RESUMO
CK1δ is a serine-threonine kinase involved in several pathological conditions including neuroinflammation and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and Amyotrophic Lateral Sclerosis. Specifically, it seems that an inhibition of CK1δ could have a neuroprotective effect in these conditions. Here, a series of [1,2,4]triazolo[1,5-a][1,3,5]triazines were developed as ATP-competitive CK1δ inhibitors. Both positions 2 and 5 have been explored leading to a total of ten compounds exhibiting IC50s comprised between 29.1 µM and 2.08 µM. Three of the four most potent compounds (IC50 < 3 µM) bear a thiophene ring at the 2 position. All compounds have been submitted to computational studies that identified the chain composed of at least 2 atoms (e.g., nitrogen and carbon atoms) at the 5 position as crucial to determine a key bidentate hydrogen bond with Leu85 of CK1δ. Most potent compounds have been tested in vitro, resulting passively permeable to the blood-brain barrier and, safe and slight neuroprotective on a neuronal cell model. These results encourage to further structural optimize the series to obtain more potent CK1δ inhibitors as possible neuroprotective agents to be tested on models of the above-mentioned neurodegenerative diseases.
Assuntos
Caseína Quinase Idelta , Inibidores de Proteínas Quinases , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/síntese química , Humanos , Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/metabolismo , Relação Estrutura-Atividade , Estrutura Molecular , Triazinas/química , Triazinas/farmacologia , Triazinas/síntese química , Relação Dose-Resposta a Droga , Triazóis/química , Triazóis/farmacologia , Triazóis/síntese química , Animais , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/síntese química , Simulação de Acoplamento MolecularRESUMO
Protein kinase CK1δ (CK1δ) is a serine-threonine/kinase that modulates different physiological processes, including the cell cycle, DNA repair, and apoptosis. CK1δ overexpression, and the consequent hyperphosphorylation of specific proteins, can lead to sleep disorders, cancer, and neurodegenerative diseases. CK1δ inhibitors showed anticancer properties as well as neuroprotective effects in cellular and animal models of Parkinson's and Alzheimer's diseases and amyotrophic lateral sclerosis. To obtain new ATP-competitive CK1δ inhibitors, three sets of benzimidazole-2-amino derivatives were synthesized (1-32), bearing different substituents on the fused benzo ring (R) and diverse pyrazole-containing acyl moieties on the 2-amino group. The best-performing derivatives were those featuring the (1H-pyrazol-3-yl)-acetyl moiety on the benzimidazol-2-amino scaffold (13-32), which showed CK1δ inhibitor activity in the low micromolar range. Among the R substituents, 5-cyano was the most advantageous, leading to a compound endowed with nanomolar potency (23, IC50 = 98.6 nM). Molecular docking and dynamics studies were performed to point out the inhibitor-kinase interactions.
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Rare diseases are particular pathological conditions affecting a limited number of people and few drugs are known to be effective as therapeutic treatment. Gaucher disease, caused by a deficiency of the lysosomal enzyme glucocerebrosidase, belongs to this class of disorders, and it is considered the most common among the Lysosomal Storage Diseases. The two main therapeutic approaches are the Enzyme Replacement Therapy (ERT) and the Substrate Reduction Therapy (SRT). ERT, consisting in replacing the defective enzyme by administering a recombinant enzyme, is effective in alleviating the visceral symptoms, hallmarks of the most common subtype of the disease whereas it has no effects when symptoms involve CNS, since the recombinant protein is unable to significantly cross the Blood Brain Barrier. The SRT strategy involves inhibiting glucosylceramide synthase (GCS), the enzyme responsible for the production of the associated storage molecule. The rational design of new inhibitors of GCS has been hampered by the lack of either the crystal structure of the enzyme or an in-silico model of the active site which could provide important information regarding the interactions of potential inhibitors with the target, but, despite this, interesting results have been obtained and are herein reviewed.
