RESUMO
We describe the discovery of histone deacetylase (HDACs) 1, 2, and 3 inhibitors with ethyl ketone as the zinc-binding group. These HDACs 1, 2, and 3 inhibitors have good enzymatic and cellular activity. Their serum shift in cellular potency has been minimized, and selectivity against hERG has been improved. They are also highly selective over HDACs 6 and 8. These inhibitors contain a variety of substituted heterocycles on the imidazole or oxazole scaffold. Compounds 31 and 48 stand out due to their good potency, high selectivity over HDACs 6 and 8, reduced hERG activity, optimized serum shift in cellular potency, and good rat and dog PK profiles.
Assuntos
Canal de Potássio ERG1/metabolismo , HIV-1/fisiologia , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Cetonas/química , Animais , Cães , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Imidazóis/química , Oxazóis/química , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Relação Estrutura-Atividade , Ativação Viral/efeitos dos fármacosRESUMO
Structure- and ligand-based virtual-screening methods (docking, 2D- and 3D-similarity searching) were analyzed for their effectiveness in virtual screening against FFAR2. To evaluate the performance of these methods, retrospective virtual screening was performed. Statistical quality of the methods was evaluated by BEDROC and RIE. The results revealed that electrostatic similarity search protocol using EON (ET combo) outperformed all other protocols with outstanding enrichment of >95% in top 1% and 2% of the dataset with an AUC of 0.958. Interestingly, the hit lists that are obtained from different virtual-screening methods are generally highly complementary to hits found from electrostatic similarity searching. These results suggest that considering electrostatic similarity searching first increases the chance of identifying more (and more diverse) active compounds from a virtual-screening campaign. Accordingly, prospective virtual screening using electrostatic similarity searching was used to identify novel FFAR2 ligands. The discovered compounds provide new chemical matter starting points for the initiation of a medicinal chemistry campaign.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Receptores de Superfície Celular/agonistas , Anti-Inflamatórios não Esteroides/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Células HEK293 , Humanos , Ligantes , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The 2016 D3R Grand Challenge 2 includes both pose and affinity or ranking predictions. This article is focused exclusively on affinity predictions submitted to the D3R challenge from a collaborative effort of the modeling and informatics group. Our submissions include ranking of 102 ligands covering 4 different chemotypes against the FXR ligand binding domain structure, and the relative binding affinity predictions of the two designated free energy subsets of 15 and 18 compounds. Using all the complex structures prepared in the same way allowed us to cover many types of workflows and compare their performances effectively. We evaluated typical workflows used in our daily structure-based design modeling support, which include docking scores, force field-based scores, QM/MM, MMGBSA, MD-MMGBSA, and MacroModel interaction energy estimations. The best performing methods for the two free energy subsets are discussed. Our results suggest that affinity ranking still remains very challenging; that the knowledge of more structural information does not necessarily yield more accurate predictions; and that visual inspection and human intervention are considerably important for ranking. Knowledge of the mode of action and protein flexibility along with visualization tools that depict polar and hydrophobic maps are very useful for visual inspection. QM/MM-based workflows were found to be powerful in affinity ranking and are encouraged to be applied more often. The standardized input and output enable systematic analysis and support methodology development and improvement for high level blinded predictions.
Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Receptores Citoplasmáticos e Nucleares/metabolismo , Termodinâmica , Fluxo de Trabalho , Sítios de Ligação , Desenho Assistido por Computador , Bases de Dados de Proteínas , Desenho de Fármacos , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/química , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
We describe the performance of multiple pose prediction methods for the D3R 2016 Grand Challenge. The pose prediction challenge includes 36 ligands, which represent 4 chemotypes and some miscellaneous structures against the FXR ligand binding domain. In this study we use a mix of fully automated methods as well as human-guided methods with considerations of both the challenge data and publicly available data. The methods include ensemble docking, colony entropy pose prediction, target selection by molecular similarity, molecular dynamics guided pose refinement, and pose selection by visual inspection. We evaluated the success of our predictions by method, chemotype, and relevance of publicly available data. For the overall data set, ensemble docking, visual inspection, and molecular dynamics guided pose prediction performed the best with overall mean RMSDs of 2.4, 2.2, and 2.2 Å respectively. For several individual challenge molecules, the best performing method is evaluated in light of that particular ligand. We also describe the protein, ligand, and public information data preparations that are typical of our binding mode prediction workflow.
