RESUMO
Psychedelic substances such as lysergic acid diethylamide (LSD) and psilocybin show potential for the treatment of various neuropsychiatric disorders1-3. These compounds are thought to mediate their hallucinogenic and therapeutic effects through the serotonin (5-hydroxytryptamine (5-HT)) receptor 5-HT2A (ref. 4). However, 5-HT1A also plays a part in the behavioural effects of tryptamine hallucinogens5, particularly 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT), a psychedelic found in the toxin of Colorado River toads6. Although 5-HT1A is a validated therapeutic target7,8, little is known about how psychedelics engage 5-HT1A and which effects are mediated by this receptor. Here we map the molecular underpinnings of 5-MeO-DMT pharmacology through five cryogenic electron microscopy (cryo-EM) structures of 5-HT1A, systematic medicinal chemistry, receptor mutagenesis and mouse behaviour. Structure-activity relationship analyses of 5-methoxytryptamines at both 5-HT1A and 5-HT2A enable the characterization of molecular determinants of 5-HT1A signalling potency, efficacy and selectivity. Moreover, we contrast the structural interactions and in vitro pharmacology of 5-MeO-DMT and analogues to the pan-serotonergic agonist LSD and clinically used 5-HT1A agonists. We show that a 5-HT1A-selective 5-MeO-DMT analogue is devoid of hallucinogenic-like effects while retaining anxiolytic-like and antidepressant-like activity in socially defeated animals. Our studies uncover molecular aspects of 5-HT1A-targeted psychedelics and therapeutics, which may facilitate the future development of new medications for neuropsychiatric disorders.
Assuntos
5-Metoxitriptamina , Ansiolíticos , Antidepressivos , Metoxidimetiltriptaminas , Receptor 5-HT1A de Serotonina , Receptor 5-HT2A de Serotonina , Animais , Humanos , Masculino , Camundongos , 5-Metoxitriptamina/análogos & derivados , 5-Metoxitriptamina/química , 5-Metoxitriptamina/farmacologia , 5-Metoxitriptamina/uso terapêutico , Ansiolíticos/química , Ansiolíticos/farmacologia , Ansiolíticos/uso terapêutico , Antidepressivos/química , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Microscopia Crioeletrônica , Alucinógenos , Dietilamida do Ácido Lisérgico/química , Dietilamida do Ácido Lisérgico/farmacologia , Metoxidimetiltriptaminas/química , Metoxidimetiltriptaminas/farmacologia , Metoxidimetiltriptaminas/uso terapêutico , Modelos Moleculares , Receptor 5-HT1A de Serotonina/química , Receptor 5-HT1A de Serotonina/genética , Receptor 5-HT1A de Serotonina/metabolismo , Receptor 5-HT1A de Serotonina/ultraestrutura , Receptor 5-HT2A de Serotonina/química , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/ultraestrutura , Agonistas do Receptor de Serotonina/química , Agonistas do Receptor de Serotonina/farmacologia , Agonistas do Receptor de Serotonina/uso terapêutico , Relação Estrutura-AtividadeRESUMO
Modular polyketide synthases (PKSs) are giant assembly lines that produce an impressive range of biologically active compounds. However, our understanding of the structural dynamics of these megasynthases, specifically the delivery of acyl carrier protein (ACP)-bound building blocks to the catalytic site of the ketosynthase (KS) domain, remains severely limited. Using a multipronged structural approach, we report details of the inter-domain interactions after C-C bond formation in a chain-branching module of the rhizoxin PKS. Mechanism-based crosslinking of an engineered module was achieved using a synthetic substrate surrogate that serves as a Michael acceptor. The crosslinked protein allowed us to identify an asymmetric state of the dimeric protein complex upon C-C bond formation by cryo-electron microscopy (cryo-EM). The possible existence of two ACP binding sites, one of them a potential "parking position" for substrate loading, was also indicated by AlphaFold2 predictions. NMR spectroscopy showed that a transient complex is formed in solution, independent of the linker domains, and photochemical crosslinking/mass spectrometry of the standalone domains allowed us to pinpoint the interdomain interaction sites. The structural insights into a branching PKS module arrested after C-C bond formation allows a better understanding of domain dynamics and provides valuable information for the rational design of modular assembly lines.