Assuntos
Inibidores Enzimáticos , Doença de Gaucher , Doença de Gaucher/tratamento farmacológico , Humanos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/síntese química , Glucosiltransferases/antagonistas & inibidores , Glucosiltransferases/metabolismo , Química Farmacêutica , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/metabolismo , Glucosilceramidase/química , Terapia de Reposição de Enzimas , Estrutura MolecularRESUMO
Adenosine receptors are largely distributed in our organism and are promising therapeutic targets for the treatment of many pathologies. In this perspective, investigating the structural features of the ligands leading to affinity and/or selectivity is of great interest. In this work, we have focused on a small series of pyrazolo-triazolo-pyrimidine antagonists substituted in positions 2, 5, and N8, where bulky acyl moieties at the N5 position and small alkyl groups at the N8 position are associated with affinity and selectivity at the A3 adenosine receptor even if a good affinity toward the A2B adenosine receptor has also been observed. Conversely, a free amino function at the 5 position induces high affinity at the A2A and A1 receptors with selectivity vs. the A3 subtype. A molecular modeling study suggests that differences in affinity toward A1, A2A, and A3 receptors could be ascribed to two residues: one in the EL2, E168 in human A2A/E172 in human A1, that is occupied by the hydrophobic residue V169 in the human A3 receptor; and the other in TM6, occupied by H250/H251 in human A2A and A1 receptors and by a less bulky S247 in the A3 receptor. In the end, these findings could help to design new subtype-selective adenosine receptor ligands.
Assuntos
Antagonistas de Receptores Purinérgicos P1 , Receptores Purinérgicos P1 , Humanos , Relação Estrutura-Atividade , Antagonistas de Receptores Purinérgicos P1/farmacologia , Modelos Moleculares , Pirimidinas/farmacologia , Pirimidinas/químicaRESUMO
The A3 adenosine receptor is an interesting target whose role in cancer is controversial. In this work, a structural investigation at the 2-position of the [1,2,4]triazolo[1,5-c]pyrimidine nucleus was performed, finding new potent and selective A3 adenosine receptor antagonists such as the ethyl 2-(4-methoxyphenyl)-5-(methylamino)-[1,2,4]triazolo[1,5-c]pyrimidine-8-carboxylate (20, DZ123) that showed a Ki value of 0.47â nM and an exceptional selectivity profile over the other adenosine receptor subtypes. Computational studies were performed to rationalize the affinity and the selectivity profile of the tested compounds at the A3 adenosine receptor and the A1 and A2A adenosine receptors. Compound 20 was tested on both A3 adenosine receptor positive cell lines (CHO-A3 AR transfected, THP1 and HCT16) and on A3 negative cancer cell lines, showing no effect in the latter and a pro-proliferative effect at a low concentration in the former. These interesting results pave the way to further investigation on both the mechanism involved and potential therapeutic applications.
Assuntos
Neoplasias , Receptor A3 de Adenosina , Cricetinae , Animais , Relação Estrutura-Atividade , Receptor A3 de Adenosina/metabolismo , Receptores Purinérgicos P1/química , Receptores Purinérgicos P1/metabolismo , Linhagem Celular , Pirimidinas/química , Antagonistas de Receptores Purinérgicos P1/farmacologia , Antagonistas de Receptores Purinérgicos P1/química , Células CHO , Receptor A2A de AdenosinaRESUMO
Based on a screening of a chemical library of A2A adenosine receptor (AR) antagonists, a series of di- and tri-substituted adenine derivatives were synthesized and tested for their ability to inhibit the activity of the enzyme casein kinase 1 delta (CK1δ) and to bind adenosine receptors (ARs). Some derivatives, here called "dual anta-inhibitors", demonstrated good CK1δ inhibitory activity combined with a high binding affinity, especially for the A2AAR. The N6-methyl-(2-benzimidazolyl)-2-dimethyamino-9-cyclopentyladenine (17, IC50 = 0.59 µM and KiA2A = 0.076 µM) showed the best balance of A2AAR affinity and CK1δ inhibitory activity. Computational studies were performed to simulate, at the molecular level, the protein-ligand interactions involving the compounds of our series. Hence, the dual anta-inhibitor 17 could be considered the lead compound of new therapeutic agents endowed with synergistic effects for the treatment of chronic neurodegenerative and cancer diseases.
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Traditionally, molecular recognition between the orthosteric site of adenosine receptors and their endogenous ligand occurs with a 1 : 1 stoichiometry. Inspired by previous mechanistic insights derived from supervised molecular dynamics (SuMD) simulations, which suggested an alternative 2 : 1 binding stoichiometry, we synthesized BRA1, a bis-ribosyl adenosine derivative, tested its ability to bind to and activate members of the adenosine receptor family, and rationalized its activity through molecular modeling.
