RESUMO
The endoplasmic reticulum (ER) is a dynamic structure, playing multiple roles including calcium storage, protein synthesis and lipid metabolism. During cellular stress, variations in ER homeostasis and its functioning occur. This condition is referred as ER stress and generates a cascade of signaling events termed unfolded protein response (UPR), activated as adaptative response to mitigate the ER stress condition. In this regard, calcium levels play a pivotal role in ER homeostasis and therefore in cell fate regulation since calcium signaling is implicated in a plethora of physiological processes, but also in disease conditions such as neurodegeneration, cancer and metabolic disorders. A large body of emerging evidence highlighted the functional role of TRP channels and their ability to promote cell survival or death depending on endoplasmic reticulum stress resolution, making them an attractive target. Thus, in this review we focused on the TRP channels' correlation to UPR-mediated ER stress in disease pathogenesis, providing an overview of their implication in the activation of this cellular response.
Assuntos
Cálcio , Estresse do Retículo Endoplasmático , Cálcio/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Resposta a Proteínas não Dobradas , Retículo Endoplasmático/metabolismo , Sinalização do CálcioRESUMO
Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug-protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC50 1.25 ± 0.26 µM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 µM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.
Assuntos
Canais de Cátion TRPM/antagonistas & inibidores , Animais , Descoberta de Drogas/métodos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Relação Estrutura-AtividadeRESUMO
Different molecular mechanisms contribute to the development of multidrug resistance in cancer, including increased drug efflux, enhanced cellular repair mechanisms and alterations of drug metabolism or drug targets. ABCG2 is a member of the ATP-binding cassette superfamily transporters that promotes drug efflux, inducing chemotherapeutic resistance in malignant cells. In this context, the development of selective ABCG2 inhibitors might be a suitable strategy to improve chemotherapy efficacy. Thus, through a multidisciplinary approach, we identified a new ABCG2 selective inhibitor (8), highlighting its ability to increase mitoxantrone cytotoxicity in both hepatocellular carcinoma (EC50from 8.67 ± 2.65 to 1.25 ± 0.80 µM) and transfected breast cancer cell lines (EC50from 9.92 ± 2.32 to 2.45 ± 1.40 µM). Moreover, mitoxantrone co-administration in both transfected and non-transfected HEK293 revealed that compound 8 notably lowered the mitoxantrone EC50, demonstrating its efficacy along with the importance of the ABCG2 extrusion pump overexpression in MDR reversion. These results were corroborated by evaluating the effect of inhibitor 8 on mitoxantrone cell uptake in multicellular tumor spheroids and via proteomic experiments.
Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/química , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Células HEK293 , Mitoxantrona/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , ProteômicaRESUMO
The transient receptor potential melastatin subtype 8 (TRPM8) is a cold sensor in humans, activated by low temperatures (>10, <28 °C), but also a polymodal ion channel, stimulated by voltage, pressure, cooling compounds (menthol, icilin), and hyperosmolarity. An increased number of experimental results indicate the implication of TRPM8 channels in cold thermal transduction and pain detection, transmission, and maintenance in different tissues and organs. These channels also have a repercussion on different kinds of life-threatening tumors and other pathologies, which include urinary and respiratory tract dysfunctions, dry eye disease, and obesity. This compendium firstly covers newly described papers on the expression of TRPM8 channels and their correlation with pathological states. An overview on the structural knowledge, after cryo-electron microscopy success in solving different TRPM8 structures, as well as some insights obtained from mutagenesis studies, will follow. Most recently described families of TRPM8 modulators are also covered, along with a section of molecules that have reached clinical trials. To finalize, authors provide an outline of the potential prospects in the TRPM8 field.
