Publications by authors named "Bryan D Moyer"

31 Publications

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

PAC1 receptor blockade reduces central nociceptive activity: new approach for primary headache?

Pain 2020 07;161(7):1670-1681

Basic and Clinical Neuroscience, Institute of Psychiatry, Psychology and Neuroscience, King's College London, United Kingdom.

Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) may play an important role in primary headaches. Preclinical evidence suggests that PACAP38 modulates trigeminal nociceptive activity mainly through PAC1 receptors while clinical studies report that plasma concentrations of PACAP38 are elevated in spontaneous attacks of cluster headache and migraine and normalize after treatment with sumatriptan. Intravenous infusion of PACAP38 induces migraine-like attacks in migraineurs and cluster-like attacks in cluster headache patients. A rodent-specific PAC1 receptor antibody Ab181 was developed, and its effect on nociceptive neuronal activity in the trigeminocervical complex was investigated in vivo in an electrophysiological model relevant to primary headaches. Ab181 is potent and selective at the rat PAC1 receptor and provides near-maximum target coverage at 10 mg/kg for more than 48 hours. Without affecting spontaneous neuronal activity, Ab181 effectively inhibits stimulus-evoked activity in the trigeminocervical complex. Immunohistochemical analysis revealed its binding in the trigeminal ganglion and sphenopalatine ganglion but not within the central nervous system suggesting a peripheral site of action. The pharmacological approach using a specific PAC1 receptor antibody could provide a novel mechanism with a potential clinical efficacy in the treatment of primary headaches.
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http://dx.doi.org/10.1097/j.pain.0000000000001858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302332PMC
July 2020

Engineering Na1.7 Inhibitory JzTx-V Peptides with a Potency and Basicity Profile Suitable for Antibody Conjugation To Enhance Pharmacokinetics.

ACS Chem Biol 2019 04 27;14(4):806-818. Epub 2019 Mar 27.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel Na1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed Na1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNa1.7 IC = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable Na in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained Na1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a Na1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage Na1.7 in vivo.
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http://dx.doi.org/10.1021/acschembio.9b00183DOI Listing
April 2019

Discovery of Tarantula Venom-Derived Na1.7-Inhibitory JzTx-V Peptide 5-Br-Trp24 Analogue AM-6120 with Systemic Block of Histamine-Induced Pruritis.

J Med Chem 2018 11 22;61(21):9500-9512. Epub 2018 Oct 22.

Therapeutic Discovery, Amgen Research , Amgen Inc. , 1120 Veterans Blvd , South San Francisco , California 94080 , United States.

Inhibitors of the voltage-gated sodium channel Na1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified Na1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived Na1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic Na1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic Na1.7 inhibitors.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00736DOI Listing
November 2018

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

Engineering Antibody Reactivity for Efficient Derivatization to Generate Na1.7 Inhibitory GpTx-1 Peptide-Antibody Conjugates.

ACS Chem Biol 2017 09 28;12(9):2427-2435. Epub 2017 Aug 28.

Therapeutic Discovery, ‡Neuroscience, and §Pharmacokinetics and Drug Metabolism, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

The voltage-gated sodium channel Na1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional Na1.7 targeting antibodies are not known. In this report, we describe Na1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified Na1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the Na1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked Na1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not Na1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit Na1.7 in engineered and neuronal cells.
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http://dx.doi.org/10.1021/acschembio.7b00542DOI Listing
September 2017

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

Evaluation of recombinant monoclonal antibody SVmab1 binding to Na 1.7 target sequences and block of human Na 1.7 currents.

F1000Res 2016 25;5:2764. Epub 2016 Nov 25.

Neuroscience, Amgen Inc., Thousand Oaks, USA.

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na 1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na 1.7 DII voltage sensor domain and block human Na 1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na 1.7 DII voltage sensor domain via ELISA, and does not bind Na 1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na 1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na 1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na 1.7 target sequences or specifically inhibit Na 1.7 current.
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http://dx.doi.org/10.12688/f1000research.9918.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5155501PMC
November 2016

Selective antagonism of TRPA1 produces limited efficacy in models of inflammatory- and neuropathic-induced mechanical hypersensitivity in rats.