Assuntos
Adenosina , Simulação de Dinâmica Molecular , Adenosina/química , Receptores Purinérgicos P1 , LigantesRESUMO
Polypharmacology is a new trend in amyotrophic lateral sclerosis (ALS) therapy and an effective way of addressing a multifactorial etiology involving excitotoxicity, mitochondrial dysfunction, oxidative stress, and microglial activation. Inspired by a reported clinical trial, we converted a riluzole (1)-rasagiline (2) combination into single-molecule multi-target-directed ligands. By a ligand-based approach, the highly structurally integrated hybrids 3-8 were designed and synthesized. Through a target- and phenotypic-based screening pipeline, we identified hit compound 6. It showed monoamine oxidase A (MAO-A) inhibitory activity (IC50 = 6.9 µM) rationalized by in silico studies as well as in vitro brain permeability. By using neuronal and non-neuronal cell models, including ALS-patient-derived cells, we disclosed for 6 a neuroprotective/neuroinflammatory profile similar to that of the parent compounds and their combination. Furthermore, the unexpected MAO inhibitory activity of 1 (IC50 = 8.7 µM) might add a piece to the puzzle of its anti-ALS molecular profile.
Assuntos
Esclerose Lateral Amiotrófica , Fármacos Neuroprotetores , Esclerose Lateral Amiotrófica/tratamento farmacológico , Humanos , Indanos , Ligantes , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Riluzol/farmacologia , Riluzol/uso terapêuticoRESUMO
Amyotrophic lateral sclerosis (ALS) is a degenerating disease involving the motor neurons, which causes a progressive loss of movement ability, usually leading to death within 2 to 5 years from the diagnosis. Much effort has been put into research for an effective therapy for its eradication, but still, no cure is available. The only two drugs approved for this pathology, Riluzole and Edaravone, are onlyable to slow down the inevitable disease progression. As assessed in the literature, drug targets such as protein kinases have already been extensively examined as potential drug targets for ALS, with some molecules already in clinical trials. Here, we focus on the involvement of another very important and studied class of biological entities, G protein-coupled receptors (GPCRs), in the onset and progression of ALS. This workaimsto give an overview of what has been already discovered on the topic, providing useful information and insights that can be used by scientists all around the world who are putting efforts into the fight against this very important neurodegenerating disease.
Assuntos
Esclerose Lateral Amiotrófica , Esclerose Lateral Amiotrófica/tratamento farmacológico , Edaravone/uso terapêutico , Humanos , Neurônios Motores , Receptores Acoplados a Proteínas G , Riluzol/uso terapêuticoRESUMO
BACKGROUND: GSK-3ß activity has been strictly related to neuroinflammation and neurodegeneration. Alzheimer's disease is the most studied neurodegenerative disease, but GSK-3ß seems to be involved in almost all neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, and the autoimmune disease multiple sclerosis. OBJECTIVE: This review aims to help researchers both working on this research topic or not to have a comprehensive overview of GSK-3ß in the context of neuroinflammation and neurodegeneration. METHODS: Literature has been searched using PubMed and SciFinder databases by inserting specific keywords. A total of more than 500 articles have been discussed. RESULTS: First of all, the structure and regulation of the kinase were briefly discussed, and then, specific GSK-3ß implications in neuroinflammation and neurodegenerative diseases were illustrated with the help of figures, to conclude with a comprehensive overview on the most important GSK-3ß and multitarget inhibitors. The structure and IC50 values at the target kinase have been reported for all the discussed compounds. CONCLUSION: GSK-3ß is involved in several signaling pathways in neurons, glial cells and immune cells. The fine regulation and interconnection of all these pathways are at the base of the rationale use of GSK-ß inhibitors in neuroinflammation and neurodegeneration. Some compounds are now under clinical trials. Despite this, the compounds' pharmacodynamic and ADME/Tox profiles were often not fully characterized which is deleterious in such a complex system.
Assuntos
Doenças Neurodegenerativas , Doença de Parkinson , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neuroinflamatórias , Doença de Parkinson/metabolismo , Transdução de SinaisRESUMO
Fragment-Based Drug Discovery (FBDD) has become, in recent years, a consolidated approach in the drug discovery process, leading to several drug candidates under investigation in clinical trials and some approved drugs. Among these successful applications of the FBDD approach, kinases represent a class of targets where this strategy has demonstrated its real potential with the approved kinase inhibitor Vemurafenib. In the Kinase family, protein kinase CK1 isoform δ (CK1δ) has become a promising target in the treatment of different neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. In the present work, we set up and applied a computational workflow for the identification of putative fragment binders in large virtual databases. To validate the method, the selected compounds were tested in vitro to assess the CK1δ inhibition.
Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Caseína Quinase Idelta/química , Descoberta de Drogas/métodos , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Sítios de Ligação , Humanos , Conformação Molecular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Relação Estrutura-Atividade , Fluxo de TrabalhoRESUMO
A3 adenosine receptors were found to have a role in different pathological states, such as glaucoma, renal fibrosis, neuropathic pain and cancer. Consequently, it is important to utilize any molecular tool which could help to study these conditions. In the present study we continue our search for potent A3 adenosine receptor ligands which could be successively conjugated to other molecules with the aim of obtaining more potent (e.g. allosteric ligand conjugation) or detectable ligands (e.g. fluorescent molecule or biotin conjugation). Specifically, different aminoester moieties were introduced at the 5 position of the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine core. The ester functionalization represents the candidate for the subsequent conjugation. All the reported compounds are potent hA3 adenosine receptor antagonists and some of them exhibited high selectivity against the other adenosine receptors. The main structural terms of ligand recognition and selectivity were disclosed by molecular modelling studies. Molecular docking results led to the characterization of an alternative binding mode for antagonists at the orthosteric binding site of the hA3 adenosine receptor, evaluated and assessed by classical molecular dynamics simulations.
RESUMO
Protein kinase CK1δ expression and activity is involved in different pathological situations that include neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1δ has become a possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1δ inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines was developed. Some crucial interactors have been identified, such as the presence of a free amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular modeling studies identified the key interactions involved in the inhibitor-protein recognition process that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1 protein kinase is involved in various pathologies in particular of the central nervous system, the interest in the development of new inhibitors permeable to the blood-brain barrier represents today an important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC50 = 0.18 µM) that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and represents an optimal starting point to both studies the therapeutic value of protein kinase CK1δ inhibition and to develop new more potent derivatives.
Assuntos
Caseína Quinase Idelta/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Triazóis/química , Sítios de Ligação , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Caseína Quinase Idelta/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Humanos , Cinética , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Eletricidade Estática , Relação Estrutura-Atividade , Termodinâmica , Triazinas/química , Triazóis/metabolismo , Triazóis/farmacologiaRESUMO
The A3 adenosine receptor (AR) is a G protein-coupled receptor (GPCR) overexpressed in the membrane of specific cancer cells. Thus, the development of nanosystems targeting this receptor could be a strategy to both treat and diagnose cancer. Iron-filled carbon nanotubes (CNTs) are an optimal platform for theranostic purposes, and the use of a magnetic field can be exploited for cancer magnetic cell sorting and thermal therapy. In this work, we have conjugated an A3 AR ligand on the surface of iron-filled CNTs with the aim of targeting cells overexpressing A3 ARs. In particular, two conjugates bearing PEG linkers of different length were designed. A docking analysis of A3 AR showed that neither CNT nor linker interferes with ligand binding to the receptor; this was confirmed by inâ vitro preliminary radioligand competition assays on A3 AR. Encouraged by this result, magnetic cell sorting was applied to a mixture of cells overexpressing or not the A3 AR in which our compound displayed indiscriminate binding to all cells. Despite this, it is the first time that a GPCR ligand has been anchored to a magnetic nanosystem, thus it opens the door to new applications for cancer treatment.
Assuntos
Separação Celular/métodos , Nanotubos de Carbono/química , Receptor A3 de Adenosina/metabolismo , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Humanos , Ferro/química , Fenômenos Magnéticos , Pirazóis/síntese química , Pirazóis/química , Pirimidinas/síntese química , Pirimidinas/química , Triazóis/síntese química , Triazóis/químicaRESUMO
Recent studies have highlighted the key role of Casein kinase 1 δ (CK1δ) in the development of several neurodegenerative pathologies, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). So far, CK1δ inhibitors are noncovalent ATP competitive ligands and no drugs are currently available for this molecular target, hence the interest in developing new CK1δ inhibitors. The study aims to identify new inhibitors able to bind the enzyme; by a dual approach in silico/in vitro, the virtual screening has been performed on an in-house chemical library, which was previously designed and synthesized for other targets. The work can, therefore, be seen in the scaffold repurposing logic. The proposed strategy has led to the identification of two hits, having a novel scaffold in the landscape of CK1δ inhibitors and with an activity in the micromolar range.
RESUMO
Compounds able to simultaneously bind a biological target and be conjugated to a second specific moiety are attractive tools for the development of multi-purpose ligands useful as multi-target ligands, receptor probes or drug delivery systems, with both therapeutic and diagnostic applications. The human A3 adenosine receptor is a G protein-coupled receptor involved in many physio-pathological conditions, e.g. cancer and inflammation, thus representing a promising research target. In this work, two series of conjugable hA3AR antagonists, based on the pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine nucleus, were developed. The introduction of an aromatic ring at the 5 position of the scaffold, before (phenylacetamido moiety) or after (1,2,3-triazole obtained by click chemistry) the conjugation is aimed to increase affinity and selectivity towards the hA3AR receptor. As expected, conjugable compounds showed good affinity towards the hA3AR. In order to prove their potential in the development of hA3AR ligands for different purposes, compounds were also functionalized with fluorescent probes. Unfortunately, conjugation decreased affinity and selectivity for the target as compared to the hA2AAR. Computational studies identified specific non-conserved residues of the extracellular loops which constitute a structural barrier able to discriminate between ligands, giving insights into the rational development of new highly selective ligands.
Assuntos
Corantes Fluorescentes/farmacologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Receptor A3 de Adenosina/metabolismo , Relação Dose-Resposta a Droga , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ligantes , Simulação de Dinâmica Molecular , Estrutura Molecular , Antagonistas de Receptores Purinérgicos P1/síntese química , Antagonistas de Receptores Purinérgicos P1/química , Relação Estrutura-AtividadeRESUMO
Research on the adenosine receptors has been supported by the continuous discovery of new chemical probes characterized by more and more affinity and selectivity for the single adenosine receptor subtypes (A1, A2A, A2B and A3 adenosine receptors). Furthermore, the development of new techniques for the detection of G protein-coupled receptors (GPCR) requires new specific probes. In fact, if in the past radioligands were the most important GPCR probes for detection, compound screening and diagnostic purposes, nowadays, increasing importance is given to fluorescent and covalent ligands. In fact, advances in techniques such as fluorescence resonance energy transfer (FRET) and fluorescent polarization, as well as new applications in flow cytometry and different fluorescence-based microscopic techniques, are at the origin of the extensive research of new fluorescent ligands for these receptors. The resurgence of covalent ligands is due in part to a change in the common thinking in the medicinal chemistry community that a covalent drug is necessarily more toxic than a reversible one, and in part to the useful application of covalent ligands in GPCR structural biology. In this review, an updated collection of available chemical probes targeting adenosine receptors is reported.
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Human A3 adenosine receptor hA3AR has been implicated in gastrointestinal cancer, where its cellular expression has been found increased, thus suggesting its potential as a molecular target for novel anticancer compounds. Observation made in our previous work indicated the importance of the carbonyl group of amide in the indolylpyrimidylpiperazine (IPP) for its human A2A adenosine receptor (hA2AAR) subtype binding selectivity over the other AR subtypes. Taking this observation into account, we structurally modified an indolylpyrimidylpiperazine (IPP) scaffold, 1 (a non-selective adenosine receptors' ligand) into a modified IPP (mIPP) scaffold by switching the position of the carbonyl group, resulting in the formation of both ketone and tertiary amine groups in the new scaffold. Results showed that such modification diminished the A2A activity and instead conferred hA3AR agonistic activity. Among the new mIPP derivatives (3-6), compound 4 showed potential as a hA3AR partial agonist, with an Emax of 30% and EC50 of 2.89 ± 0.55 µM. In the cytotoxicity assays, compound 4 also exhibited higher cytotoxicity against both colorectal and liver cancer cells as compared to normal cells. Overall, this new series of compounds provide a promising starting point for further development of potent and selective hA3AR partial agonists for the treatment of gastrointestinal cancers.