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1.
Neuron ; 109(2): 273-284.e4, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33152265

RESUMO

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.


Assuntos
Medição da Dor/métodos , Canal de Cátion TRPA1/agonistas , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Células HEK293 , Humanos , Ligantes , Masculino , Medição da Dor/efeitos dos fármacos , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Canal de Cátion TRPA1/química
2.
J Exp Med ; 218(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33620419

RESUMO

Despite the development of effective therapies, a substantial proportion of asthmatics continue to have uncontrolled symptoms, airflow limitation, and exacerbations. Transient receptor potential cation channel member A1 (TRPA1) agonists are elevated in human asthmatic airways, and in rodents, TRPA1 is involved in the induction of airway inflammation and hyperreactivity. Here, the discovery and early clinical development of GDC-0334, a highly potent, selective, and orally bioavailable TRPA1 antagonist, is described. GDC-0334 inhibited TRPA1 function on airway smooth muscle and sensory neurons, decreasing edema, dermal blood flow (DBF), cough, and allergic airway inflammation in several preclinical species. In a healthy volunteer Phase 1 study, treatment with GDC-0334 reduced TRPA1 agonist-induced DBF, pain, and itch, demonstrating GDC-0334 target engagement in humans. These data provide therapeutic rationale for evaluating TRPA1 inhibition as a clinical therapy for asthma.


Assuntos
Asma/tratamento farmacológico , Inflamação Neurogênica/tratamento farmacológico , Dor/tratamento farmacológico , Prurido/tratamento farmacológico , Piridinas/farmacologia , Piridinas/uso terapêutico , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Canal de Cátion TRPA1/antagonistas & inibidores , Adolescente , Adulto , Animais , Estudos de Coortes , Modelos Animais de Doenças , Cães , Método Duplo-Cego , Feminino , Cobaias , Voluntários Saudáveis , Humanos , Isotiocianatos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Dor/induzido quimicamente , Prurido/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Canal de Cátion TRPA1/deficiência , Resultado do Tratamento , Adulto Jovem
3.
Methods Mol Biol ; 2025: 51-68, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31267448

RESUMO

The expression analysis of recombinant proteins is a challenging step in any high-throughput protein production pipeline. Often multiple expression systems and a variety of expression construct designs are considered for the production of a protein of interest. There is a strong need to triage constructs rapidly and systematically. This chapter describes a semiautomated method for the simultaneous purification and characterization of proteins expressed from multiple samples of expression cultures from the E. coli, baculovirus expression vector system, and mammalian transient expression systems. This method assists in the selection of the most promising expression construct(s) or the most favorable expression condition(s) to move forward into large-scale protein production.


Assuntos
Proteínas Recombinantes/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes/genética
4.
Science ; 350(6267): aac5464, 2015 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-26680203

RESUMO

Voltage-gated sodium (Nav) channels propagate action potentials in excitable cells. Accordingly, Nav channels are therapeutic targets for many cardiovascular and neurological disorders. Selective inhibitors have been challenging to design because the nine mammalian Nav channel isoforms share high sequence identity and remain recalcitrant to high-resolution structural studies. Targeting the human Nav1.7 channel involved in pain perception, we present a protein-engineering strategy that has allowed us to determine crystal structures of a novel receptor site in complex with isoform-selective antagonists. GX-936 and related inhibitors bind to the activated state of voltage-sensor domain IV (VSD4), where their anionic aryl sulfonamide warhead engages the fourth arginine gating charge on the S4 helix. By opposing VSD4 deactivation, these compounds inhibit Nav1.7 through a voltage-sensor trapping mechanism, likely by stabilizing inactivated states of the channel. Residues from the S2 and S3 helices are key determinants of isoform selectivity, and bound phospholipids implicate the membrane as a modulator of channel function and pharmacology. Our results help to elucidate the molecular basis of voltage sensing and establish structural blueprints to design selective Nav channel antagonists.


Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/química , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Tiadiazóis/química , Tiadiazóis/farmacologia , Sequência de Aminoácidos , Membrana Celular/química , Cristalização/métodos , Cristalografia por Raios X , Análise Mutacional de DNA , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Percepção da Dor/efeitos dos fármacos , Engenharia de Proteínas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
5.
PLoS One ; 9(10): e109366, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25285661

RESUMO

Inhibiting NAD biosynthesis by blocking the function of nicotinamide phosphoribosyl transferase (NAMPT) is an attractive therapeutic strategy for targeting tumor metabolism. However, the development of drug resistance commonly limits the efficacy of cancer therapeutics. This study identifies mutations in NAMPT that confer resistance to a novel NAMPT inhibitor, GNE-618, in cell culture and in vivo, thus demonstrating that the cytotoxicity of GNE-618 is on target. We determine the crystal structures of six NAMPT mutants in the apo form and in complex with various inhibitors and use cellular, biochemical and structural data to elucidate two resistance mechanisms. One is the surprising finding of allosteric modulation by mutation of residue Ser165, resulting in unwinding of an α-helix that binds the NAMPT substrate 5-phosphoribosyl-1-pyrophosphate (PRPP). The other mechanism is orthosteric blocking of inhibitor binding by mutations of Gly217. Furthermore, by evaluating a panel of diverse small molecule inhibitors, we unravel inhibitor structure activity relationships on the mutant enzymes. These results provide valuable insights into the design of next generation NAMPT inhibitors that offer improved therapeutic potential by evading certain mechanisms of resistance.


Assuntos
Antineoplásicos/farmacologia , Citocinas/antagonistas & inibidores , Citocinas/química , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Nicotinamida Fosforribosiltransferase/química , Domínio Catalítico , Linhagem Celular Tumoral , Citocinas/genética , Humanos , Modelos Moleculares , Mutação , Nicotinamida Fosforribosiltransferase/genética
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