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1.
Int J Antimicrob Agents ; 56(2): 106020, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32862840

RESUMO

The emergence of SARS-coronavirus-2 (SARS-CoV-2) has led to a global pandemic disease referred to as coronavirus disease 19 (COVID-19). Hydroxychloroquine (CLQ-OH)/azithromycin (ATM) combination therapy is currently being tested for the treatment of COVID-19, with promising results. However, the molecular mechanism of action of this combination is not yet established. Using molecular dynamics (MD) simulations, this study shows that the drugs act in synergy to prevent any close contact between the virus and the plasma membrane of host cells. Unexpected molecular similarity is shown between ATM and the sugar moiety of GM1, a lipid raft ganglioside acting as a host attachment cofactor for respiratory viruses. Due to this mimicry, ATM interacts with the ganglioside-binding domain of SARS-CoV-2 spike protein. This binding site shared by ATM and GM1 displays a conserved amino acid triad Q-134/F-135/N-137 located at the tip of the spike protein. CLQ-OH molecules are shown to saturate virus attachment sites on gangliosides in the vicinity of the primary coronavirus receptor, angiotensin-converting enzyme-2 (ACE-2). Taken together, these data show that ATM is directed against the virus, whereas CLQ-OH is directed against cellular attachment cofactors. We conclude that both drugs act as competitive inhibitors of SARS-CoV-2 attachment to the host-cell membrane. This is consistent with a synergistic antiviral mechanism at the plasma membrane level, where therapeutic intervention is likely to be most efficient. This molecular mechanism may explain the beneficial effects of CLQ-OH/ATM combination therapy in patients with COVID-19. Incidentally, the data also indicate that the conserved Q-134/F-135/N-137 triad could be considered as a target for vaccine strategies.


Assuntos
Azitromicina/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Gangliosídeo G(M1)/metabolismo , Hidroxicloroquina/farmacologia , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Ligação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Dinâmica Molecular , Pandemias , Peptidil Dipeptidase A/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos/efeitos dos fármacos , Alinhamento de Sequência
2.
In Vivo ; 34(5): 3023-3026, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32871846

RESUMO

BACKGROUND/AIM: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One drug that has attracted interest is the antiparasitic compound ivermectin, a macrocyclic lactone derived from the bacterium Streptomyces avermitilis. We carried out a docking study to determine if ivermectin might be able to attach to the SARS-CoV-2 spike receptor-binding domain bound with ACE2. MATERIALS AND METHODS: We used the program AutoDock Vina Extended to perform the docking study. RESULTS: Ivermectin docked in the region of leucine 91 of the spike and histidine 378 of the ACE2 receptor. The binding energy of ivermectin to the spike-ACE2 complex was -18 kcal/mol and binding constant was 5.8 e-08. CONCLUSION: The ivermectin docking we identified may interfere with the attachment of the spike to the human cell membrane. Clinical trials now underway should determine whether ivermectin is an effective treatment for SARS-Cov2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Ivermectina/química , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Betacoronavirus/química , Betacoronavirus/patogenicidade , Sítios de Ligação/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Histidina/química , Humanos , Ivermectina/uso terapêutico , Leucina/química , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral/virologia , Streptomyces/química
3.
Nat Commun ; 11(1): 4417, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887884