Assuntos
Temperatura Baixa , Canais de Cátion TRPM , Sensação Térmica , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/genética , Síndromes do Olho Seco/metabolismo , Humanos , Obesidade/tratamento farmacológico , Obesidade/genética , Obesidade/metabolismo , Doenças Respiratórias/tratamento farmacológico , Doenças Respiratórias/genética , Doenças Respiratórias/metabolismo , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Doenças Urológicas/tratamento farmacológico , Doenças Urológicas/genética , Doenças Urológicas/metabolismoRESUMO
Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a Ca2+ non-selective ion channel implicated in a variety of pathological conditions, including cancer, inflammatory and neuropathic pain. In previous works we identified a family of chiral, highly hydrophobic ß-lactam derivatives, and began to intuit a possible effect of the stereogenic centers on the antagonist activity. To investigate the influence of configuration on the TRPM8 antagonist properties, here we prepare and characterize four possible diastereoisomeric derivatives of 4-benzyl-1-[(3'-phenyl-2'-dibenzylamino)prop-1'-yl]-4-benzyloxycarbonyl-3-methyl-2-oxoazetidine. In microfluorography assays, all isomers were able to reduce the menthol-induced cell Ca2+ entry to larger or lesser extent. Potency follows the order 3R,4R,2'R > 3S,4S,2'R â 3R,4R,2'S > 3S,4S,2'S, with the most potent diastereoisomer showing a half inhibitory concentration (IC50) in the low nanomolar range, confirmed by Patch-Clamp electrophysiology experiments. All four compounds display high receptor selectivity against other members of the TRP family. Furthermore, in primary cultures of rat dorsal root ganglion (DRG) neurons, the most potent diastereoisomers do not produce any alteration in neuronal excitability, indicating their high specificity for TRPM8 channels. Docking studies positioned these ß-lactams at different subsites by the pore zone, suggesting a different mechanism than the known N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide (AMTB) antagonist.
Assuntos
Neurônios/metabolismo , Fenilalanina/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , beta-Lactamas/farmacologia , Animais , Células Cultivadas , Gânglios Espinais/metabolismo , Simulação de Acoplamento Molecular , Neurônios/efeitos dos fármacos , Fenilalanina/análogos & derivados , Fenilalanina/química , Ratos , Relação Estrutura-Atividade , beta-Lactamas/químicaRESUMO
In this paper, we report studies concerning four variants of the G-quadruplex forming anti-HIV-integrase aptamer T30923, in which specific 2'-deoxyguanosines have been singly replaced by 8-methyl-2'-deoxyguanosine residues, with the aim to exploit the methyl group positioned in the G-quadruplex grooves as a steric probe to investigate the interaction aptamer/target. Although, the various modified aptamers differ in the localization of the methyl group, NMR, circular dichroism (CD), electrophoretic and molecular modeling data suggest that all of them preserve the ability to fold in a stable dimeric parallel G-quadruplex complex resembling that of their natural counterpart T30923. However, the biological data have shown that the T30923 variants are characterized by different efficiencies in inhibiting the HIV-integrase, thus suggesting the involvement of the G-quadruplex grooves in the aptamer/target interaction.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Quadruplex G , Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , Oligonucleotídeos/farmacologia , Dicroísmo Circular , Dimerização , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Temperatura de TransiçãoRESUMO
Several peptides play an important role in physiological and pathological conditions into the cardiovascular system. In addition to well-known vasoactive agents such as angiotensin II, endothelin, serotonin or natriuretic peptides, the vasoconstrictor Urotensin-II (Uro-II) and the vasodilators Urocortins (UCNs) and Adrenomedullin (AM) have been implicated in the control of vascular tone and blood pressure as well as in cardiovascular disease states including congestive heart failure, atherosclerosis, coronary artery disease, and pulmonary and systemic hypertension. Therefore these peptides, together with their receptors, become important therapeutic targets in cardiovascular diseases (CVDs). Circulating levels of these agents in the blood are markedly modified in patients with specific CVDs compared with those in healthy patients, becoming also potential biomarkers for these pathologies. This review will provide an overview of current knowledge about the physiological roles of Uro-II, UCN and AM in the cardiovascular system and their implications in cardiovascular diseases. It will further focus on the structural modifications carried out on original peptide sequences in the search of analogues with improved physiochemical properties as well as in the delivery methods. Finally, we have overviewed the possible application of these peptides and/or their precursors as biomarkers of CVDs.
