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1.
Cell ; 186(10): 2160-2175.e17, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37137306

RESUMO

The serotonin transporter (SERT) removes synaptic serotonin and is the target of anti-depressant drugs. SERT adopts three conformations: outward-open, occluded, and inward-open. All known inhibitors target the outward-open state except ibogaine, which has unusual anti-depressant and substance-withdrawal effects, and stabilizes the inward-open conformation. Unfortunately, ibogaine's promiscuity and cardiotoxicity limit the understanding of inward-open state ligands. We docked over 200 million small molecules against the inward-open state of the SERT. Thirty-six top-ranking compounds were synthesized, and thirteen inhibited; further structure-based optimization led to the selection of two potent (low nanomolar) inhibitors. These stabilized an outward-closed state of the SERT with little activity against common off-targets. A cryo-EM structure of one of these bound to the SERT confirmed the predicted geometry. In mouse behavioral assays, both compounds had anxiolytic- and anti-depressant-like activity, with potencies up to 200-fold better than fluoxetine (Prozac), and one substantially reversed morphine withdrawal effects.


Assuntos
Ibogaína , Inibidores Seletivos de Recaptação de Serotonina , Proteínas da Membrana Plasmática de Transporte de Serotonina , Bibliotecas de Moléculas Pequenas , Animais , Camundongos , Fluoxetina/farmacologia , Ibogaína/química , Ibogaína/farmacologia , Conformação Molecular , Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/química , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/ultraestrutura , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia
2.
Nature ; 600(7890): 759-764, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34880501

RESUMO

The σ2 receptor has attracted intense interest in cancer imaging1, psychiatric disease2, neuropathic pain3-5 and other areas of biology6,7. Here we determined the crystal structure of this receptor in complex with the clinical candidate roluperidone2 and the tool compound PB288. These structures templated a large-scale docking screen of 490 million virtual molecules, of which 484 compounds were synthesized and tested. We identified 127 new chemotypes with affinities superior to 1 µM, 31 of which had affinities superior to 50 nM. The hit rate fell smoothly and monotonically with docking score. We optimized three hits for potency and selectivity, and achieved affinities that ranged from 3 to 48 nM, with up to 250-fold selectivity versus the σ1 receptor. Crystal structures of two ligands bound to the σ2 receptor confirmed the docked poses. To investigate the contribution of the σ2 receptor in pain, two potent σ2-selective ligands and one potent σ1/σ2 non-selective ligand were tested for efficacy in a mouse model of neuropathic pain. All three ligands showed time-dependent decreases in mechanical hypersensitivity in the spared nerve injury model9, suggesting that the σ2 receptor has a role in nociception. This study illustrates the opportunities for rapid discovery of in vivo probes through structure-based screens of ultra large libraries, enabling study of underexplored areas of biology.


Assuntos
Neuralgia , Receptores sigma , Animais , Ligantes , Camundongos , Neuralgia/tratamento farmacológico , Receptores sigma/metabolismo , Relação Estrutura-Atividade
3.
Artigo em Inglês | MEDLINE | ID: mdl-28507115

RESUMO

White and opaque cells of Candida albicans have the same genome but differ in gene expression patterns, metabolic profiles, and host niche preferences. We tested whether these differences, which include the differential expression of drug transporters, resulted in different sensitivities to 27 antifungal agents. The analysis was performed in two different strain backgrounds; although there was strain-to-strain variation, only terbinafine hydrochloride and caspofungin showed consistent, 2-fold differences between white and opaque cells across both strains.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/microbiologia , Regulação Fúngica da Expressão Gênica , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Caspofungina , Equinocandinas/farmacologia , Proteínas Fúngicas/genética , Lipopeptídeos/farmacologia , Testes de Sensibilidade Microbiana , Naftalenos/farmacologia , Fenótipo , Terbinafina
4.
J Pain ; 25(1): 53-63, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37482234

