reduction in nociceptive behavior.
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http://dx.doi.org/10.1016/j.bmcl.2017.06.054DOI Listing
August 2017

The discovery of benzoxazine sulfonamide inhibitors of Na1.7: Tools that bridge efficacy and target engagement.

Bioorg Med Chem Lett 2017 08 1;27(15):3477-3485. Epub 2017 Jun 1.

Department of Medicinal Chemistry, Amgen Inc., 360 Binney Street, Cambridge, MA 02142, United States.

The voltage-gated sodium channel Na1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of Na1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat Na1.7 versus human Na1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse Na1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.
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http://dx.doi.org/10.1016/j.bmcl.2017.05.070DOI Listing
August 2017

Pharmacologic Characterization of AMG8379, a Potent and Selective Small Molecule Sulfonamide Antagonist of the Voltage-Gated Sodium Channel Na1.7.

J Pharmacol Exp Ther 2017 07 4;362(1):146-160. Epub 2017 May 4.

Department of Neuroscience (T.J.K., R.Y., S.A, C.P.I., M.J., D.J., J.H.L., S.G.L., J.Li., D.L., J.Lu., D.M., D.O., K.T., J.W., V.Y., D.X.D.Z., R.T.F., B.D.M.), Department of Medicinal Chemistry (M.M.W.), and Department of Pharmacokinetics and Drug Metabolism (X.B., V.B., J.R.), Amgen Inc., Cambridge, Massachusetts and Thousand Oaks, California

Potent and selective antagonists of the voltage-gated sodium channel Na1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human Na1.7 channels with an IC of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other Na family members, including Na1.4 expressed in muscle and Na1.5 expressed in the heart, as well as TTX-resistant Na channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple Na1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective Na1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
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http://dx.doi.org/10.1124/jpet.116.239590DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency, Pharmacokinetics, and Metabolic Properties to Obtain Atropisomeric Quinolinone (AM-0466) that Affords Robust in Vivo Activity.

J Med Chem 2017 07 20;60(14):5990-6017. Epub 2017 Apr 20.

Department of Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Because of its strong genetic validation, Na1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as Na1.7 inhibitors that demonstrate high levels of selectivity over other Na isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of Na1.7, which demonstrate nanomolar inhibition of Na1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a Na1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01850DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency and Pharmacokinetics While Mitigating Metabolic Liabilities.

J Med Chem 2017 07 20;60(14):5969-5989. Epub 2017 Apr 20.

Department of Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as Na1.7 inhibitors that demonstrate high levels of selectivity over other Na isoforms. The optimization of a series of internal Na1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01851DOI Listing
July 2017

Sulfonamides as Selective Na1.7 Inhibitors: Optimizing Potency and Pharmacokinetics to Enable in Vivo Target Engagement.

ACS Med Chem Lett 2016 Dec 21;7(12):1062-1067. Epub 2016 Sep 21.

Department of Medicinal Chemistry, Department of Molecular Engineering, Department of Pharmacokinetics and Drug Metabolism, Department of Neuroscience, and Department of Biologics, Amgen, Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, and One Amgen Center Drive, Thousand Oaks, California 91320, United States.

Human genetic evidence has identified the voltage-gated sodium channel Na1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide as a potent and selective inhibitor of Na1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log ) while maintaining Na1.7 potency led to the identification of quinazoline (AM-2099). Compound demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.
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http://dx.doi.org/10.1021/acsmedchemlett.6b00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5150675PMC
December 2016

Application of a Parallel Synthetic Strategy in the Discovery of Biaryl Acyl Sulfonamides as Efficient and Selective NaV1.7 Inhibitors.

J Med Chem 2016 09 29;59(17):7818-39. Epub 2016 Aug 29.