Assuntos
Desenho Assistido por Computador , Desenho de Fármacos , Descoberta de Drogas , Simulação de Acoplamento Molecular , Receptores Citoplasmáticos e Nucleares/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Ligantes , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , TermodinâmicaRESUMO
High-throughput screening (HTS) has enabled millions of compounds to be assessed for biological activity, but challenges remain in the prioritization of hit series. While biological, absorption, distribution, metabolism, excretion, and toxicity (ADMET), purity, and structural data are routinely used to select chemical matter for further follow-up, the scarcity of historical ADMET data for screening hits limits our understanding of early hit compounds. Herein, we describe a process that utilizes a battery of in-house quantitative structure-activity relationship (QSAR) models to generate in silico ADMET profiles for hit series to enable more complete characterizations of HTS chemical matter. These profiles allow teams to quickly assess hit series for desirable ADMET properties or suspected liabilities that may require significant optimization. Accordingly, these in silico data can direct ADMET experimentation and profoundly impact the progression of hit series. Several prospective examples are presented to substantiate the value of this approach.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Preparações Farmacêuticas/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Simulação por Computador , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Preparações Farmacêuticas/metabolismo , Farmacocinética , Farmacologia , Relação Quantitativa Estrutura-AtividadeRESUMO
Pharmacological mitigation of injuries caused by high-dose ionizing radiation is an unsolved medical problem. A specific nonlipid agonist of the type 2 G protein coupled receptor for lysophosphatidic acid (LPA2) 2-[4-(1,3-dioxo-1H,3H-benzoisoquinolin-2-yl)butylsulfamoyl]benzoic acid (DBIBB) when administered with a postirradiation delay of up to 72 hr reduced mortality of C57BL/6 mice but not LPA2 knockout mice. DBIBB mitigated the gastrointestinal radiation syndrome, increased intestinal crypt survival and enterocyte proliferation, and reduced apoptosis. DBIBB enhanced DNA repair by augmenting the resolution of γ-H2AX foci, increased clonogenic survival of irradiated IEC-6 cells, attenuated the radiation-induced death of human CD34(+) hematopoietic progenitors and enhanced the survival of the granulocyte/macrophage lineage. DBIBB also increased the survival of mice suffering from the hematopoietic acute radiation syndrome after total-body irradiation. DBIBB represents a drug candidate capable of mitigating acute radiation syndrome caused by high-dose γ-radiation to the hematopoietic and gastrointestinal system.
Assuntos
Apoptose/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Naftalimidas/farmacologia , Receptores de Ácidos Lisofosfatídicos/agonistas , Sulfonamidas/farmacologia , Síndrome Aguda da Radiação/metabolismo , Síndrome Aguda da Radiação/patologia , Síndrome Aguda da Radiação/prevenção & controle , Animais , Apoptose/efeitos da radiação , Sítios de Ligação , Caspase 8/metabolismo , Linhagem Celular , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Raios gama , Histonas/metabolismo , Humanos , Lisofosfolipídeos/química , Lisofosfolipídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Simulação de Acoplamento Molecular , Naftalimidas/química , Naftalimidas/uso terapêutico , Estrutura Terciária de Proteína , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Sulfonamidas/química , Sulfonamidas/uso terapêuticoRESUMO
Lysophosphatidic acid (LPA) is a growth factor-like mediator and a ligand for multiple GPCR. The LPA2 GPCR mediates antiapoptotic and mucosal barrier-protective effects in the gut. We synthesized sulfamoyl benzoic acid (SBA) analogues that are the first specific agonists of LPA2, some with subnanomolar activity. We developed an experimental SAR that is supported and rationalized by computational docking analysis of the SBA compounds into the LPA2 ligand-binding pocket.