RESUMO

Most reports agree that aging negatively impacts pain processing and that the prevalence of chronic pain increases significantly with age. To improve current therapies, it is critical that aged animals be included in preclinical studies. Here we compared sensitivities to pain and itch-provoking stimuli in naïve and injured young and aged mice. Surprisingly, we found that in the absence of injury, aged male and female mice are significantly less responsive to mechanical stimuli and, in females, also to noxious thermal (heat) stimuli. In both older male and female mice, compared to younger (6-month-old mice), we also recorded reduced pruritogen-evoked scratching. On the other hand, after nerve injury, aged mice nevertheless developed significant mechanical hypersensitivity. Interestingly, however, and in contrast to young mice, aged mice developed both ipsilateral and contralateral postinjury mechanical allodynia. In a parallel immunohistochemical analysis of microglial and astrocyte markers, we found that the ipsilateral to the contralateral ratio of nerve injury-induced expression decreased with age. That observation is consistent with our finding of contralateral hypersensitivity after nerve injury in the aged but not the young mice. We conclude that aging has opposite effects on baseline versus postinjury pain and itch processing. PERSPECTIVE: Aged male and female mice (22-24 months) are less sensitive to mechanical, thermal (heat), and itch-provoking stimuli than are younger mice (6 months).


Assuntos
Dor , Prurido , Masculino , Feminino , Camundongos , Animais , Hiperalgesia/etiologia
5.
bioRxiv ; 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38328157

RESUMO

Large library docking can reveal unexpected chemotypes that complement the structures of biological targets. Seeking new agonists for the cannabinoid-1 receptor (CB1R), we docked 74 million tangible molecules, prioritizing 46 high ranking ones for de novo synthesis and testing. Nine were active by radioligand competition, a 20% hit-rate. Structure-based optimization of one of the most potent of these (Ki = 0.7 uM) led to '4042, a 1.9 nM ligand and a full CB1R agonist. A cryo-EM structure of the purified enantiomer of '4042 ('1350) in complex with CB1R-Gi1 confirmed its docked pose. The new agonist was strongly analgesic, with generally a 5-10-fold therapeutic window over sedation and catalepsy and no observable conditioned place preference. These findings suggest that new cannabinoid chemotypes may disentangle characteristic cannabinoid side-effects from their analgesia, supporting the further development of cannabinoids as pain therapeutics.

6.
Nat Commun ; 14(1): 8067, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38057319

RESUMO

The lipid prostaglandin E2 (PGE2) mediates inflammatory pain by activating G protein-coupled receptors, including the prostaglandin E2 receptor 4 (EP4R). Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce nociception by inhibiting prostaglandin synthesis, however, the disruption of upstream prostanoid biosynthesis can lead to pleiotropic effects including gastrointestinal bleeding and cardiac complications. In contrast, by acting downstream, EP4R antagonists may act specifically as anti-inflammatory agents and, to date, no selective EP4R antagonists have been approved for human use. In this work, seeking to diversify EP4R antagonist scaffolds, we computationally dock over 400 million compounds against an EP4R crystal structure and experimentally validate 71 highly ranked, de novo synthesized molecules. Further, we show how structure-based optimization of initial docking hits identifies a potent and selective antagonist with 16 nanomolar potency. Finally, we demonstrate favorable pharmacokinetics for the discovered compound as well as anti-allodynic and anti-inflammatory activity in several preclinical pain models in mice.


Assuntos
Dinoprostona , Receptores de Prostaglandina , Humanos , Camundongos , Animais , Fagocitose , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Dor/tratamento farmacológico , Anti-Inflamatórios não Esteroides/farmacologia
7.
J Med Chem ; 65(5): 4201-4217, 2022 03 10.
Artigo em Inglês | MEDLINE | ID: mdl-35195401

RESUMO

The 5-HT5A receptor (5-HT5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT5AR homology model identified 5 mid-µM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT5AR activity revealed that 5-HT5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT5AR.


Assuntos
Antagonistas da Serotonina , Serotonina , Humanos , Ligantes , Dor , Receptores de Serotonina , Antagonistas da Serotonina/farmacologia
8.
Science ; 377(6614): eabn7065, 2022 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-36173843

RESUMO

Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit '9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, '7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine.


Assuntos
Agonistas de Receptores Adrenérgicos alfa 2 , Analgésicos não Narcóticos , Descoberta de Drogas , Manejo da Dor , Dor , Agonistas de Receptores Adrenérgicos alfa 2/química , Agonistas de Receptores Adrenérgicos alfa 2/farmacologia , Agonistas de Receptores Adrenérgicos alfa 2/uso terapêutico , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/farmacologia , Analgésicos não Narcóticos/uso terapêutico , Animais , Dexmedetomidina/química , Dexmedetomidina/farmacologia , Dexmedetomidina/uso terapêutico , Desenho de Fármacos , Descoberta de Drogas/métodos , Humanos , Ligantes , Camundongos , Simulação de Acoplamento Molecular/métodos , Relação Estrutura-Atividade
9.
Elife ; 102021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34652270