RESUMO

COVID-19 was declared a pandemic on March 11 by WHO, due to its great threat to global public health. The coronavirus main protease (Mpro, also called 3CLpro) is essential for processing and maturation of the viral polyprotein, therefore recognized as an attractive drug target. Here we show that a clinically approved anti-HCV drug, Boceprevir, and a pre-clinical inhibitor against feline infectious peritonitis (corona) virus (FIPV), GC376, both efficaciously inhibit SARS-CoV-2 in Vero cells by targeting Mpro. Moreover, combined application of GC376 with Remdesivir, a nucleotide analogue that inhibits viral RNA dependent RNA polymerase (RdRp), results in sterilizing additive effect. Further structural analysis reveals binding of both inhibitors to the catalytically active side of SARS-CoV-2 protease Mpro as main mechanism of inhibition. Our findings may provide critical information for the optimization and design of more potent inhibitors against the emerging SARS-CoV-2 virus.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/virologia , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/virologia , Prolina/análogos & derivados , Inibidores de Proteases/farmacologia , Pirrolidinas/farmacologia , Proteínas não Estruturais Virais/antagonistas & inibidores , Animais , Antivirais/farmacologia , Betacoronavirus/enzimologia , Sítios de Ligação/efeitos dos fármacos , Domínio Catalítico , Chlorocebus aethiops , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Modelos Moleculares , Pandemias , Prolina/farmacologia , RNA Replicase/antagonistas & inibidores , RNA Replicase/química , RNA Replicase/metabolismo , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/efeitos dos fármacos
4.
Protein Cell ; 11(10): 723-739, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32754890

RESUMO

Emerging and re-emerging RNA viruses occasionally cause epidemics and pandemics worldwide, such as the on-going outbreak of the novel coronavirus SARS-CoV-2. Herein, we identified two potent inhibitors of human DHODH, S312 and S416, with favorable drug-likeness and pharmacokinetic profiles, which all showed broad-spectrum antiviral effects against various RNA viruses, including influenza A virus, Zika virus, Ebola virus, and particularly against SARS-CoV-2. Notably, S416 is reported to be the most potent inhibitor so far with an EC50 of 17 nmol/L and an SI value of 10,505.88 in infected cells. Our results are the first to validate that DHODH is an attractive host target through high antiviral efficacy in vivo and low virus replication in DHODH knock-out cells. This work demonstrates that both S312/S416 and old drugs (Leflunomide/Teriflunomide) with dual actions of antiviral and immuno-regulation may have clinical potentials to cure SARS-CoV-2 or other RNA viruses circulating worldwide, no matter such viruses are mutated or not.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Oxirredutases/antagonistas & inibidores , Pandemias , Pneumonia Viral/tratamento farmacológico , Vírus de RNA/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Infecções por Coronavirus/virologia , Crotonatos/farmacologia , Síndrome da Liberação de Citocina/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Técnicas de Inativação de Genes , Humanos , Vírus da Influenza A/efeitos dos fármacos , Leflunomida/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico , Oseltamivir/uso terapêutico , Oxirredutases/metabolismo , Pneumonia Viral/virologia , Ligação Proteica/efeitos dos fármacos , Pirimidinas/biossíntese , Vírus de RNA/fisiologia , Relação Estrutura-Atividade , Toluidinas/farmacologia , Ubiquinona/metabolismo , Replicação Viral/efeitos dos fármacos
5.
Chaos ; 30(6): 061102, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32611087

RESUMO

There is an urgent necessity of effective medication against severe acute respiratory syndrome coronavirus 2 (SARS CoV-2), which is producing the COVID-19 pandemic across the world. Its main protease (Mpro) represents an attractive pharmacological target due to its involvement in essential viral functions. The crystal structure of free Mpro shows a large structural resemblance with the main protease of SARS CoV (nowadays known as SARS CoV-1). Here, we report that average SARS CoV-2 Mpro is 1900% more sensitive than SARS CoV-1 Mpro in transmitting tiny structural changes across the whole protein through long-range interactions. The largest sensitivity of Mpro to structural perturbations is located exactly around the catalytic site Cys-145 and coincides with the binding site of strong inhibitors. These findings, based on a simplified representation of the protein as a residue network, may help in designing potent inhibitors of SARS CoV-2 Mpro.