Assuntos
Adrenomedulina/uso terapêutico , Produtos Biológicos/uso terapêutico , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/tratamento farmacológico , Urocortinas/uso terapêutico , Urotensinas/uso terapêutico , Animais , Biomarcadores/metabolismo , HumanosRESUMO
The transient receptor potential melastatin subtype 8 (TRPM8) is a nonselective, multimodal ion channel, activated by low temperatures (<28 °C), pressure, and cooling compounds (menthol, icilin). Experimental evidences indicated a role of TRPM8 in cold thermal transduction, different life-threatening tumors, and other pathologies, including migraine, urinary tract dysfunction, dry eye disease, and obesity. Hence, the modulation of the TRPM8 channel could be essential in order to understand its implications in these pathologies and for therapeutic intervention. This short review will cover recent progress on the TRPM8 agonists and antagonists, describing newly reported chemotypes, and their application in the pharmacological characterization of TRPM8 in health and disease. The recently described structures of the TRPM8 channel alone or complexed with known agonists and PIP2 are also discussed.
Assuntos
Moduladores de Transporte de Membrana/química , Canais de Cátion TRPM/agonistas , Animais , Sítios de Ligação , Humanos , Moduladores de Transporte de Membrana/farmacologia , Ligação Proteica , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismoRESUMO
BACKGROUND: Transient Receptor Potential Melastatin-8 (TRPM8) is a non-selective cation channel activated by cold temperature and by cooling agents. Several studies have proved that this channel is involved in pain perception. Although some studies indicate that TRPM8 inhibition is necessary to reduce acute and chronic pain, it is also reported that TRPM8 activation produces analgesia. These conflicting results could be explained by extracellular Ca2+-dependent desensitization that is induced by an excessive activation. Likely, this effect is due to phosphatidylinositol 4,5-bisphosphate (PIP2) depletion that leads to modification of TRPM8 channel activity, shifting voltage dependence towards more positive potentials. This phenomenon needs further evaluation and confirmation that would allow us to understand better the role of this channel and to develop new therapeutic strategies for controlling pain. EXPERIMENTAL APPROACH: To understand the role of TRPM8 in pain perception, we tested two specific TRPM8-modulating compounds, an antagonist (IGM-18) and an agonist (IGM-5), in either acute or chronic animal pain models using male Sprague-Dawley rats or CD1 mice, after systemic or topical routes of administration. RESULTS: IGM-18 and IGM-5 were fully characterized in vivo. The wet-dog shake test and the body temperature measurements highlighted the antagonist activity of IGM-18 on TRPM8 channels. Moreover, IGM-18 exerted an analgesic effect on formalin-induced orofacial pain and chronic constriction injury-induced neuropathic pain, demonstrating the involvement of TRPM8 channels in these two pain models. Finally, the results were consistent with TRPM8 downregulation by agonist IGM-5, due to its excessive activation. CONCLUSIONS: TRPM8 channels are strongly involved in pain modulation, and their selective antagonist is able to reduce both acute and chronic pain.