Department of Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

The majority of potent and selective hNaV1.7 inhibitors possess common pharmacophoric features that include a heteroaryl sulfonamide headgroup and a lipophilic aromatic tail group. Recently, reports of similar aromatic tail groups in combination with an acyl sulfonamide headgroup have emerged, with the acyl sulfonamide bestowing levels of selectivity over hNaV1.5 comparable to the heteroaryl sulfonamide. Beginning with commercially available carboxylic acids that met selected pharmacophoric requirements in the lipophilic tail, a parallel synthetic approach was applied to rapidly generate the derived acyl sulfonamides. A biaryl acyl sulfonamide hit from this library was elaborated, optimizing for potency and selectivity with attention to physicochemical properties. The resulting novel leads are potent, ligand and lipophilic efficient, and selective over hNaV1.5. Representative lead 36 demonstrates selectivity over other human NaV isoforms and good pharmacokinetics in rodents. The biaryl acyl sulfonamides reported herein may also offer ADME advantages over known heteroaryl sulfonamide inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00425DOI Listing
September 2016

Optimization of a Novel Quinazolinone-Based Series of Transient Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in Vivo Activity.

J Med Chem 2016 Mar 4;59(6):2794-809. Epub 2016 Mar 4.

Departments of Medicinal Chemistry, ‡Lead Discovery, §Molecular Structure, and ∥Pharmacokinetics and Drug Metabolism, Amgen, Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, United States.

There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00039DOI Listing
March 2016

Development and implementation of a high-throughput AlphaLISA assay for identifying inhibitors of EZH2 methyltransferase.

Assay Drug Dev Technol 2013 Apr 14;11(3):152-62. Epub 2013 Feb 14.

Amgen, Inc., Cambridge, Massachusetts 02142, USA.

The methylation state of lysine residues within histone H3 is a major determinant of active and inactive regions of the genome. Enhancer of Zeste homolog 2 (EZH2) is a histone lysine methyltransferase that is part of the polycomb repressive complex 2 (PRC2). Elevated EZH2 expression levels have been linked to hypertrimethylation of histone H3 lysine 27 (H3K27), repression of tumor repressor genes, and the onset of several types of cancers. We used the AlphaLISA technology to develop a high-throughput assay for identifying small molecule inhibitors of EZH2. AlphaLISA Acceptor Beads coated with antibodies directed against methylated H3K27 provided a sensitive method of detecting EZH2 activity through measurement of K27 methylation of a biotinylated H3-based peptide substrate. Optimized assay conditions resulted in a robust assay (Z'>0.7) which was successfully implemented in a high-throughput screening campaign. Small molecule inhibitors identified by this method may serve as powerful tools to further elucidate the potential importance of EZH2 in the development and treatment of cancer.
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http://dx.doi.org/10.1089/adt.2012.481DOI Listing
April 2013

Differential selectivity of JAK2 inhibitors in enzymatic and cellular settings.

Exp Hematol 2013 May 19;41(5):491-500. Epub 2013 Jan 19.

Amgen, Thousand Oaks, California, USA.

Small molecule inhibitors of Janus kinase (JAK) family members (JAK1, JAK2, JAK3, and Tyk2) are currently being pursued as potential new modes of therapy for a variety of diseases, including the inhibition of JAK2 for the treatment of myeloproliferative disorders. Selective inhibition within the JAK family can be beneficial in avoiding undesirable side effects (e.g., immunosuppression) caused by parallel inhibition of other JAK members. In an effort to design an assay paradigm for the development of JAK2 selective inhibitors, we investigated whether compound selectivity differed between cellular and purified enzyme environments. A set of JAK2 inhibitors was tested in a high-throughput JAK family cell assay suite and in corresponding purified enzyme assays. The high-throughput JAK cell assay suite comprises Ba/F3 cells individually expressing translocated ETS leukemia (TEL) fusions of each JAK family member (TEL-JAK Ba/F3) and an AlphaScreen phosphorylated-STAT5 (pSTAT5) immunoassay. Compound potencies from the TEL-JAK Ba/F3 pSTAT5 assays were similar to those determined in downstream cell proliferation measurements and more physiologically relevant cytokine-induced pSTAT5 PBMC assays. However, compound selectivity data between cell and purified enzyme assays were discrepant because of different potency shifts between cell and purified enzyme values for each JAK family member. For any JAK small molecule development program, our results suggest that relying solely on enzyme potency and selectivity data may be misleading. Adopting the high-throughput TEL-JAK Ba/F3 pSTAT5 cell assay suite in lead development paradigms should provide a more meaningful understanding of selectivity and facilitate the development of more selective JAK inhibitors.
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http://dx.doi.org/10.1016/j.exphem.2013.01.005DOI Listing
May 2013