Mol Pain 2016 29;12. Epub 2016 Nov 29.

Department of Neuroscience, Amgen Inc, Thousand Oaks, CA, USA.

The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund's adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.
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http://dx.doi.org/10.1177/1744806916677761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131813PMC
October 2017

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

Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the NaV1.7 Inhibitory Peptide GpTx-1.

J Med Chem 2016 Mar 10;59(6):2704-17. Epub 2016 Mar 10.

Therapeutic Discovery and ‡Neuroscience, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01947DOI Listing
March 2016

Sustained inhibition of the NaV1.7 sodium channel by engineered dimers of the domain II binding peptide GpTx-1.

Bioorg Med Chem Lett 2015 Nov 16;25(21):4866-4871. Epub 2015 Jun 16.

Therapeutic Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel NaV1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to NaV1.7, we have mapped the binding site to transmembrane segments 1-4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using NaV1.5/NaV1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting NaV1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.
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http://dx.doi.org/10.1016/j.bmcl.2015.06.033DOI Listing
November 2015

Engineering potent and selective analogues of GpTx-1, a tarantula venom peptide antagonist of the Na(V)1.7 sodium channel.

J Med Chem 2015 Mar 19;58(5):2299-314. Epub 2015 Feb 19.

Departments of Therapeutic Discovery, ‡Neuroscience, and §Pharmacokinetics & Drug Metabolism, Amgen Inc. , One Amgen Center Drive, Thousand Oaks, California 91320, United States.

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.
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http://dx.doi.org/10.1021/jm501765vDOI Listing
March 2015

Voltage-gated sodium channel function and expression in injured and uninjured rat dorsal root ganglia neurons.

Int J Neurosci 2016 7;126(2):182-92. Epub 2015 Apr 7.

a Department of Neuroscience, Amgen Inc. , Thousand Oaks, CA, USA.

The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.
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http://dx.doi.org/10.3109/00207454.2015.1004172DOI Listing
September 2016

Global Nav1.7 knockout mice recapitulate the phenotype of human congenital indifference to pain.

PLoS One 2014 4;9(9):e105895. Epub 2014 Sep 4.

Department of Neuroscience, Amgen Inc., Cambridge, Massachusetts, United States of America.

Clinical genetic studies have shown that loss of Nav1.7 function leads to the complete loss of acute pain perception. The global deletion is reported lethal in mice, however, and studies of mice with promoter-specific deletions of Nav1.7 have suggested that the role of Nav1.7 in pain transduction depends on the precise form of pain. We developed genetic and animal husbandry strategies that overcame the neonatal-lethal phenotype and enabled construction of a global Nav1.7 knockout mouse. Knockouts were anatomically normal, reached adulthood, and had phenotype wholly analogous to human congenital indifference to pain (CIP): compared to littermates, knockouts showed no defects in mechanical sensitivity or overall movement yet were completely insensitive to painful tactile, thermal, and chemical stimuli and were anosmic. Knockouts also showed no painful behaviors resulting from peripheral injection of nonselective sodium channel activators, did not develop complete Freund's adjuvant-induced thermal hyperalgesia, and were insensitive to intra-dermal histamine injection. Tetrodotoxin-sensitive sodium current recorded from cell bodies of isolated sensory neurons and the mechanically-evoked spiking of C-fibers in a skin-nerve preparation each were reduced but not eliminated in tissue from knockouts compared to littermates. Results support a role for Nav1.7 that is conserved between rodents and humans and suggest several possibly translatable biomarkers for the study of Nav1.7-targeted therapeutics. Results further suggest that Nav1.7 may retain its key role in persistent as well as acute forms of pain.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0105895PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4154897PMC
April 2015

Expression of genes encoding multi-transmembrane proteins in specific primate taste cell populations.

PLoS One 2009 Dec 4;4(12):e7682. Epub 2009 Dec 4.

Senomyx, Inc, San Diego, California, United States of America.

Background: Using fungiform (FG) and circumvallate (CV) taste buds isolated by laser capture microdissection and analyzed using gene arrays, we previously constructed a comprehensive database of gene expression in primates, which revealed over 2,300 taste bud-associated genes. Bioinformatics analyses identified hundreds of genes predicted to encode multi-transmembrane domain proteins with no previous association with taste function. A first step in elucidating the roles these gene products play in gustation is to identify the specific taste cell types in which they are expressed.