Assuntos
Betacoronavirus/metabolismo , Domínio Catalítico/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Pneumonia Viral/tratamento farmacológico , Inibidores de Proteases/farmacologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Cisteína Endopeptidases/efeitos dos fármacos , Desenho de Fármacos , Humanos , Pandemias , Vírus da SARS/metabolismo , Proteínas não Estruturais Virais/efeitos dos fármacos
6.
Int J Mol Sci ; 21(14)2020 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-32708196

RESUMO

(1) Background: Virtual screening studies on the therapeutically relevant proteins of the severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) require a detailed characterization of their druggable binding sites, and, more generally, a convenient pocket mapping represents a key step for structure-based in silico studies; (2) Methods: Along with a careful literature search on SARS-CoV-2 protein targets, the study presents a novel strategy for pocket mapping based on the combination of pocket (as performed by the well-known FPocket tool) and docking searches (as performed by PLANTS or AutoDock/Vina engines); such an approach is implemented by the Pockets 2.0 plug-in for the VEGA ZZ suite of programs; (3) Results: The literature analysis allowed the identification of 16 promising binding cavities within the SARS-CoV-2 proteins and the here proposed approach was able to recognize them showing performances clearly better than those reached by the sole pocket detection; and (4) Conclusions: Even though the presented strategy should require more extended validations, this proved successful in precisely characterizing a set of SARS-CoV-2 druggable binding pockets including both orthosteric and allosteric sites, which are clearly amenable for virtual screening campaigns and drug repurposing studies. All results generated by the study and the Pockets 2.0 plug-in are available for download.


Assuntos
Antivirais/química , Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Proteínas Virais/química , Sítios de Ligação/efeitos dos fármacos , Reposicionamento de Medicamentos , Humanos , Simulação de Acoplamento Molecular , Pandemias , Ligação Proteica/efeitos dos fármacos , Conformação Proteica
7.
BMC Mol Cell Biol ; 21(1): 49, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32611313

RESUMO

BACKGROUND: Following the recent outbreak of the new coronavirus pandemic (Covid-19), the rapid determination of the structure of the homo-trimeric spike glycoprotein has prompted the study reported here. The aims were to identify potential "druggable" binding pockets in the protein and, if located, to virtual screen pharmaceutical agents currently in use for predicted affinity to these pockets which might be useful to restrict, reduce, or inhibit the infectivity of the virion. RESULTS: Our analyses of this structure have revealed a key potentially druggable pocket where it might be viable to bind pharmaceutical agents to inhibit its ability to infect human cells. This pocket is found at the inter-chain interface that exists between two domains prior to the virion binding to human Angiotensin Converting Enzyme 2 (ACE2) protein. One of these domains is the highly mobile receptor binding domain, which must move into position to interact with ACE2, which is an essential feature for viral entry to the host cell. Virtual screening with a library of purchasable drug molecules has identified pharmaceuticals currently in use as prescription and over the counter medications that, in silico, readily bind into this pocket. CONCLUSIONS: This study highlights possible drugs already in use as pharmaceuticals that may act as agents to interfere with the movements of the domains within this protein essential for the infectivity processes and hence might slow, or even halt, the infection of host cells by this new coronavirus. As these are existing pharmaceuticals already approved for use in humans, this knowledge could accelerate their roll-out, through repurposing, for affected individuals and help guide the efforts of other researchers in finding effective treatments for the disease.


Assuntos
Antivirais/farmacologia , Sítios de Ligação/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Reposicionamento de Medicamentos , Pneumonia Viral/tratamento farmacológico , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequência de Aminoácidos , Betacoronavirus/efeitos dos fármacos , Humanos , Pandemias , Peptidil Dipeptidase A/metabolismo , Domínios Proteicos/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/química , Internalização do Vírus/efeitos dos fármacos
8.
Mol Pharmacol ; 98(2): 143-155, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32616523