Structure-Based Design of Potent and Selective CK1γ Inhibitors.

ACS Med Chem Lett 2012 Dec 18;3(12):1059-64. Epub 2012 Oct 18.

Departments of Medicinal Chemistry; Pharmacokinetics and Drug Metabolism; Oncology Research; and Molecular Structure, Amgen Inc. , 360 Binney Street, Cambridge, Massachusetts 02142, United States.

Aberrant activation of the Wnt pathway is believed to drive the development and growth of some cancers. The central role of CK1γ in Wnt signal transduction makes it an attractive target for the treatment of Wnt-pathway dependent cancers. We describe a structure-based approach that led to the discovery of a series of pyridyl pyrrolopyridinones as potent and selective CK1γ inhibitors. These compounds exhibited good enzyme and cell potency, as well as selectivity against other CK1 isoforms. A single oral dose of compound 13 resulted in significant inhibition of LRP6 phosphorylation in a mouse tumor PD model.
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http://dx.doi.org/10.1021/ml300278fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025826PMC
December 2012

The discovery and optimization of a novel class of potent, selective, and orally bioavailable anaplastic lymphoma kinase (ALK) inhibitors with potential utility for the treatment of cancer.

J Med Chem 2012 Jul 10;55(14):6523-40. Epub 2012 Jul 10.

Amgen Inc., 360 Binney Street, Cambridge, MA 02142, USA.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure-activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.
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http://dx.doi.org/10.1021/jm3005866DOI Listing
July 2012

Discovery of potent and highly selective thienopyridine Janus kinase 2 inhibitors.

J Med Chem 2011 Dec 16;54(24):8440-50. Epub 2011 Nov 16.

Department of Medicinal Chemistry, Amgen, Inc., 360 Binney Street, Cambridge, Massachusetts 02142, USA.

Developing Janus kinase 2 (Jak2) inhibitors has become a significant focus for small molecule drug discovery programs in recent years due to the identification of a Jak2 gain-of-function mutation in the majority of patients with myeloproliferative disorders (MPD). Here, we describe the discovery of a thienopyridine series of Jak2 inhibitors that culminates with compounds showing 100- to >500-fold selectivity over the related Jak family kinases in enzyme assays. Selectivity for Jak2 was also observed in TEL-Jak cellular assays, as well as in cytokine-stimulated peripheral blood mononuclear cell (PBMC) and whole blood assays. X-ray cocrystal structures of 8 and 19 bound to the Jak2 kinase domain aided structure-activity relationship efforts and, along with a previously reported small molecule X-ray cocrystal structure of the Jak1 kinase domain, provided structural rationale for the observed high levels of Jak2 selectivity.
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http://dx.doi.org/10.1021/jm200911rDOI Listing
December 2011

High-throughput TR-FRET assays for identifying inhibitors of LSD1 and JMJD2C histone lysine demethylases.

J Biomol Screen 2012 Jan 21;17(1):27-38. Epub 2011 Aug 21.

Amgen, Inc., Cambridge, MA 02142, USA.