Assuntos
Benzoatos/química , Receptores de Ácidos Lisofosfatídicos/agonistas , Sítios de Ligação , Técnicas de Química Sintética , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Simulação de Acoplamento Molecular , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Modulation of autotaxin (ATX), the lysophospholipase D enzyme that produces lysophosphatidic acid, with small-molecule inhibitors is a promising strategy for blocking the ATX-lysophosphatidic acid signaling axis. Although discovery campaigns have been successful in identifying ATX inhibitors, many of the reported inhibitors target the catalytic cleft of ATX. A recent study provided evidence for an additional inhibitory surface in the hydrophobic binding pocket of ATX, confirming prior studies that relied on enzyme kinetics and differential inhibition of substrates varying in size. Multiple hits from previous high-throughput screening for ATX inhibitors were obtained with aromatic sulfonamide derivatives interacting with the hydrophobic pocket. Here, we describe the development of a ligand-based strategy and its application in virtual screening, which yielded novel high-potency inhibitors that target the hydrophobic pocket of ATX. Characterization of the structure-activity relationship of these new inhibitors forms the foundation of a new pharmacophore model of the hydrophobic pocket of ATX.
Assuntos
Diester Fosfórico Hidrolases/química , Sulfonamidas/química , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and â¼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.
Assuntos
Antineoplásicos/química , Benzamidas/química , Benzenoacetamidas/química , Hidrazinas/química , Indóis/química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Sulfonamidas/química , Tetrazóis/química , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Benzenoacetamidas/farmacologia , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Ensaios de Triagem em Larga Escala , Humanos , Hidrazinas/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Indóis/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Mutação , Invasividade Neoplásica , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/farmacologia , Tetrazóis/farmacologiaRESUMO
Due to its antiapoptotic action, derivatives of the lipid mediator lysophosphatidic acid (LPA) provide potential therapeutic utility in diseases associated with programmed cell death. Apoptosis is one of the major pathophysiological processes elicited by radiation injury to the organism. Consequently, therapeutic explorations applying compounds that mimic the antiapoptotic action of LPA have begun. Here we present a brief account of our decade-long drug discovery effort aimed at developing LPA mimics with a special focus on specific agonists of the LPA(2) receptor subtype, which was found to be highly effective in protecting cells from apoptosis. We describe new evidence that 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), a prototypic nonlipid agonist specific to the LPA(2) receptor subtype, rescues apoptotically condemned cells in vitro and in vivo from injury caused by high-dose γ-irradiation. GRI977143 shows the features of a radiomitigator because it is effective in rescuing the lives of mice from deadly levels of radiation when administered 24h after radiation exposure. Our findings suggest that by specifically activating LPA(2) receptors GRI977143 activates the ERK1/2 prosurvival pathway, effectively reduces Bax translocation to the mitochondrion, attenuates the activation of initiator and effector caspases, reduces DNA fragmentation, and inhibits PARP-1 cleavage associated with γ-irradiation-induced apoptosis. GRI977143 also inhibits bystander apoptosis elicited by soluble proapoptotic mediators produced by irradiated cells. Thus, GRI977143 can serve as a prototype scaffold for lead optimization paving the way to more potent analogs amenable for therapeutic exploration. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Assuntos
Lesões por Radiação/metabolismo , Lesões por Radiação/prevenção & controle , Receptores de Ácidos Lisofosfatídicos/metabolismo , Síndrome Aguda da Radiação/tratamento farmacológico , Síndrome Aguda da Radiação/patologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Efeito Espectador/efeitos dos fármacos , Efeito Espectador/efeitos da radiação , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos da radiação , Citoproteção/efeitos dos fármacos , Citoproteção/efeitos da radiação , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Embrião de Mamíferos/citologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibroblastos/efeitos da radiação , Raios gama , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Knockout , Compostos Organofosforados/farmacologia , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Lesões por Radiação/patologia , Análise de SobrevidaRESUMO
Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA2 G protein-coupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L5547924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro. Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA(1&2) double-knockout mice reconstituted with the LPA2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA2, Na⺠- H⺠exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA2-specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células CHO , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cricetinae , Fragmentação do DNA/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas com Domínio LIM/metabolismo , Lisofosfolipídeos/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Knockout , Invasividade Neoplásica , Fosfoproteínas/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Complexo de Endopeptidases do Proteassoma , Ratos , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trocadores de Sódio-Hidrogênio/metabolismo , Fatores de Transcrição/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
A structurally diverse dataset of 119 compounds was used to develop and validate a 2D binary QSAR model for the LPA(3) receptor. The binary QSAR model was generated using an activity threshold of greater than 15% inhibition at 10 microM. The overall accuracy of the model on the training set was 82%. It had accuracies of 55% for active and 91% for inactive compounds, respectively. The model was validated using an external test set of 10 compounds. The accuracy on the external test set was 60% overall, identifying three out of seven actives and all three inactive compounds. This model was combined with similarity searching to rapidly screen libraries and select 14 candidate LPA(3) antagonists. Experimental assays confirmed 13 of these (93%) met the 15% inhibition threshold defining actives. The successful application of the model to select candidates for screening demonstrates the power of this binary QSAR model to prioritize compound selection for experimental consideration.