RESUMO

The two-pore domain potassium channel (K2P-channel) THIK-1 has several predicted protein kinase A (PKA) phosphorylation sites. In trying to elucidate whether THIK-1 is regulated via PKA, we expressed THIK-1 channels in a mammalian cell line (CHO cells) and used the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) as a pharmacological tool to induce activation of PKA. Using the whole-cell patch-clamp recording, we found that THIK-1 currents were inhibited by application of IBMX with an IC50 of 120 µM. Surprisingly, intracellular application of IBMX or of the second messenger cAMP via the patch pipette had no effect on THIK-1 currents. In contrast, extracellular application of IBMX produced a rapid and reversible inhibition of THIK-1. In patch-clamp experiments with outside-out patches, THIK-1 currents were also inhibited by extracellular application of IBMX. Expression of THIK-1 channels in Xenopus oocytes was used to compare wild-type channels with mutated channels. Mutation of the putative PKA phosphorylation sites did not change the inhibitory effect of IBMX on THIK-1 currents. Mutational analysis of all residues of the (extracellular) helical cap of THIK-1 showed that mutation of the arginine residue at position 92, which is in the linker between cap helix 2 and pore helix 1, markedly reduced the inhibitory effect of IBMX. This flexible linker region, which is unique for each K2P-channel subtype, may be a possible target of channel-specific blockers. SIGNIFICANCE STATEMENT: The potassium channel THIK-1 is strongly expressed in the central nervous system. We studied the effect of 3-isobutyl-1-methyl-xanthine (IBMX) on THIK-1 currents. IBMX inhibits breakdown of cAMP and thus activates protein kinase A (PKA). Surprisingly, THIK-1 current was inhibited when IBMX was applied from the extracellular side of the membrane, but not from the intracellular side. Our results suggest that IBMX binds directly to the channel and that the inhibition of THIK-1 current was not related to activation of PKA.


Assuntos
1-Metil-3-Isobutilxantina/farmacologia , Canais de Potássio de Domínios Poros em Tandem/química , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Animais , Arginina/genética , Sítios de Ligação/efeitos dos fármacos , Células CHO , Cricetulus , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Mutação , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/genética , Ratos , Xenopus
9.
Nature ; 582(7810): 129-133, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494073

RESUMO

Mitochondria take up Ca2+ through the mitochondrial calcium uniporter complex to regulate energy production, cytosolic Ca2+ signalling and cell death1,2. In mammals, the uniporter complex (uniplex) contains four core components: the pore-forming MCU protein, the gatekeepers MICU1 and MICU2, and an auxiliary subunit, EMRE, essential for Ca2+ transport3-8. To prevent detrimental Ca2+ overload, the activity of MCU must be tightly regulated by MICUs, which sense changes in cytosolic Ca2+ concentrations to switch MCU on and off9,10. Here we report cryo-electron microscopic structures of the human mitochondrial calcium uniporter holocomplex in inhibited and Ca2+-activated states. These structures define the architecture of this multicomponent Ca2+-uptake machinery and reveal the gating mechanism by which MICUs control uniporter activity. Our work provides a framework for understanding regulated Ca2+ uptake in mitochondria, and could suggest ways of modulating uniporter activity to treat diseases related to mitochondrial Ca2+ overload.


Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Microscopia Crioeletrônica , Sítios de Ligação/efeitos dos fármacos , Cálcio/metabolismo , Cálcio/farmacologia , Canais de Cálcio/ultraestrutura , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
10.
Nature ; 584(7820): 298-303, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32555460

RESUMO

Metabotropic γ-aminobutyric acid receptors (GABAB) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis1. GABAB belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits2. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABAB. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.


Assuntos
Microscopia Crioeletrônica , Receptores de GABA-B/química , Receptores de GABA-B/ultraestrutura , Regulação Alostérica/efeitos dos fármacos , Apoproteínas/química , Apoproteínas/metabolismo , Apoproteínas/ultraestrutura , Sítios de Ligação/efeitos dos fármacos , Agonistas dos Receptores de GABA-B/química , Agonistas dos Receptores de GABA-B/metabolismo , Agonistas dos Receptores de GABA-B/farmacologia , Humanos , Modelos Moleculares , Domínios Proteicos/efeitos dos fármacos , Multimerização Proteica/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade
11.
Proc Natl Acad Sci U S A ; 117(16): 8890-8899, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32245806