Lysine demethylase 1 (LSD1) and Jumonji C domain-containing oxygenase D2C (JMJD2C) participate in regulating the methylation status of histone H3 lysine residues. In some contexts, LSD1 and JMJD2C activity causes enhanced cellular proliferation, which may lead to tumorigenesis. The authors explored the utility of time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays, which employed peptides consisting of the first 21 amino acids of histone H3 in which lysine 4 (H3K4) or lysine 9 (H3K9) was methylated (me) to quantify LSD1 and JMJD2C activity. The LSD1 assay monitored demethylation of the H3K4me1 peptide using an antibody that recognizes H3K4me1 but not the unmethylated peptide product. The JMJD2C assay measured demethylation of H3K9me3 with an antibody that selectively recognizes H3K9me2. The optimized conditions resulted in robust assays (Z' > 0.7) that required only 3 to 6 nM of enzyme in a reaction volume of 6 to 10 µL. These assays were used to compare the activity of different LSD1 constructs and to determine the apparent K(m) of each JMJD2C substrate. Finally, both assays were used in a high-throughput setting for identifying demethylase inhibitors. Compounds discovered by these TR-FRET methods may lead to powerful tools for ascertaining the roles of demethylases in a cellular context and ultimately for potential cancer treatments.
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http://dx.doi.org/10.1177/1087057111418228DOI Listing
January 2012

Comparison of 2 cell-based phosphoprotein assays to support screening and development of an ALK inhibitor.

J Biomol Screen 2011 Feb;16(2):164-73

Amgen, Inc., Cambridge, MA 02142, USA.

Anaplastic lymphoma kinase (ALK) when expressed as a fusion protein with nucleophosmin (NPM) has been implicated as a driving oncogene in a subset of lymphomas. Recent reports of ALK expression in a number of other cancers have raised the possibility that an ALK inhibitor may benefit patients with these diseases as well. In a campaign to identify and develop a selective ALK inhibitor, 2 assays were devised to measure the phosphorylation of tyrosine residue 1604 of ALK (pY(1604) ALK). Amplified Luminescent Proximity Homogeneous Assay (AlphaScreen(®)) and phosflow platforms were used to detect modulation of pY(1604) ALK to determine the relative potency of a set of small-molecule inhibitors. Prior to making use of these assays in diverse settings, the authors attempted to ensure their equivalence with a direct comparison of their performance. The pY(1604) ALK assays correlated well both with each other and with assays of ALK enzyme activity or ALK-dependent cell proliferation. The AlphaScreen(®) assay was amenable to automation and enabled rapid, high-throughput compound assessment in an NPM-ALK-driven cell line, whereas the phosflow assay enabled the authors to characterize the activity of compounds with respect to their impact on targeted enzymes and pathways. Results show that both AlphaScreen(®) and phosflow ALK assays exhibited diverse characteristics that made them desirable for different applications but were determined to be equally sensitive and robust in the detection of inhibition of pY(1604) ALK.
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http://dx.doi.org/10.1177/1087057110394657DOI Listing
February 2011

Small molecules with potent osteogenic-inducing activity in osteoblast cells.

Bioorg Med Chem Lett 2009 Mar 15;19(5):1442-5. Epub 2009 Jan 15.

Department of Metabolic Disorders, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

A chemical screen of 45,000 compounds from a diverse collection led to the identification of two series of small molecules with potent osteogenic activity in mouse MC3T3-E1 osteoblast cells. The first chemical group was characterized by an amino benzothiazole core (AMG0892 series) and the second group by a naphthyl amide core (AMG0309 series). Using alkaline phosphatase (ALP), osteocalcin (OCL) and calcium as markers of osteoblast differentiation and mineralization, both chemical series showed EC(50)s in the 0.01-0.2 microM range and were consistent for all three markers. Compounds inhibited cell proliferation, had no effect on apoptosis and showed evidence for CREB pathway activity. The present compounds represent some of the most potent osteogenic small molecules reported to date and provide new tools for elucidating signaling mechanisms in osteoblasts.
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http://dx.doi.org/10.1016/j.bmcl.2009.01.025DOI Listing
March 2009

Kinetic characterization and identification of a novel inhibitor of hypoxia-inducible factor prolyl hydroxylase 2 using a time-resolved fluorescence resonance energy transfer-based assay technology.