RESUMO

Peripheral nerve injury-induced neuropathic pain is a chronic and debilitating condition characterized by mechanical hypersensitivity. We previously identified microglial activation via release of colony-stimulating factor 1 (CSF1) from injured sensory neurons as a mechanism contributing to nerve injury-induced pain. Here, we show that intrathecal administration of CSF1, even in the absence of injury, is sufficient to induce pain behavior, but only in male mice. Transcriptional profiling and morphologic analyses after intrathecal CSF1 showed robust immune activation in male but not female microglia. CSF1 also induced marked expansion of lymphocytes within the spinal cord meninges, with preferential expansion of regulatory T-cells (Tregs) in female mice. Consistent with the hypothesis that Tregs actively suppress microglial activation in females, Treg deficient (Foxp3DTR) female mice showed increased CSF1-induced microglial activation and pain hypersensitivity equivalent to males. We conclude that sexual dimorphism in the contribution of microglia to pain results from Treg-mediated suppression of microglial activation and pain hypersensitivity in female mice.


Assuntos
Fator Estimulador de Colônias de Macrófagos/genética , Microglia/metabolismo , Neuralgia/genética , Linfócitos T Reguladores/fisiologia , Animais , Feminino , Injeções Espinhais , Fator Estimulador de Colônias de Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Macrófagos/metabolismo , Masculino , Camundongos , Fatores Sexuais
10.
PLoS One ; 15(2): e0226289, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32015563

RESUMO

Calcium binding proteins are expressed throughout the central and peripheral nervous system and disruption of their activity has major consequences in a wide array of cellular processes, including transmission of nociceptive signals that are processed at the level of the spinal cord. We previously reported that the calcium binding protein, hippocalcin-like 4 (Hpcal4), is heavily expressed in interneurons of the superficial dorsal horn, and that its expression is significantly downregulated in a TR4 mutant mouse model that exhibits major pain and itch deficits due to loss of a subpopulation of excitatory interneurons. That finding suggested that Hpcal4 may be a contributor to the behavioral phenotype of the TR4 mutant mouse. To address this question, here we investigated the behavioral consequences of global deletion of Hpcal4 in a battery of acute and persistent pain and itch tests. Unexpectedly, with the exception of a mild reduction in acute baseline thermal responses, Hpcal4-deficient mice exhibit no major deficits in pain or itch responses, under normal conditions or in the setting of tissue or nerve injury. Taken together, our results indicate that the neural calcium sensor Hpcal4 likely makes a limited contribution to pain and itch processing.


Assuntos
Neurocalcina/metabolismo , Dor/metabolismo , Prurido/metabolismo , Animais , Escala de Avaliação Comportamental , Comportamento Animal , Cloroquina/administração & dosagem , Cloroquina/farmacologia , Técnicas de Inativação de Genes , Histamina/administração & dosagem , Histamina/farmacologia , Temperatura Alta , Interneurônios/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurocalcina/genética , Prurido/induzido quimicamente , Nervo Isquiático/lesões , Corno Dorsal da Medula Espinal/metabolismo
11.
Genetics ; 203(4): 1679-92, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27280690

RESUMO

The human fungal pathogen Candida albicans can reversibly switch between two cell types named "white" and "opaque," each of which is stable through many cell divisions. These two cell types differ in their ability to mate, their metabolic preferences and their interactions with the mammalian innate immune system. A highly interconnected network of eight transcriptional regulators has been shown to control switching between these two cell types. To identify additional regulators of the switch, we systematically and quantitatively measured white-opaque switching rates of 196 strains, each deleted for a specific transcriptional regulator. We identified 19 new regulators with at least a 10-fold effect on switching rates and an additional 14 new regulators with more subtle effects. To investigate how these regulators affect switching rates, we examined several criteria, including the binding of the eight known regulators of switching to the control region of each new regulatory gene, differential expression of the newly found genes between cell types, and the growth rate of each mutant strain. This study highlights the complexity of the transcriptional network that regulates the white-opaque switch and the extent to which switching is linked to a variety of metabolic processes, including respiration and carbon utilization. In addition to revealing specific insights, the information reported here provides a foundation to understand the highly complex coupling of white-opaque switching to cellular physiology.


Assuntos
Candida albicans/genética , Genes Fúngicos Tipo Acasalamento/genética , Elementos Reguladores de Transcrição/genética , Fatores de Transcrição/genética , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Epigênese Genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes/genética , Genes de Troca , Humanos , Fatores de Transcrição/biossíntese
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