Assuntos
Analgésicos , Percepção da Dor/efeitos dos fármacos , Dor , Canais de Cátion TRPM , Analgésicos/química , Analgésicos/farmacologia , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Dor/tratamento farmacológico , Dor/metabolismo , Dor/patologia , Dor/fisiopatologia , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismoRESUMO
Bovine lactoferrin C-lobe is able to prevent both influenza virus hemagglutination and cell infection. In particular, it was demonstrated that the fragment 418SKHSSLDCVLRP429 is a potent antiviral peptide. Therefore, we tried to increase the stability of this fragment through side-chain lactam cyclization of the peptide, S[KHSSLD]CVLRP (1). However, classic strategy involving solid-supported cyclization of the linear precursor, containing orthogonal allyl/alloc-based protection for the key amino and carboxyl residues, did not provide the desired cyclic peptide. Here, we report the identification of problematic stretches during the sequence assembly process and the optimization of the different parameters involved in the construction of 1. Results indicated a significant influence of ß-protecting group of both aspartic acid and adjacent cysteine residues on the formation of side products. Therefore, the identification of suitable ß-protecting groups of these residues allowed us to optimize the synthesis of designed lactam-bridged cyclic peptide.
Assuntos
Lactamas/química , Lactoferrina/síntese química , Peptídeos Cíclicos/química , Animais , Ácido Aspártico/química , Bovinos , Ciclização , Cisteína/química , Lactoferrina/químicaRESUMO
An acid- and oxidant-promoted intramolecular cyclization of a tetrahydro-ß-carboline-based dipeptide has been developed to prepare new indole-fused aminoacetals. This approach involves N-acyliminium formation from readily available precursors and cyclization under mild reaction conditions. The diastereoselectivity in the formation of the products is influenced by the specific substituents of the starting reagents, which has been rationalized analyzing the energy profile of the related reactions and the relative stability of the proposed structures based on DFT computational methods.
RESUMO
The p53-MDM2 interaction is a well-known protein-protein contact, and its disruption is a key event for p53 activation and induction of its oncosuppressor response. The design of small molecules that can block the p53-MDM2 interaction and reactivate the p53 function is a promising strategy for cancer therapy. To date, several compounds have been identified as p53-MDM2 inhibitors, and X-ray structures of MDM2 complexed with several ligands are available in the Brookhaven Protein Data Bank. These data have been exploited to compile a hierarchical virtual screening protocol. The first steps were aimed at selecting a focused library, which was submitted in parallel to docking and pharmacophore model alignment. Selected compounds were subjected to inhibition assays of both cellular vitality (MTT) and p53-MDM2 interaction (ELISA and co-immunoprecipitation), disclosing four nanomolar inhibitors.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Humanos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica/efeitos dos fármacos , Conformação Proteica em alfa-Hélice , Proteínas Proto-Oncogênicas c-mdm2/química , Relação Estrutura-Atividade , Proteína Supressora de Tumor p53/química , Interface Usuário-ComputadorRESUMO
The urotensin II receptor (UTR) has long been studied mainly for its involvement in the cardiovascular homeostasis both in health and disease state. Two endogenous ligands activate UTR, i.e. urotensin II (U-II) and urotensin II-related peptide (URP). Extensive expression of the two ligands uncovers the diversified pathophysiological effects mediated by the urotensinergic system such as cardiovascular disorders, smooth muscle cell proliferation, renal disease, diabetes, and tumour growth. As newly reported, U-II and URP have distinct effects on transcriptional activity, cell proliferation, and myocardial contractile activities supporting the idea that U-II and URP interact with UTR in a distinct manner (biased agonism). To shed light on the origin of the divergent activities of the two endogenous ligands, we performed a conformational study on URP by solution NMR in sodium dodecyl sulfate micelle solution and compared the obtained NMR structure of URP with that of hU-II previously determined. Finally, we undertook docking studies between URP, hU-II, and an UT receptor model.