Methodology/principal Findings: Using double label in situ hybridization analyses, we identified seven new genes expressed in specific taste cell types, including sweet, bitter, and umami cells (TRPM5-positive), sour cells (PKD2L1-positive), as well as other taste cell populations. Transmembrane protein 44 (TMEM44), a protein with seven predicted transmembrane domains with no homology to GPCRs, is expressed in a TRPM5-negative and PKD2L1-negative population that is enriched in the bottom portion of taste buds and may represent developmentally immature taste cells. Calcium homeostasis modulator 1 (CALHM1), a component of a novel calcium channel, along with family members CALHM2 and CALHM3; multiple C2 domains; transmembrane 1 (MCTP1), a calcium-binding transmembrane protein; and anoctamin 7 (ANO7), a member of the recently identified calcium-gated chloride channel family, are all expressed in TRPM5 cells. These proteins may modulate and effect calcium signalling stemming from sweet, bitter, and umami receptor activation. Synaptic vesicle glycoprotein 2B (SV2B), a regulator of synaptic vesicle exocytosis, is expressed in PKD2L1 cells, suggesting that this taste cell population transmits tastant information to gustatory afferent nerve fibers via exocytic neurotransmitter release.

Conclusions/significance: Identification of genes encoding multi-transmembrane domain proteins expressed in primate taste buds provides new insights into the processes of taste cell development, signal transduction, and information coding. Discrete taste cell populations exhibit highly specific gene expression patterns, supporting a model whereby each mature taste receptor cell is responsible for sensing, transmitting, and coding a specific taste quality.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0007682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2780358PMC
December 2009

Genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes.

PLoS One 2009 Jul 28;4(7):e6395. Epub 2009 Jul 28.

Senomyx, Inc, San Diego, California, United States of America.

Efforts to unravel the mechanisms underlying taste sensation (gustation) have largely focused on rodents. Here we present the first comprehensive characterization of gene expression in primate taste buds. Our findings reveal unique new insights into the biology of taste buds. We generated a taste bud gene expression database using laser capture microdissection (LCM) procured fungiform (FG) and circumvallate (CV) taste buds from primates. We also used LCM to collect the top and bottom portions of CV taste buds. Affymetrix genome wide arrays were used to analyze gene expression in all samples. Known taste receptors are preferentially expressed in the top portion of taste buds. Genes associated with the cell cycle and stem cells are preferentially expressed in the bottom portion of taste buds, suggesting that precursor cells are located there. Several chemokines including CXCL14 and CXCL8 are among the highest expressed genes in taste buds, indicating that immune system related processes are active in taste buds. Several genes expressed specifically in endocrine glands including growth hormone releasing hormone and its receptor are also strongly expressed in taste buds, suggesting a link between metabolism and taste. Cell type-specific expression of transcription factors and signaling molecules involved in cell fate, including KIT, reveals the taste bud as an active site of cell regeneration, differentiation, and development. IKBKAP, a gene mutated in familial dysautonomia, a disease that results in loss of taste buds, is expressed in taste cells that communicate with afferent nerve fibers via synaptic transmission. This database highlights the power of LCM coupled with transcriptional profiling to dissect the molecular composition of normal tissues, represents the most comprehensive molecular analysis of primate taste buds to date, and provides a foundation for further studies in diverse aspects of taste biology.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006395PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712080PMC
July 2009

Voltage-gated sodium channels in taste bud cells.

BMC Neurosci 2009 Mar 12;10:20. Epub 2009 Mar 12.

Senomyx, Inc, 4767 Nexus Centre Drive, San Diego, CA 92121, USA.

Background: Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown.

Results: We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore.

Conclusion: SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.
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http://dx.doi.org/10.1186/1471-2202-10-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2660338PMC
March 2009

Small molecule activator of the human epithelial sodium channel.

J Biol Chem 2008 May 6;283(18):11981-94. Epub 2008 Mar 6.

Senomyx, Inc., San Diego, California 92121, USA.