Anal Biochem 2009 Jan 14;384(2):213-23. Epub 2008 Oct 14.

Amgen Inc., One Amgen Center Drive, HTS-Molecular Pharmacology, Thousand Oaks, CA 91320-1789, USA.

The human hypoxia-inducible factor prolyl hydroxylases 1, 2, and 3 (HIF-PHD1, -2, and -3) are thought to act as proximal sensors of cellular hypoxia by virtue of their mechanism-based dependence on molecular oxygen. These 2-oxoglutarate (2-OG) and non-heme iron-dependent oxygenases constitutively hydroxylate HIF, resulting in high-affinity binding to Von Hippel-Lindau protein (pVHL). Some reported affinities for the HIF-PHDs for 2-OG and iron approach the estimated physiological concentrations for these cofactors, suggesting that the system as described is not catalytically optimal. Here we report the enzymatic characterization of full-length recombinant human HIF-PHD2 using a novel and sensitive catalytic assay. We demonstrated submicromolar affinities for 2-OG and ferrous iron and HIF-PHD2 Km values for oxygen that are greater than atmospheric oxygen levels, suggesting that molecular oxygen is indeed the key regulator of this pathway. In addition, we observed enhancement of HIF-PHD2 catalytic activity in the presence of ascorbic acid with only minor modifications of HIF-PHD2 requirements for 2-OG, and a detailed pH study demonstrated optimal HIF-PHD2 catalytic activity at pH 6.0. Lastly, we used this sensitive and facile assay to rapidly perform a large high-throughput screen of a chemical library to successfully identify and characterize novel 2-OG competitive inhibitors of HIF-PHD2.
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http://dx.doi.org/10.1016/j.ab.2008.09.052DOI Listing
January 2009

3-amino-7-phthalazinylbenzoisoxazoles as a novel class of potent, selective, and orally available inhibitors of p38alpha mitogen-activated protein kinase.

J Med Chem 2008 Oct 26;51(20):6280-92. Epub 2008 Sep 26.

Department of Chemistry Research and Discovery, Amgen Inc., Thousand Oaks, California 91320, USA.

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.
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http://dx.doi.org/10.1021/jm8005405DOI Listing
October 2008

Discovery of amido-benzisoxazoles as potent c-Kit inhibitors.

Bioorg Med Chem Lett 2008 Sep 31;18(18):5115-7. Epub 2008 Jul 31.

Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320-1799, USA.

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.
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http://dx.doi.org/10.1016/j.bmcl.2008.07.111DOI Listing
September 2008

Discovery of a potent and selective c-Kit inhibitor for the treatment of inflammatory diseases.

Bioorg Med Chem Lett 2008 Jul 28;18(14):4137-41. Epub 2008 May 28.

Department of Medicinal Chemistry, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

A potent and selective c-Kit inhibitor 20 was identified through a structure-activity relationship study. In an in vivo mouse model of mast cell activation, 20 blocked the SCF-induced histamine release with an EC(50) of 26 nM.
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http://dx.doi.org/10.1016/j.bmcl.2008.05.089DOI Listing
July 2008

Discovery of aryl aminoquinazoline pyridones as potent, selective, and orally efficacious inhibitors of receptor tyrosine kinase c-Kit.

J Med Chem 2008 Jun 1;51(11):3065-8. Epub 2008 May 1.

Department of Medicinal Chemistry, Amgen Inc, Thousand Oaks, CA 91320-1799, USA.

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.
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http://dx.doi.org/10.1021/jm800188gDOI Listing
June 2008

High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1.

Assay Drug Dev Technol 2007 Feb;5(1):105-15

Amgen Inc., Thousand Oaks, CA 91320-1799, USA.

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.
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http://dx.doi.org/10.1089/adt.2006.047DOI Listing
February 2007

High-content screening analysis of the p38 pathway: profiling of structurally related p38alpha kinase inhibitors using cell-based assays.