Assuntos
Hormônios Peptídicos/agonistas , Hormônios Peptídicos/química , Receptores Acoplados a Proteínas G/metabolismo , Urotensinas/agonistas , Urotensinas/química , Sequência de Aminoácidos , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Acoplamento Molecular , Hormônios Peptídicos/síntese química , Hormônios Peptídicos/metabolismo , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Dodecilsulfato de Sódio/química , Relação Estrutura-Atividade , Urotensinas/metabolismoRESUMO
The frog skin peptide temporin L (TL, 13-residues long) has a wide and potent spectrum of antimicrobial activity, but it is also toxic on mammalian cells at its microbicidal concentrations. Previous studies have indicated that its analogue [Pro(3)]TL has a slightly reduced hemolytic activity and a stable helical conformation along residues 6-13. Here, to expand our knowledge on the relationship between the extent/position of α-helix in TL and its biological activities, we systematically replaced single amino acids within the α-helical domain of [Pro(3)]TL with the corresponding d isomers, known as helix breakers. Structure-activity relationship studies of these analogues, by means of CD and NMR spectroscopy analyses as well as antimicrobial and hemolytic assays were performed. Besides increasing our understanding on the structural elements that are responsible for cell selectivity of TL, this study revealed that a single l to d amino acid substitution can preserve strong anti-Candida activity of [Pro(3)]TL, without giving a toxic effect towards human cells.
Assuntos
Aminoácidos/química , Candida/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Acinetobacter baumannii/metabolismo , Substituição de Aminoácidos , Peptídeos Catiônicos Antimicrobianos , Candida albicans/metabolismo , Dicroísmo Circular , Eritrócitos/efeitos dos fármacos , Escherichia coli/metabolismo , Fluoresceínas/química , Humanos , Espectroscopia de Ressonância Magnética/métodos , Micelas , Testes de Sensibilidade Microbiana , Conformação Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas aeruginosa/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Staphylococcus/metabolismo , Staphylococcus aureus/metabolismo , Yersinia pseudotuberculosis/metabolismoRESUMO
G protein-coupled receptor kinase 2 (GRK2) plays a central role in the cellular transduction network. In particular, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. Thereby, its inhibition offers a potential therapeutic solution to several pathological conditions. In the present study, we performed a SAR study and a NMR conformational analysis of peptides derived from HJ loop of GRK2 and able to selectively inhibit GRK2. From Ala-scan and D-Ala point replacement, we found that Arg residues don't affect the inhibitory properties, while a D-amino acid at position 5 is key to the activity. Conformational analysis identified two ß-turns that involve N-terminal residues, followed by a short extended region. These information can help the design of peptides and peptido-mimetics with enhanced GRK2 inhibition properties.
Assuntos
Quinase 2 de Receptor Acoplado a Proteína G , Peptídeos , Peptídeos/metabolismo , Fosforilação , Estrutura Secundária de ProteínaRESUMO
Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have previously identified the compound termed urantide (H-Asp-c[Pen-Phe-DTrp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor (UTR) antagonist described to date. Urantide may have potential clinical value in the treatment of atherosclerosis. In the present study, we studied the conformational preferences of urantide in DPC micelles and developed a urantide/UTR interaction model. This model can help the design of novel peptides and small molecules as UTR antagonists.
Assuntos
Simulação de Acoplamento Molecular , Fragmentos de Peptídeos/química , Receptores Acoplados a Proteínas G/química , Urotensinas/química , Sítios de Ligação , Desenho Assistido por Computador , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Conformação Proteica , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Urotensinas/metabolismo , Urotensinas/farmacologiaRESUMO
Gain-of-function (GoF) variants in KCNT1 channels cause severe, drug-resistant forms of epilepsy. Quinidine is a known KCNT1 blocker, but its clinical use is limited due to severe drawbacks. To identify novel KCNT1 blockers, a homology model of human KCNT1 was built and used to screen an in-house library of compounds. Among the 20 molecules selected, five (CPK4, 13, 16, 18, and 20) showed strong KCNT1-blocking ability in an in vitro fluorescence-based assay. Patch-clamp experiments confirmed a higher KCNT1-blocking potency of these compounds when compared to quinidine, and their selectivity for KCNT1 over hERG and Kv7.2 channels. Among identified molecules, CPK20 displayed the highest metabolic stability; this compound also blocked KCNT2 currents, although with a lower potency, and counteracted GoF effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T). The present results provide solid rational basis for future design of novel compounds to counteract KCNT1-related neurological disorders.