RESUMO

Eastern equine encephalitis virus (EEEV), a mosquito-borne icosahedral alphavirus found mainly in North America, causes human and equine neurotropic infections. EEEV neurovirulence is influenced by the interaction of the viral envelope protein E2 with heparan sulfate (HS) proteoglycans from the host's plasma membrane during virus entry. Here, we present a 5.8-Å cryoelectron microscopy (cryo-EM) structure of EEEV complexed with the HS analog heparin. "Peripheral" HS binding sites were found to be associated with the base of each of the E2 glycoproteins that form the 60 quasi-threefold spikes (q3) and the 20 sites associated with the icosahedral threefold axes (i3). In addition, there is one HS site at the vertex of each q3 and i3 spike (the "axial" sites). Both the axial and peripheral sites are surrounded by basic residues, suggesting an electrostatic mechanism for HS binding. These residues are highly conserved among EEEV strains, and therefore a change in these residues might be linked to EEEV neurovirulence.


Assuntos
Desenho de Fármacos , Vírus da Encefalite Equina do Leste/ultraestrutura , Encefalomielite Equina/tratamento farmacológico , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/ultraestrutura , Animais , Antivirais/farmacologia , Antivirais/uso terapêutico , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Sulfatos de Condroitina/farmacologia , Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste/metabolismo , Encefalomielite Equina/virologia , Proteoglicanas de Heparan Sulfato/análogos & derivados , Heparina/metabolismo , Humanos , Mesocricetus , Estrutura Molecular , Relação Estrutura-Atividade , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Ligação Viral/efeitos dos fármacos
12.
Inorg Chem ; 59(5): 2711-2718, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32049511

RESUMO

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.


Assuntos
Auranofina/química , Auranofina/farmacologia , Compostos de Metilmercúrio/química , Compostos de Metilmercúrio/farmacologia , Selenocisteína/química , Tiorredoxinas/antagonistas & inibidores , Tiorredoxinas/química , Animais , Sítios de Ligação/efeitos dos fármacos , Ratos , Selenocisteína/metabolismo , Tiorredoxinas/metabolismo
13.
Int J Mol Sci ; 21(2)2020 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-31963646

RESUMO

Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.


Assuntos
Dissulfetos/química , Peptídeos/farmacologia , Receptores Imunológicos/metabolismo , Membro 14 de Receptores do Fator de Necrose Tumoral/metabolismo , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Peptídeos/síntese química , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptores Imunológicos/química , Membro 14 de Receptores do Fator de Necrose Tumoral/química
14.
PLoS One ; 15(1): e0218494, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31935212

RESUMO

Inhibiting vascular endothelial growth factor (VEGF) is a therapeutic option in diabetic microangiopathy. However, VEGF is needed at physiological concentrations to maintain glomerular integrity; complete VEGF blockade has deleterious effects on glomerular structure and function. Anti-VEGF therapy in diabetes raises the challenge of reducing VEGF-induced pathology without accelerating endothelial cell injury. Heparan sulfate (HS) act as a co-receptor for VEGF. Calcium dobesilate (CaD) is a small molecule with vasoprotective properties that has been used for the treatment of diabetic microangiopathy. Preliminary evidence suggests that CaD interferes with HS binding sites of fibroblast growth factor. We therefore tested the hypotheses that (1) CaD inhibits VEGF signaling in endothelial cells, (2) that this effect is mediated via interference between CaD and HS, and (3) that CaD ameliorates diabetic nephropathy in a streptozotocin-induced diabetic mouse model by VEGF inhibition. We found that CaD significantly inhibited VEGF165-induced endothelial cell migration, proliferation, and permeability. CaD significantly inhibited VEGF165-induced phosphorylation of VEGFR-2 and suppressed the activity of VEGFR-2 mediated signaling cascades. The effects of CaD in vitro were abrogated by heparin, suggesting the involvement of heparin-like domain in the interaction with CaD. In addition, VEGF121, an isoform which does not bind to heparin, was not inhibited by CaD. Using the proximity ligation approach, we detected inhibition of interaction in situ between HS and VEGF and between VEGF and VEGFR-2. Moreover, CaD reduced VEGF signaling in mice diabetic kidneys and ameliorated diabetic nephropathy and neuropathy, suggesting CaD as a VEGF inhibitor without the negative effects of complete VEGF blockade and therefore could be useful as a strategy in treating diabetic nephropathy.