The epithelial sodium channel (ENaC), a heterotrimeric complex composed of alpha, beta, and gamma subunits, belongs to the ENaC/degenerin family of ion channels and forms the principal route for apical Na(+) entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the beta subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of alphabetaG37Sgamma hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na(+) flux across epithelial membranes is clinically desirable.
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http://dx.doi.org/10.1074/jbc.M708001200DOI Listing
May 2008

Endoplasmic reticulum degradation impedes olfactory G-protein coupled receptor functional expression.

BMC Cell Biol 2004 Sep 15;5:34. Epub 2004 Sep 15.

Senomyx, Inc, 11099 North Torrey Pines Road, La Jolla, CA 92037, USA. senomyx.com

Background: Research on olfactory G-protein coupled receptors (GPCRs) has been severely impeded by poor functional expression in heterologous systems. Previously, we demonstrated that inefficient olfactory receptor (OR) expression at the plasma membrane is attributable, in part, to degradation of endoplasmic reticulum (ER)-retained ORs by the ubiquitin-proteasome system and sequestration of ORs in ER aggregates that are degraded by autophagy. Thus, experiments were performed to test the hypothesis that attenuation of ER degradation improves OR functional expression in heterologous cells.

Results: To develop means to increase the functional expression of ORs, we devised an approach to measure activation of the mOREG OR (Unigene # Mm.196680; Olfr73) through coupling to an olfactory cyclic nucleotide-gated cation channel (CNG). This system, which utilizes signal transduction machinery coupled to OR activation in native olfactory sensory neurons, was used to demonstrate that degradation, both by the ubiquitin-proteasome system and autophagy, limits mOREG functional expression. The stimulatory effects of proteasome and autophagy inhibitors on mOREG function required export from the ER and trafficking through the biosynthetic pathway.

Conclusions: These findings demonstrate that poor functional expression of mOREG in heterologous cells is improved by blocking proteolysis. Inhibition of ER degradation may improve the function of other ORs and assist future efforts to elucidate the molecular basis of odor discrimination.
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http://dx.doi.org/10.1186/1471-2121-5-34DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC520810PMC
September 2004

Endoplasmic reticulum retention, degradation, and aggregation of olfactory G-protein coupled receptors.

Traffic 2003 Jun;4(6):416-33

Senomyx, Inc., 11099 North Torrey Pines Road, La Jolla, CA 92037, USA.

The mammalian olfactory G-protein coupled receptor family is comprised of hundreds of proteins that mediate odorant binding and initiate signal transduction cascades leading to the sensation of smell. However, efforts to functionally express olfactory receptors and identify specific odorant ligand-olfactory receptor interactions have been severely impeded by poor olfactory receptor surface expression in heterologous systems. Therefore, experiments were performed to elucidate the cellular mechanism(s) responsible for inefficient olfactory receptor cell surface expression. We determined that the mouse odorant receptors mI7 and mOREG are not selected for export from the ER and therefore are not detectable at the Golgi apparatus or plasma membrane. Specifically, olfactory receptors interact with the ER chaperone calnexin, are excluded from ER export sites, do not accumulate in ER-Golgi transport intermediates at 15 degrees C, and contain endoglycosidase H-sensitive oligosaccharides, consistent with olfactory receptor exclusion from post-ER compartments. A labile pool of ER-retained olfactory receptors are post-translationally modified by polyubiquitination and targeted for degradation by the proteasome. In addition, olfactory receptors are sequestered into ER aggregates that are degraded by autophagy. Collectively, these data demonstrate that poor surface expression of olfactory receptors in heterologous cells is attributable to a combination of ER retention due to inefficient folding and poor coupling to ER export machinery, aggregation, and degradation via both proteasomal and autophagic pathways.
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http://dx.doi.org/10.1034/j.1600-0854.2003.00097.xDOI Listing
June 2003

Analysis of CFTR trafficking and polarization using green fluorescent protein and confocal microscopy.

Methods Mol Med 2002 ;70:217-27

Department of Cell Biology, Scripps Research Institute, La Jolla, CA, USA.

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http://dx.doi.org/10.1385/1-59259-187-6:217DOI Listing
September 2002

Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

J Biol Chem 2002 Mar 17;277(13):11401-9. Epub 2002 Jan 17.

Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.
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http://dx.doi.org/10.1074/jbc.M110263200DOI Listing
March 2002