Assuntos
Epilepsia , Humanos , Epilepsia/tratamento farmacológico , Epilepsia/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Bloqueadores dos Canais de Potássio/farmacologia , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Animais , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Relação Estrutura-Atividade , Células HEK293 , Simulação por Computador , Canais de Potássio Ativados por SódioRESUMO
In this paper we present the design, synthesis, and biological evaluation of a new series of peptidomimetics acting as potent anti-SARS-CoV-2 agents. Starting from our previously described Main Protease (MPro) and Papain Like Protease (PLPro) dual inhibitor, CV11, here we disclose its high inhibitory activity against cathepsin L (CTSL) (IC50 = 19.80 ± 4.44 nM), an emerging target in SARS-CoV-2 infection machinery. An in silico design, inspired by the structure of CV11, led to the development of a library of peptidomimetics showing interesting activities against CTSL and Mpro, allowing us to trace the chemical requirements for the binding to both enzymes. The screening in Vero cells infected with 5 different SARS-CoV-2 variants of concerns, highlighted sub-micromolar activities for most of the synthesized compounds (13, 15, 16, 17 and 31) in agreement with the enzymatic inhibition assays results. The compounds showed lack of activity against several different RNA viruses except for the 229E and OC43 human coronavirus strains, also characterized by a cathepsin-L dependent release into the host cells. The most promising derivatives were also evaluated for their chemical and metabolic in-vitro stability, with derivatives 15 and 17 showing a suitable profile for further preclinical characterization.
Assuntos
COVID-19 , Peptidomiméticos , Chlorocebus aethiops , Humanos , Animais , Catepsina L , SARS-CoV-2 , Peptidomiméticos/farmacologia , Inibidores de Proteases/farmacologia , Células Vero , Peptídeo Hidrolases , Antivirais/farmacologia , Simulação de Acoplamento MolecularRESUMO
Haspin is an emerging, but rather unexplored, divergent kinase involved in tumor growth by regulating the mitotic phase. In this paper, the in-silico design, synthesis, and biological characterization of a new series of substituted indoles acting as potent Haspin inhibitors are reported. The synthesized derivatives have been evaluated by FRET analysis, showing very potent Haspin inhibition. Then, a comprehensive in-cell investigation highlighted compounds 47 and 60 as the most promising inhibitors. These compounds were challenged for their synergic activity with paclitaxel in 2D and 3D cellular models, demonstrating a twofold improvement of the paclitaxel antitumor activity. Compound 60 also showed remarkable selectivity when tested in a panel of 70 diverse kinases. Finally, in-silico studies provided new insight about the chemical requirements useful to develop new Haspin inhibitors. Biological results, together with the drug-likeness profile of 47 and 60, make these derivatives deserving further studies.
Assuntos
Indóis , Indóis/farmacologia , Indóis/química , Indóis/síntese química , Humanos , Relação Estrutura-Atividade , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Linhagem Celular Tumoral , Paclitaxel/farmacologia , Paclitaxel/química , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos AntitumoraisRESUMO
The cannabinoid system is one of the most investigated neuromodulatory systems because of its involvement in multiple pathologies such as cancer, inflammation, and psychiatric diseases. Recently, the CB2 receptor has gained increased attention considering its crucial role in modulating neuroinflammation in several pathological conditions like neurodegenerative diseases. Here we describe the rational design of pyrrole-based analogues, which led to a potent and pharmacokinetically suitable CB2 full agonist particularly effective in improving cognitive functions in a scopolamine-induced amnesia murine model. Therefore, we extended our study by investigating the interconnection between CB2 activation and neurotransmission in this experimental paradigm. To this purpose, we performed a MALDI imaging analysis on mice brains, observing that the administration of our lead compound was able to revert the effect of scopolamine on different neurotransmitter tones, such as acetylcholine, serotonin, and GABA, shedding light on important networks not fully explored, so far.