Assuntos
Proteína de Transporte de Acila , Policetídeo Sintases , Policetídeo Sintases/metabolismo , Microscopia Crioeletrônica , Sítios de Ligação , Domínio Catalítico , Proteína de Transporte de Acila/metabolismoRESUMO
Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.
Assuntos
Proteína 1 de Troca de Ânion do Eritrócito , Bicarbonatos , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Bicarbonatos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sítios de Ligação , Domínios ProteicosRESUMO
As part of the infective process, Porphyromonas gingivalis must acquire heme which is indispensable for life and enables the microorganism to survive and multiply at the infection site. This oral pathogenic bacterium uses a newly discovered novel hmu heme uptake system with a leading role played by the HmuY hemophore-like protein, responsible for acquiring heme and increasing virulence of this periodontopathogen. We demonstrated that Prevotella intermedia produces two HmuY homologs, termed PinO and PinA. Both proteins were produced at higher mRNA and protein levels when the bacterium grew under low-iron/heme conditions. PinO and PinA bound heme, but preferentially under reducing conditions, and in a manner different from that of the P. gingivalis HmuY. The analysis of the three-dimensional structures confirmed differences between apo-PinO and apo-HmuY, mainly in the fold forming the heme-binding pocket. Instead of two histidine residues coordinating heme iron in P. gingivalis HmuY, PinO and PinA could use one methionine residue to fulfill this function, with potential support of additional methionine residue/s. The P. intermedia proteins sequestered heme only from the host albumin-heme complex under reducing conditions. Our findings suggest that HmuY-like family might comprise proteins subjected during evolution to significant diversification, resulting in different heme coordination modes. The newer data presented in this manuscript on HmuY homologs produced by P. intermedia sheds more light on the novel mechanism of heme uptake, could be helpful in discovering their biological function, and in developing novel therapeutic approaches.
Assuntos
Heme/genética , Hemeproteínas/genética , Periodontite/genética , Prevotella intermedia/genética , Regulação Bacteriana da Expressão Gênica/genética , Heme/química , Hemeproteínas/química , Humanos , Ferro/metabolismo , Periodontite/microbiologia , Periodontite/patologia , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.
Assuntos
Esclerose Lateral Amiotrófica/patologia , Antioxidantes/farmacologia , Azóis/farmacologia , Compostos Organosselênicos/farmacologia , Dobramento de Proteína/efeitos dos fármacos , Superóxido Dismutase-1/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Antioxidantes/uso terapêutico , Asma/tratamento farmacológico , Asma/patologia , Azóis/uso terapêutico , Cristalografia por Raios X , Cisteína/química , Dissulfetos/química , Edaravone/farmacologia , Células HEK293 , Humanos , Isoindóis , Chaperonas Moleculares/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Compostos Organosselênicos/uso terapêutico , Ligação Proteica , Multimerização Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Superóxido Dismutase-1/química , Superóxido Dismutase-1/genéticaRESUMO
Cytochrome bc1 is a proven drug target in the prevention and treatment of malaria. The rise in drug-resistant strains of Plasmodium falciparum, the organism responsible for malaria, has generated a global effort in designing new classes of drugs. Much of the design/redesign work on overcoming this resistance has been focused on compounds that are presumed to bind the Q(o) site (one of two potential binding sites within cytochrome bc1 using the known crystal structure of this large membrane-bound macromolecular complex via in silico modeling. Cocrystallization of the cytochrome bc1 complex with the 4(1H)-pyridone class of inhibitors, GSK932121 and GW844520, that have been shown to be potent antimalarial agents in vivo, revealed that these inhibitors do not bind at the Q(o) site but bind at the Q(i )site. The discovery that these compounds bind at the Q(i) site may provide a molecular explanation for the cardiotoxicity and eventual failure of GSK932121 in phase-1 clinical trial and highlight the need for direct experimental observation of a compound bound to a target site before chemical optimization and development for clinical trials. The binding of the 4(1H)-pyridone class of inhibitors to Q(i) also explains the ability of this class to overcome parasite Q(o)-based atovaquone resistance and provides critical structural information for future design of new selective compounds with improved safety profiles.