Assuntos
Dobesilato de Cálcio/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Sítios de Ligação/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Células Endoteliais/efeitos dos fármacos , Heparitina Sulfato/metabolismo , Humanos , Cinética , Camundongos , Camundongos Endogâmicos NOD/genética , Camundongos Endogâmicos NOD/crescimento & desenvolvimento , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética
15.
Mol Pharmacol ; 97(3): 226-236, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31900312

RESUMO

Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein ßγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.


Assuntos
Descoberta de Drogas/métodos , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neutrófilos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Células Cultivadas , Cristalografia por Raios X/métodos , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Fatores de Troca do Nucleotídeo Guanina/química , Humanos , Neutrófilos/efeitos dos fármacos , Fosfatos de Fosfatidilinositol/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Peixe-Zebra
16.
Biochim Biophys Acta Gen Subj ; 1864(4): 129519, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31911242

RESUMO

BACKGROUND: Fragment-based ligand design is used for the development of novel ligands that target macromolecules, most notably proteins. Central to its success is the identification of fragment binding sites that are spatially adjacent such that fragments occupying those sites may be linked to create drug-like ligands. Current experimental and computational approaches that address this problem typically identify only a limited number of sites as well as use a limited number of fragment types. METHODS: The site-identification by ligand competitive saturation (SILCS) approach is extended to the identification of fragment bindings sites, with the method termed SILCS-Hotspots. The approach involves precomputation of the SILCS FragMaps following which the identification of Hotspots, performed by identifying of all possible fragment binding sites on the full 3D structure of the protein followed by spatial clustering. RESULTS: The SILCS-Hotspots approach identifies a large number of sites on the target protein, including many sites not accessible in experimental structures due to low binding affinities and binding sites on the protein interior. The identified sites are shown to recapitulate the location of known drug-like molecules in both allosteric and orthosteric binding sites on seven proteins including the androgen receptor, the CDK2 and Erk5 kinases, PTP1B phosphatase and three GPCRs; the ß2-adrenergic, GPR40 fatty-acid binding and M2-muscarinic receptors. Analysis indicates the importance of considering all possible fragment binding sites, and not just those accessible to experimental methods, when identifying novel binding sites and performing ligand design versus just considering the most favorable sites. The approach is shown to identify a larger number of known binding sites of drug-like molecules versus the commonly used FTMap and Fpocket methods. GENERAL SIGNIFICANCE: The present results indicate the potential utility of the SILCS-Hotspots approach for fragment-based rational design of ligands, including allosteric modulators.


Assuntos
Simulação de Acoplamento Molecular , Sítio Alostérico , Sítios de Ligação/efeitos dos fármacos , Quinase 5 Dependente de Ciclina/antagonistas & inibidores , Humanos , Ligantes , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Receptor Muscarínico M2/antagonistas & inibidores , Receptores Adrenérgicos beta 2/metabolismo , Receptores Androgênicos/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores
17.
Int J Mol Sci ; 21(1)2020 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947863

RESUMO

Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular "canonical" site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/ß- sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.


Assuntos
Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Domínios Proteicos/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Sítios de Ligação/efeitos dos fármacos , Desenho de Fármacos , Humanos , Ligantes , Modelos Moleculares , Quinolonas/química , Quinolonas/farmacologia , Receptores de GABA-A/química , Ácido gama-Aminobutírico/metabolismo
18.
Sci Adv ; 6(1): eaax5819, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911943

