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Nat Commun ; 12(1): 4688, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344896


Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.

Fatores de Ribosilação do ADP/antagonistas & inibidores , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas ras/antagonistas & inibidores , Fatores de Ribosilação do ADP/metabolismo , Sítios de Ligação , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/química , Proteínas ras/metabolismo
Methods ; 92: 5-10, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-25890247


There has been a growing appreciation that G protein-coupled receptor (GPCR) functional selectivity (viz. biased signaling), in particular between G protein- and ß-arrestin-dependent signaling, can be achieved with specific ligands, and that such directed signaling represents a promising avenue for improving drug efficacy and therapy. Thus, for any given GPCRs it is important to define means to pharmacologically characterize and classify drugs for their propensity to bias signaling. Here we describe an experimental protocol and step-by-step approach to assess functional selectivity between Gαq and ß-arrestin-dependent responses using the prototypical angiotensin II (AngII) type 1 receptor (AT1R) expressed in HEK 293 cells. The protocol describes the expression of Bioluminescence Resonance Energy Transfer (BRET) sensors for either Gαq or ß-arrestin with AT1R, and the use of the operational model of pharmacological agonism to quantify ligand bias. Such methods are equally applicable to other GPCRs and their downstream signaling effectors.

Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Receptor Tipo 1 de Angiotensina/análise , Receptor Tipo 1 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Angiotensina II/metabolismo , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ligantes
J Biomol Screen ; 19(6): 890-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24464434


Protein kinases C (PKC) modulate the activity of the Kv11.1 ion channel current (hERG). However, the differential effects of specific PKC subtypes on the biophysics of the channel are unknown. The pharmaceutical tools to selectively modulate PKC subtypes are not membrane permeable and must be added directly to the intracellular solution in electrophysiology studies. Here, the PatchXpress electrophysiology robot was used to voltage clamp up to 16 cells simultaneously yet asynchronously across individual Sealchip chambers. The precision afforded by repeats of automation procedures minimized the experimental errors typical of these assays. Eight well-known PKC selective peptidomimmetics and general synthetic modulators were used to modulate the protein-protein interactions between hERG and the major PKC subtypes. We identified a specific role for the PKCε inhibitory peptidomimmetics in decreasing PKC-induced hERG τ activation (80%) and half-maximum activation voltage (90%) at steady state; a specific PKCε activator exhibited the opposite effect. Disruption of PKCß, PKCα, and PKCη interactions also showed significant effects albeit of lower magnitudes. The effect of PKCδ inhibitor was only marginal. A significant correlation was observed between the shifts in τ activation and half-maximum voltage at steady state (R(2)= 0.85). Peak current amplitudes and time constant of deactivation remained unaffected in all conditions.

Canal de Potássio ERG1/química , Proteína Quinase C/química , Acetato de Tetradecanoilforbol/química , Membrana Celular/metabolismo , Eletrofisiologia , Células HEK293 , Humanos , Cinética , Peptídeos/química , Peptidomiméticos/química , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/química , Inibidores de Proteínas Quinases/química , Robótica , Transfecção
Curr Top Med Chem ; 13(3): 367-85, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23432066


During inflammation, several Transient Receptor Potential (TRP) channels are directly or indirectly activated by inflammatory signaling molecules and microenvironmental changes including heat, oxidative conditions or low pH. In either case, specific TRP isoforms participate in chains of pro- or anti-inflammatory signaling cascades often including activation of transcription factors, protein kinases and phospholipases, which result in signal integration or amplification. In a few cases, their potentials as therapeutic targets for inflammatory conditions like pruritis, cystitis, dermatitis, asthma among other conditions are investigated pre-clinically or clinically by pioneering academic groups and industries. Significant efforts are still devoted to the understanding of the detailed physiological roles played by TRP channels during inflammation. This review intends to summarize key biological findings and reports of drug discovery activities when available, in an overview of the current status and recent developments in the field.

Anti-Inflamatórios não Esteroides/farmacologia , Descoberta de Drogas , Inflamação/metabolismo , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/química , Humanos , Terapia de Alvo Molecular
J Biomol Screen ; 18(5): 544-55, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23264449


TRPV1 was originally characterized as an integrator of various noxious stimuli such as capsaicin, heat, and protons. TRPV1-null mice exhibit a deficiency in sensing noxious heat stimuli, suggesting that TRPV1 is one of the main heat sensors on nociceptive primary afferent neurons and a candidate target for heat hypersensitivity in chronic pain. Several different potent and selective TRPV1 antagonists have been developed by more than 50 companies since the characterization of the receptor in 1997. A consequence of this competitive interest is the crowding of patentable chemical space, because very similar in vitro screening assays are used. To circumvent this issue and to expand our understanding of TRPV1 biology, we sought to take advantage of recent advancements in automated patch-clamp technology to design a novel screening cascade. This SAR-driving assay identified novel modulators that blocked the depolarization-induced activation of outwardly-rectifying TRPV1 currents independent of agonist stimulation, and we correlated the pharmacology to three other innovative assays for higher-throughput screening. Ultimately, we have identified a screening paradigm that would have good predictive value for future TRPV1 drug discovery projects and novel chemical space with a higher probability of gaining intellectual property coverage.

Capsaicina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/agonistas
Eur J Biochem ; 269(21): 5246-58, 2002 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-12392557


Homology modeling in combination with transmembrane topology predictions are used to build the atomic model of Neurospora crassa plasma membrane H+-ATPase, using as template the 2.6 A crystal structure of rabbit sarcoplasmic reticulum Ca2+-ATPase [Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. (2000) Nature 405, 647-655]. Comparison of the two calcium-binding sites in the crystal structure of Ca2+-ATPase with the equivalent region in the H+-ATPase model shows that the latter is devoid of most of the negatively charged groups required to bind the cations, suggesting a different role for this region. Using the built model, a pathway for proton transport is then proposed from computed locations of internal polar cavities, large enough to contain at least one water molecule. As a control, the same approach is applied to the high-resolution crystal structure of halorhodopsin and the proton pump bacteriorhodopsin. This revealed a striking correspondence between the positions of internal polar cavities, those of crystallographic water molecules and, in the case of bacteriorhodopsin, the residues mediating proton translocation. In our H+-ATPase model, most of these cavities are in contact with residues previously shown to affect coupling of proton translocation to ATP hydrolysis. A string of six polar cavities identified in the cytoplasmic domain, the most accurate part of the model, suggests a proton entry path starting close to the phosphorylation site. Strikingly, members of the haloacid dehalogenase superfamily, which are close structural homologs of this domain but do not share the same function, display only one polar cavity in the vicinity of the conserved catalytic Asp residue.

Modelos Moleculares , Neurospora crassa/enzimologia , ATPases Translocadoras de Prótons/química , Sequência de Aminoácidos , Animais , Bacteriorodopsinas/química , Sítios de Ligação/fisiologia , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Simulação por Computador , Cristalografia por Raios X , Halorrodopsinas/química , Imageamento Tridimensional , Transporte de Íons/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/fisiologia , ATPases Translocadoras de Prótons/genética , Prótons , Coelhos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos