RESUMO
Atypical chemokine receptor 3 (ACKR3), formerly referred to as CXCR7, is considered to be an interesting drug target. In this study, we report on the synthesis, pharmacological characterization and radiolabeling of VUF15485, a new ACKR3 small-molecule agonist, that will serve as an important new tool to study this ß-arrestin-biased chemokine receptor. VUF15485 binds with nanomolar affinity (pIC50 = 8.3) to human ACKR3, as measured in [125I]CXCL12 competition binding experiments. Moreover, in a bioluminescence resonance energy transfer-based ß-arrestin2 recruitment assay VUF15485 acts as a potent ACKR3 agonist (pEC50 = 7.6) and shows a similar extent of receptor activation compared with CXCL12 when using a newly developed, fluorescence resonance energy transfer-based ACKR3 conformational sensor. Moreover, the ACKR3 agonist VUF15485, tested against a (atypical) chemokine receptor panel (agonist and antagonist mode), proves to be selective for ACKR3. VUF15485 labeled with tritium at one of its methoxy groups ([3H]VUF15485), binds ACKR3 saturably and with high affinity (K d = 8.2 nM). Additionally, [3H]VUF15485 shows rapid binding kinetics and consequently a short residence time (<2 minutes) for binding to ACKR3. The selectivity of [3H]VUF15485 for ACKR3, was confirmed by binding studies, whereupon CXCR3, CXCR4, and ACKR3 small-molecule ligands were competed for binding against the radiolabeled agonist. Interestingly, the chemokine ligands CXCL11 and CXCL12 are not able to displace the binding of [3H]VUF15485 to ACKR3. The radiolabeled VUF15485 was subsequently used to evaluate its binding pocket. Site-directed mutagenesis and docking studies using a recently solved cryo-EM structure propose that VUF15485 binds in the major and the minor binding pocket of ACKR3. SIGNIFICANCE STATEMENT: The atypical chemokine receptor atypical chemokine receptor 3 (ACKR3) is considered an interesting drug target in relation to cancer and multiple sclerosis. The study reports on new chemical biology tools for ACKR3, i.e., a new agonist that can also be radiolabeled and a new ACKR3 conformational sensor, that both can be used to directly study the interaction of ACKR3 ligands with the G protein-coupled receptor.
Assuntos
Quimiocina CXCL12 , Receptores CXCR4 , Humanos , Receptores CXCR4/metabolismo , Quimiocina CXCL12/metabolismo , Quimiocina CXCL11/metabolismo , Transdução de Sinais , Ligantes , Ligação CompetitivaRESUMO
Biophysical screening of compound libraries for the identification of ligands that interact with a protein is efficient, but does typically not reveal if (or how) ligands may interfere with its functional properties. For this a biochemical/functional assay is required. But for proteins whose function is dependent on a conformational change, such assays are typically complex or have low throughput. Here we have explored a high-throughput second-harmonic generation (SHG) biosensor to detect fragments that induce conformational changes upon binding to a protein in real time and identify dynamic regions. Multiwell plate format SHG assays were developed for wild-type and six engineered single-cysteine mutants of acetyl choline binding protein (AChBP), a homologue to ligand gated ion channels (LGICs). They were conjugated with second harmonic-active labels via amine or maleimide coupling. To validate the assay, it was confirmed that the conformational changes induced in AChBP by nicotinic acetyl choline receptor (nAChR) agonists and antagonists were qualitatively different. A 1056 fragment library was subsequently screened against all variants and conformational modulators of AChBP were successfully identified, with hit rates from 9-22%, depending on the AChBP variant. A subset of four hits was selected for orthogonal validation and structural analysis. A time-resolved grating-coupled interferometry-based biosensor assay confirmed the interaction to be a reversible 1-step 1 : 1 interaction, and provided estimates of affinities and interaction kinetic rate constants (K D = 0.28-63 µM, k a = 0.1-6 µM-1 s-1, k d = 1 s-1). X-ray crystallography of two of the fragments confirmed their binding at a previously described conformationally dynamic site, corresponding to the regulatory site of LGICs. These results reveal that SHG has the sensitivity to identify fragments that induce conformational changes in a protein. A selection of fragment hits with a response profile different to known LGIC regulators was characterized and confirmed to bind to dynamic regions of the protein.