RESUMO

Autophagy is an evolutionarily conserved catabolic process, which plays a vital role in removing misfolded proteins and clearing damaged organelles to maintain internal environment homeostasis. Here, we uncovered the checkpoint kinase 2 (CHK2)-FOXK (FOXK1 and FOXK2) axis playing an important role in DNA damage-mediated autophagy at the transcriptional regulation layer. Mechanistically, following DNA damage, CHK2 phosphorylates FOXK and creates a 14-3-3γ binding site, which, in turn, traps FOXK proteins in the cytoplasm. Because FOXK functions as the transcription suppressor of ATGs, DNA damage-mediated FOXKs' cytoplasmic trapping induces autophagy. In addition, we found that a cancer-derived FOXK mutation induces FOXK hyperphosphorylation and enhances autophagy, resulting in chemoresistance. Cotreatment with cisplatin and chloroquine overcomes the chemoresistance caused by FOXK mutation. Overall, our study highlights a mechanism whereby DNA damage triggers autophagy by increasing autophagy genes via CHK2-FOXK-mediated transcriptional control, and misregulation of this pathway contributes to chemoresistance.


Assuntos
Autofagia/genética , Quinase do Ponto de Checagem 2/genética , Fatores de Transcrição Forkhead/genética , Neoplasias/tratamento farmacológico , Proteínas 14-3-3/genética , Células A549 , Sítios de Ligação/efeitos dos fármacos , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
19.
Eur J Med Chem ; 186: 111849, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31767137

RESUMO

Macrophage migration inhibitory factor (MIF) is a versatile protein that plays a role in inflammation, autoimmune diseases and cancers. Development of novel inhibitors will enable further exploration of MIF as a drug target. In this study, we investigated structure-activity relationships of MIF inhibitors using a MIF tautomerase activity assay to measure binding. Importantly, we notified that transition metals such as copper (II) and zinc (II) interfere with the MIF tautomerase activity under the assay conditions applied. EDTA was added to the assay buffer to avoid interference of residual heavy metals with tautomerase activity measurements. Using these assay conditions the structure-activity relationships for MIF binding of a series of triazole-phenols was explored. The most potent inhibitors in this series provided activities in the low micromolar range. Enzyme kinetic analysis indicates competitive binding that proved reversible. Binding to the enzyme was confirmed using a microscale thermophoresis (MST) assay. Molecular modelling was used to rationalize the observed structure-activity relationships. The most potent inhibitor 2d inhibited proliferation of A549 cells in a clonogenic assay. In addition, 2d attenuated MIF induced ERK phosphorylation in A549 cells. Altogether, this study provides insights in the structure-activity relationships for MIF binding of triazole-phenols and further validates this class of compounds as MIF binding agents in cell-based studies.


Assuntos
Macrófagos/efeitos dos fármacos , Fenóis/farmacologia , Triazóis/farmacologia , Células A549 , Sítios de Ligação/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Fenóis/química , Relação Estrutura-Atividade , Triazóis/química
20.
Cell Mol Life Sci ; 77(2): 351-363, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31222373

RESUMO

Cancer stem cells (CSC) are highly associated with poor prognosis in cancer patients. Our previous studies report that isorhapontigenin (ISO) down-regulates SOX2-mediated cyclin D1 induction and stem-like cell properties in glioma stem-like cells. The present study revealed that ISO could inhibit stem cell-like phenotypes and invasivity of human bladder cancer (BC) by specific attenuation of expression of CD44 but not SOX-2, at both the protein transcription and degradation levels. On one hand, ISO inhibited cd44 mRNA expression through decreases in Sp1 direct binding to its promoter region-binding site, resulting in attenuation of its transcription. On the other hand, ISO also down-regulated USP28 expression, which in turn reduced CD44 protein stability. Further studies showed that ISO treatment induced miR-4295, which specific bound to 3'-UTR activity of usp28 mRNA and inhibited its translation and expression, while miR-4295 induction was mediated by increased Dicer protein to enhance miR-4295 maturation upon ISO treatment. Our results provide the first evidence that ISO has a profound inhibitory effect on human BC stem cell-like phenotypes and invasivity through the mechanisms distinct from those previously noted in glioma stem-like cells.


Assuntos
Receptores de Hialuronatos/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Estilbenos/farmacologia , Regiões 3' não Traduzidas/efeitos dos fármacos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D1/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Células-Tronco , Transcrição Genética/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Neoplasias da Bexiga Urinária
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