Assay Drug Dev Technol 2006 Aug;4(4):397-409

Amgen Inc., Thousand Oaks, CA 91320-1799, USA.

The complexity of the p38 mitogen-activated protein kinase (MAPK) signaling pathway presents challenges to understanding the efficacy of p38 inhibitors. Biochemical recombinant kinase assays and tumor necrosis factor alpha (TNFalpha) secretion assays are typically used to evaluate p38alpha inhibitors, but they do not provide insight into proximal intracellular events. Stimulation of the pathway evokes a cascade of phosphorylation events, accompanied by movement of molecules to different cellular compartments. Herein, we describe the profiling and potency comparison of a large set of p38alpha inhibitors with a pyrimidinone, imidazopyrimidine, or triazolopyrimidine core against biochemical recombinant p38alpha kinase activity, lipopolysaccharide (LPS)-mediated TNFalpha secretion by THP-1 cells, and a set of cellular imaging assays in SW1353 chondrocytes and baby hamster kidney cells. These pathway assays included p38 phosphorylation, MAPK-activated protein kinase 2 translocation, and heat shock protein (HSP) 27 phosphorylation. We established that HSP27 phosphorylation correlates well with LPS-induced TNFalpha secretion, validating our cellular imaging assays. We also found that the choice of cells and inducer can profoundly affect cellular potency results. High-content analysis may reveal signaling details, enriching our understanding of the mechanism of action of p38alpha inhibitors.
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http://dx.doi.org/10.1089/adt.2006.4.397DOI Listing
August 2006

Design and synthesis of potent pyridazine inhibitors of p38 MAP kinase.

Bioorg Med Chem Lett 2005 May;15(9):2409-13

Chemistry Research and Discovery, Amgen, Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.
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http://dx.doi.org/10.1016/j.bmcl.2005.02.010DOI Listing
May 2005

Generation and functional characterization of arylamine N-acetyltransferase Nat1/Nat2 double-knockout mice.

Mol Pharmacol 2003 Jul;64(1):170-9

Department of Pharmacology, University of Toronto, Medical Sciences Building 1, King's College Circle, Toronto, Ontario M5S 1A8, Canada.

Arylamine N-acetyltransferases (NATs) catalyze the biotransformation of a variety of arylamine drugs and carcinogens and may play diametrically opposing roles in enhancing either the detoxification of these chemicals or their metabolic activation into DNA-binding electrophiles. To facilitate the study of these processes, we have generated a Nat1/Nat2 double-knockout mouse model by gene targeting in embryonic stem cells. Nat1/2(-/-) mice were born at the expected frequency and seemed normal and viable with no overt phenotype, indicating that these genes are not critical for development or physiological homeostasis. In wild-type mice, NAT1 and NAT2 transcripts were detectable by RT-PCR in all tissues assayed including liver, kidney, colon, brain, bladder, and spleen. NAT1 and NAT2 transcripts were completely undetectable in the Nat1/2(-/-) mice. The in vitro N-acetylation of p-aminosalicylate was detected at significant levels in liver and kidney cytosols from either wild-type inbred 'rapid acetylator' C57BL/6 mice or from outbred CD-1 mice possessing homozygous rapid, heterozygous, or homozygous 'slow acetylator' Nat2 genotypes. Activity was undetectable in cytosol preparations from Nat1/2(-/-) mice. Nat1/2(-/-) mice also displayed severely compromised in vivo pharmacokinetics of p-aminosalicylate (PAS) and sulfamethazine (SMZ), with a drastically increased plasma area under the curve for PAS and a complete absence of their acetylated metabolites (AcPAS or AcSMZ) from plasma, confirming the functional absence of these enzymes and impaired drug metabolism capacity. This knockout mouse model should be helpful in delineating the role that variation in acetylating enzymes plays in mediating interindividual differences in susceptibility to arylamine-induced chemical toxicity and/or carcinogenesis.
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http://dx.doi.org/10.1124/mol.64.1.170DOI Listing
July 2003