RESUMO
Several members of the 3',5'-cyclic nucleotide phosphodiesterase (PDE) family play an essential role in cellular processes, which has labeled them as interesting targets for various diseases. The parasitic protozoan Trypanosoma brucei, causative agent of human African trypanosomiasis, contains several cyclic AMP specific PDEs from which TbrPDEB1 is validated as a drug target. The recent discovery of selective TbrPDEB1 inhibitors has increased their potential for a novel treatment for this disease. Compounds characterized by a rigid biphenyl tetrahydrophthalazinone core structure were used as starting point for the exploration of novel TbrPDEB1 inhibitors. Using a virtual screening campaign and structure-guided design, diaryl ether substituted phthalazinones were identified as novel TbrPDEB1 inhibitors with IC50 values around 1 µM against T. brucei. This study provides important structure-activity relationship (SAR) information for the future design of effective parasite-specific PDE inhibitors.
RESUMO
Covalent binding of G protein-coupled receptors by small molecules is a useful approach for better understanding of the structure and function of these proteins. We designed, synthesized and characterized a series of 6 potential covalent ligands for the histamine H3 receptor (H3R). Starting from a 2-amino-pyrimidine scaffold, optimization of anchor moiety and warhead followed by fine-tuning of the required reactivity via scaffold hopping resulted in the isothiocyanate H3R ligand 44. It shows high reactivity toward glutathione combined with appropriate stability in water and reacts selectively with the cysteine sidechain in a model nonapeptide equipped with nucleophilic residues. The covalent interaction of 44 with H3R was validated with washout experiments and leads to inverse agonism on H3R. Irreversible binder 44 (VUF15662) may serve as a useful tool compound to stabilize the inactive H3R conformation and to study the consequences of prolonged inhibition of the H3R.
Assuntos
Isotiocianatos/síntese química , Receptores Histamínicos H3/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Agonismo Inverso de Drogas , Células HEK293 , Agonistas dos Receptores Histamínicos/síntese química , Agonistas dos Receptores Histamínicos/química , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/síntese química , Antagonistas dos Receptores Histamínicos/química , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Isotiocianatos/química , Isotiocianatos/farmacologia , Ligantes , Receptores Histamínicos H3/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Kinases are attractive anticancer targets due to their central role in the growth, survival, and therapy resistance of tumor cells. This review explores the two primary kinase classes, the eukaryotic protein kinases (ePKs) and the atypical protein kinases (aPKs), and provides a structure-centered comparison of their sequences, structures, hydrophobic spines, mutation and SNP hotspots, and inhibitor interaction patterns. Despite the limited sequence similarity between these two classes, atypical kinases commonly share the archetypical kinase fold but lack conserved eukaryotic kinase motifs and possess altered hydrophobic spines. Furthermore, atypical kinase inhibitors explore only a limited number of binding modes both inside and outside the orthosteric binding site. The distribution of genetic variations in both classes shows multiple ways they can interfere with kinase inhibitor binding. This multilayered review provides a research framework bridging the eukaryotic and atypical kinase classes.
Assuntos
Neoplasias/enzimologia , Proteínas Quinases/classificação , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Humanos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/genética , Polimorfismo de Nucleotídeo Único , Conformação Proteica em Folha beta , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Proteínas Quinases/genética , Relação Estrutura-AtividadeRESUMO
Despite the high diversity of histamine H3 receptor (H3R) antagonist/inverse agonist structures, partial or full H3R agonists have typically been imidazole derivatives. An in-house screening campaign intriguingly afforded the non-imidazole 4-(3-azetidin-1-yl)pyrimidin-2-amine 11b as a partial H3R agonist. Here, the design, synthesis, and structure-activity relationships of 11b analogues are described. This series yields several non-imidazole full agonists with potencies varying with the alkyl substitution pattern on the basic amine following the in vitro evaluation of H3R agonism using a cyclic adenosine monophosphate response element-luciferase reporter gene assay. The key compound VUF16839 (14d) combines nanomolar on-target activity (pKi = 8.5, pEC50 = 9.5) with weak activity on cytochrome P450 enzymes and good metabolic stability. The proposed H3R binding mode of 14d indicates key interactions similar to those attained by histamine. In vivo evaluation of 14d in a social recognition test in mice revealed an amnesic effect at 5 mg/kg intraperitoneally. The excellent in vitro and in vivo pharmacological profiles and the non-imidazole structure of 14d make it a promising tool compound in H3R research.
Assuntos
Aminas/síntese química , Aminas/farmacologia , Agonistas dos Receptores Histamínicos/síntese química , Agonistas dos Receptores Histamínicos/farmacologia , Aminas/química , Animais , Comportamento Animal/efeitos dos fármacos , Células HEK293 , Agonistas dos Receptores Histamínicos/química , Humanos , Memória/efeitos dos fármacos , Camundongos , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Conformação Proteica , Comportamento SocialRESUMO
We report the evaluation of 265 compounds from a PDE-focused library for their antischistosomal activity, assessed in vitro using Schistosoma mansoni. Of the tested compounds, 171 (64%) displayed selective in vitro activity, with 16 causing worm hypermotility/spastic contractions and 41 inducing various degrees of worm killing at 100⯵M, with the surviving worms displaying sluggish movement, worm unpairing and complete absence of eggs. The compounds that did not affect worm viability (nâ¯=â¯72) induced a complete cessation of ovipositing. 82% of the compounds had an impact on male worms whereas female worms were barely affected. In vivo evaluation in S. mansoni-infected mice with the in vitro 'hit' NPD-0274â¯at 20â¯mg/kg/day orally for 5 days resulted in worm burden reductions of 29% and intestinal tissue egg load reduction of 35% at 10 days post-treatment. Combination of praziquantel (PZQ) at 10â¯mg/kg/day for 5 days with NPD-0274 or NPD-0298 resulted in significantly higher worm killing than PZQ alone, as well as a reduction in intestinal tissue egg load, disappearance of immature eggs and an increase in the number of dead eggs.
Assuntos
Anti-Helmínticos/farmacologia , Imidazóis/farmacologia , Schistosoma mansoni/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas , 3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Animais , Anti-Helmínticos/química , Descoberta de Drogas , Fibroblastos/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/química , Masculino , Camundongos , Contagem de Ovos de Parasitas , Praziquantel/farmacologiaRESUMO
Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, Trypanosoma brucei PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, 8 (NPD-008) and 9 (NPD-039), were potent ( Ki = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC50 = 5.5 and 6.7 µM, respectively). Treatment of parasites with 8 caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of T. brucei cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , Inibidores de Fosfodiesterase/química , Inibidores de Fosfodiesterase/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Trypanosoma brucei brucei/enzimologia , 3',5'-AMP Cíclico Fosfodiesterases/química , Amidas/química , Amidas/farmacologia , Domínio Catalítico , Concentração Inibidora 50 , Modelos Moleculares , Terapia de Alvo Molecular , Proteínas de Protozoários/química , Relação Estrutura-AtividadeRESUMO
EPHA4 belongs to the largest subfamily of receptor tyrosine kinases. In addition to its function during development, overexpression of EPHA4 in tumors has been correlated with increased proliferation, migration and poor survival. Several genome-wide transcription profiling studies have demonstrated high EPHA4 expression in Sézary syndrome (SS), a leukemic variant of cutaneous CD4+ T-cell lymphoma (CTCL) with an aggressive clinical course and poor prognosis. In this study we set out to explore the functional role of EPHA4 in SS. Both high EPHA4 mRNA and protein expression was found in circulating SS-cells of patients compared to healthy CD4+ T-cells. However, using a phosphospecific EPHA4 antibody, phosphorylation of the EPHA4 kinase domain was not detected in either circulating or skin residing SS cells. Moreover, treatment with the phosphatase inhibitor pervanadate did not result in detectable phosphorylation of the EPHA4 kinase domain, in either SS cells or in healthy CD4+ T-cells. Thus, the results from our study confirm high EPHA4 expression in SS cells both on the mRNA and protein levels, making EPHA4 a good diagnostic marker. However, the overexpressed EPHA4 does not appear to be functionally active and its overexpression might be secondary to other oncogenic drivers in SS, like STAT3 and TWIST1.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Proteínas Nucleares/metabolismo , Receptor EphA4/metabolismo , Fator de Transcrição STAT3/metabolismo , Síndrome de Sézary/metabolismo , Neoplasias Cutâneas/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Idoso , Western Blotting , Linfócitos T CD4-Positivos/patologia , Estudos de Casos e Controles , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Proteínas Nucleares/genética , Fosforilação , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor EphA4/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT3/genética , Síndrome de Sézary/genética , Síndrome de Sézary/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , Proteína 1 Relacionada a Twist/genéticaRESUMO
Cell division in Escherichia coli involves a set of essential proteins that assembles at midcell to form the so-called divisome. The divisome regulates the invagination of the inner membrane, cell wall synthesis, and inward growth of the outer membrane. One of the divisome proteins, FtsQ, plays a central but enigmatic role in cell division. This protein associates with FtsB and FtsL, which, like FtsQ, are bitopic inner membrane proteins with a large periplasmic domain (denoted FtsQp, FtsBp, and FtsLp) that is indispensable for the function of each protein. Considering the vital nature and accessible location of the FtsQBL complex, it is an attractive target for protein-protein interaction inhibitors intended to block bacterial cell division. In this study, we expressed FtsQp, FtsBp, and FtsLp individually and in combination. Upon co-expression, FtsQp was co-purified with FtsBp and FtsLp from E. coli extracts as a stable trimeric complex. FtsBp was also shown to interact with FtsQp in the absence of FtsLp albeit with lower affinity. Interactions were mapped at the C terminus of the respective domains by site-specific cross-linking. The binding affinity and 1:1:1 stoichiometry of the FtsQpBpLp complex and the FtsQpBp subcomplex were determined in complementary surface plasmon resonance, analytical ultracentrifugation, and native mass spectrometry experiments.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citologia , Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Sequência de Aminoácidos , Técnicas Biossensoriais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Reagentes de Ligações Cruzadas/metabolismo , Proteínas Imobilizadas/metabolismo , Luz , Espectrometria de Massas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Peptídeos/química , Peptídeos/metabolismo , Periplasma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Solubilidade , Relação Estrutura-Atividade , UltracentrifugaçãoRESUMO
There are no commercially available, small, receptor-specific P2X1 ligands. There are several synthetic derivatives of the natural agonist ATP and some structurally-complex antagonists including compounds such as PPADS, NTP-ATP, suramin and its derivatives (e.g. NF279, NF449). NF449 is the most potent and selective ligand, but potencies of many others are not particularly high and they can also act at other P2X, P2Y and non-purinergic receptors. While there is clearly scope for further work on P2X1 receptor pharmacology, screening can be difficult owing to rapid receptor desensitisation. To reduce desensitisation substitutions can be made within the N-terminus of the P2X1 receptor, but these could also affect ligand properties. An alternative is the use of fluorescent voltage-sensitive dyes that respond to membrane potential changes resulting from channel opening. Here we utilised this approach in conjunction with fragment-based drug-discovery. Using a single concentration (300 µM) we identified 46 novel leads from a library of 1443 fragments (hit rate = 3.2%). These hits were independently validated by measuring concentration-dependence with the same voltage-sensitive dye, and by visualising the competition of hits with an Alexa-647-ATP fluorophore using confocal microscopy; confocal yielded kon (1.142 × 10(6) M(-1) s(-1)) and koff (0.136 s(-1)) for Alexa-647-ATP (Kd = 119 nM). The identified hit fragments had promising structural diversity. In summary, the measurement of functional responses using voltage-sensitive dyes was flexible and cost-effective because labelled competitors were not needed, effects were independent of a specific binding site, and both agonist and antagonist actions were probed in a single assay. The method is widely applicable and could be applied to all P2X family members, as well as other voltage-gated and ligand-gated ion channels. This article is part of the Special Issue entitled 'Fluorescent Tools in Neuropharmacology'.
Assuntos
Fluorescência , Receptores Purinérgicos P2X1/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Benzenossulfonatos/farmacocinética , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/genética , Carbocianinas/farmacocinética , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Células HEK293 , Humanos , Ligantes , Microscopia Confocal , Agonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores Purinérgicos P2X1/genética , Transfecção , Imagens com Corantes Sensíveis à VoltagemRESUMO
In this work we describe the optimization of a lead compound based on the quinazoline template to give a new series of potent pyrido[3,2-d]pyrimidines as histamine H4 receptor antagonists. The pyrido[3,2-d]pyrimidine ligands have significantly reduced hERG binding compared to clinical stage compound PF-3893787 while showing good affinities at the human and rodent histamine receptors. The receptor residence time of several of these new compounds was determined for the human H4R and compared with JNJ7777120 and PF-3893787. The pyrido[3,2-d]pyrimidines showed residence times lower than JNJ7777120 but comparable to the residence time of PF-3893787. Overall, the pyrido[3,2-d]pyrimidines show an excellent in vitro profile that warrants their further investigation in relevant models of human disease.
Assuntos
Canais de Potássio Éter-A-Go-Go/metabolismo , Antagonistas dos Receptores Histamínicos/química , Piridinas/química , Pirimidinas/química , Pirrolidinas/química , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Linhagem Celular , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Meia-Vida , Antagonistas dos Receptores Histamínicos/síntese química , Antagonistas dos Receptores Histamínicos/farmacocinética , Humanos , Indóis/química , Indóis/farmacocinética , Cinética , Camundongos , Piperazinas/química , Piperazinas/farmacocinética , Ligação Proteica , Piridinas/síntese química , Piridinas/farmacocinética , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Pirrolidinas/farmacocinética , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H4 , Relação Estrutura-AtividadeRESUMO
Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of Trypanosoma brucei, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC50 values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (20b): T. brucei rhodesiense IC50 = 60 nM, T. brucei brucei IC50 = 520 nM, T. cruzi = 7.6 µM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of 20b was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target.
Assuntos
Catecóis/síntese química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/antagonistas & inibidores , Pirazóis/síntese química , Pirazolonas/síntese química , Tetrazóis/síntese química , Tripanossomicidas/síntese química , Trypanosoma brucei brucei/efeitos dos fármacos , Sítios de Ligação , Catecóis/química , Catecóis/farmacologia , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/química , Desenho de Fármacos , Simulação de Acoplamento Molecular , Pirazóis/química , Pirazóis/farmacologia , Pirazolonas/química , Pirazolonas/farmacologia , Relação Estrutura-Atividade , Tetrazóis/química , Tetrazóis/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Trypanosoma brucei rhodesiense/enzimologiaRESUMO
We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the ß-2 adrenoreceptor (ADRB2), the adenosine A(2A) receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts.
Assuntos
Bases de Dados Factuais , Desenho de Fármacos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Receptores A2 de Adenosina/química , Receptores Adrenérgicos beta 2/química , Receptores de Lisoesfingolipídeo/química , Agonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/química , Agonistas de Receptores Adrenérgicos beta 2/química , Antagonistas de Receptores Adrenérgicos beta 2/química , Animais , Células CHO , Cricetinae , Cricetulus , Agonismo Parcial de Drogas , Células HEK293 , Ensaios de Triagem em Larga Escala , Humanos , Ligantes , Estrutura Molecular , Inibidores da Fosfodiesterase 5/química , Piperazinas/química , Piperazinas/metabolismo , Purinas/química , Purinas/metabolismo , Ensaio Radioligante , Receptores A2 de Adenosina/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Lisoesfingolipídeo/agonistas , Receptores de Lisoesfingolipídeo/metabolismo , Citrato de Sildenafila , Processos Estocásticos , Sulfonas/química , Sulfonas/metabolismoRESUMO
The chemokine receptor CXCR7 is an atypical G protein-coupled receptor as it preferentially signals through the ß-arrestin pathway rather than through G proteins. CXCR7 is thought to be of importance in cancer and the development of CXCR7-targeting ligands is of huge importance to further elucidate the pharmacology and the therapeutic potential of CXCR7. In the present study, we synthesized 24 derivatives based on a compound scaffold patented by Chemocentryx and obtained CXCR7 ligands with pK(i) values ranging from 5.3 to 8.1. SAR studies were supported by computational 3D Fingerprint studies, revealing several important affinity descriptors. Two key compounds (29 and 30, VUF11207 and VUF11403) were found to be high-potency ligands that induce recruitment of ß-arrestin2 and subsequent internalization of CXCR7, making them important tool compounds in future CXCR7 research.
Assuntos
Amidas/química , Técnicas de Química Sintética , Modelos Moleculares , Receptores CXCR/agonistas , Estireno/química , Estireno/farmacologia , Células HEK293 , Humanos , Conformação Molecular , Relação Quantitativa Estrutura-Atividade , Estireno/síntese químicaRESUMO
The in silico identification, optimization and crystallographic characterization of a 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine scaffold as an inhibitor for the EPHA4 receptor tyrosine kinase is described. A database containing commercially available compounds was subjected to an in silico screening procedure which was focused on finding novel, EPHA4 hinge binding fragments. This resulted in the identification of 6,7,8,9-tetrahydro-3H-pyrazolo[3,4-c]isoquinolin-1-amine derivatives as EPHA4 inhibitors. Hit exploration yielded a compound with 2 µM (IC(50)) affinity for the EPHA4 receptor tyrosine kinase domain. Soaking experiments into a crystal of the EPHA4 kinase domain gave a 2.11Å X-ray structure of the EPHA4 - inhibitor complex, which confirmed the binding mode of the scaffold as proposed by the initial in silico work. The results underscore the strength of fragment based in silico screening as a tool for the discovery of novel lead compounds as small molecule kinase inhibitors.
Assuntos
Descoberta de Drogas/métodos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor EphA4/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Biologia Computacional , Modelos Moleculares , Conformação Proteica , Receptor EphA4/química , Receptor EphA4/metabolismoRESUMO
The Eph family of receptor tyrosine kinases regulates diverse cellular processes while the over-expression of a member of this family, EphA4, has been reported in a variety of malignant carcinomas. To gain insight into molecular mechanisms and to facilitate structure-based inhibitor design, we solved the crystal structure of the native EphA4 kinase domain in both the apo and dasatinib bound forms. Analysis of the two structures provides insight into structural features of inhibitor binding and revealed a hydrophobic back-pocket in the ATP- binding site of EphA4 which was previously unidentified. The structures suggest a route towards development of novel and specific inhibitors.
Assuntos
Antineoplásicos/química , Pirimidinas/química , Receptor EphA4/química , Tiazóis/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dasatinibe , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Receptor EphA4/antagonistas & inibidoresRESUMO
The histamine H(4) receptor (H(4)R) is a G protein-coupled receptor (GPCR) that plays an important role in inflammation. Similar to the homologous histamine H(3) receptor (H(3)R), two acidic residues in the H(4)R binding pocket, D(3.32) and E(5.46), act as essential hydrogen bond acceptors of positively ionizable hydrogen bond donors in H(4)R ligands. Given the symmetric distribution of these complementary pharmacophore features in H(4)R and its ligands, different alternative ligand binding mode hypotheses have been proposed. The current study focuses on the elucidation of the molecular determinants of H(4)R-ligand binding modes by combining (3D) quantitative structure-activity relationship (QSAR), protein homology modeling, molecular dynamics simulations, and site-directed mutagenesis studies. We have designed and synthesized a series of clobenpropit (N-(4-chlorobenzyl)-S-[3-(4(5)-imidazolyl)propyl]isothiourea) derivatives to investigate H(4)R-ligand interactions and ligand binding orientations. Interestingly, our studies indicate that clobenpropit (2) itself can bind to H(4)R in two distinct binding modes, while the addition of a cyclohexyl group to the clobenpropit isothiourea moiety allows VUF5228 (5) to adopt only one specific binding mode in the H(4)R binding pocket. Our ligand-steered, experimentally supported protein modeling method gives new insights into ligand recognition by H(4)R and can be used as a general approach to elucidate the structure of protein-ligand complexes.
Assuntos
Antagonistas dos Receptores Histamínicos/química , Imidazóis/química , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Tioureia/análogos & derivados , Linhagem Celular Tumoral , Antagonistas dos Receptores Histamínicos/síntese química , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Imidazóis/síntese química , Imidazóis/farmacologia , Ligantes , Conformação Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/genética , Receptores Histamínicos H3/genética , Receptores Histamínicos H3/metabolismo , Receptores Histamínicos H4 , Estereoisomerismo , Tioureia/síntese química , Tioureia/química , Tioureia/farmacologiaRESUMO
This study describes the evaluation, validation, and use of contactless postcolumn fractionation of bioactive mixtures with acetylcholine binding protein (AChBP) affinity analysis with help of a spotter technology. The high-resolution fractionation tailors the fractionation frequency to the chromatographic peaks. Postcolumn reagents for AChBP bioaffinity profiling are mixed prior to droplet ejection into 1536-well plates. After an incubation step, microplate reader analysis is used to determine bioactive compounds in a mixture. For ligands tested, a good correlation was found for IC(50)s determined in flow injection analysis mode when compared with traditional radioligand binding assays. After the evaluation and validation, bioaffinity profiling of actual mixtures was performed. The advantage of this "atline" technology using postcolumn bioaffinity analysis when compared to continuous flow online postcolumn bioaffinity profiling is the possibility to choose postcolumn incubation times freely without compromising resolution due to diffusion effects.
Assuntos
Proteínas de Transporte , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Misturas Complexas/análise , Análise de Injeção de Fluxo/métodos , Ensaios de Triagem em Larga Escala , Compostos Bicíclicos Heterocíclicos com Pontes/análise , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fracionamento Químico/métodos , Cromatografia de Afinidade/instrumentação , Misturas Complexas/química , Análise de Injeção de Fluxo/instrumentação , Ligantes , Espectrometria de Massas/métodos , Nanotecnologia/métodos , Nicotina/análise , Nicotina/metabolismo , Agonistas Nicotínicos/análise , Agonistas Nicotínicos/metabolismo , Ligação Proteica , Piridinas/análise , Piridinas/metabolismo , Ensaio Radioligante , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos TestesRESUMO
Small-molecule ligands for the CXCR3 chemokine receptor receive considerable attention as a means to interrogate the roles of CXCR3 in vitro and in vivo and/or to potentially treat various conditions such as inflammatory diseases and cancer. We have synthesized and explored a novel class of small-molecule antagonists for CXCR3. A medium-throughput screen revealed an adamantane-guanidine as a hit. The guanidine unit was replaced by a small quaternary ammonium group, leading to ca. 5-fold increase in affinity. Substitution of the adamantane group by a myrtenyl moiety further increased affinity, while the benzyl group was decorated with an additional (substituted) aryl ring. This led to the identification of several bisaryl-based ligands with CXCR3 affinities of around 100 nM and with the ability to antagonize the functional activity of CXCL10.