Assuntos
Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/química , Algoritmos , Bases de Dados Factuais , Estrutura Molecular , Receptores de Ácidos Lisofosfatídicos/genética , Reprodutibilidade dos TestesRESUMO
Compound 5 ([5-(3-nitrophenoxy)-1,3-dioxo-1,3-dihydro-2-isoindol-2-yl]acetic acid) was identified as a weak selective LPA(3) antagonist (IC(50)=4504 nM) in a virtual screening effort to optimize a dual LPA(2 and 3) antagonist. Structure-based drug design techniques were used to prioritize similarity search matches of compound 5. This strategy rapidly identified 10 novel antagonists. The two most efficacious compounds identified inhibit activation of the LPA(3) receptor by 200 nM LPA with IC(50) values of 752 nM and 2992 nM. These compounds additionally define changes to our previously reported pharmacophore that will improve its ability to identify more potent and selective LPA(3) receptor antagonists. The results of the combined computational and experimental screening are reported.
Assuntos
Isoindóis/química , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Sítios de Ligação , Simulação por Computador , Desenho de Fármacos , Isoindóis/síntese química , Isoindóis/farmacologia , Conformação Molecular , Receptores de Ácidos Lisofosfatídicos/metabolismo , Relação Estrutura-AtividadeRESUMO
Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric alpha-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein-coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.
Assuntos
Neoplasias da Mama/patologia , Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/uso terapêutico , Complexos Multienzimáticos/antagonistas & inibidores , Organofosfonatos/uso terapêutico , Fosfodiesterase I/antagonistas & inibidores , Pirofosfatases/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Diester Fosfórico HidrolasesRESUMO
Lysophosphatidic acid (LPA) is a ligand for LPA(1-3) of the endothelial differentiation gene family G-protein-coupled receptors, and LPA(4-8) is related to the purinergic family G-protein-coupled receptor. Because the structure-activity relationship (SAR) of GPR92/LPA(5) is limited and whether LPA is its preferred endogenous ligand has been questioned in the literature, in this study we applied a combination of computational and experimental site-directed mutagenesis of LPA(5) residues predicted to interact with the headgroup of LPA. Four residues involved in ligand recognition in LPA(5) were identified as follows: R2.60N mutant abolished receptor activation, whereas H4.64E, R6.62A, and R7.32A greatly reduced receptor activation. We also investigated the SAR of LPA(5) using LPA analogs and other non-lysophospholipid ligands. SAR revealed that the rank order of agonists is alkyl glycerol phosphate > LPA > farnesyl phosphates >> N-arachidonoylglycine. These results confirm LPA(5) to be a bona fide lysophospholipid receptor. We also evaluated several compounds with previously established selectivity for the endothelial differentiation gene receptors and found several that are LPA(5) agonists. A pharmacophore model of LPA(5) binding requirements was developed for in silico screening, which identified two non-lipid LPA(5) antagonists. Because LPA(5) transcripts are abundant in human platelets, we tested its antagonists on platelet activation and found that these non-lipid LPA(5) antagonists inhibit platelet activation. The present results suggest that selective inhibition of LPA(5) may provide a basis for future anti-thrombotic therapies.
Assuntos
Ativação Plaquetária/fisiologia , Receptores de Ácidos Lisofosfatídicos/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação/genética , Sinalização do Cálcio , Humanos , Técnicas In Vitro , Ligantes , Lisofosfolipídeos/química , Lisofosfolipídeos/metabolismo , Lisofosfolipídeos/farmacologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ativação Plaquetária/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos/agonistas , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Ácidos Lisofosfatídicos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
In the present study, we utilized virtual screening to identify LPA(3) antagonists. We have developed a three-point structure-based pharmacophore model based on known LPA(3) antagonists. This model was used to mine the NCI database. Docking, pharmacophore development, and database mining produced new, non-lipid leads. Experimental testing of seven computationally selected pharmacophore hits produced one potentiator and three antagonists, one of which displays both LPA(3) selectivity and nanomolar potency. Similarity searching in the ChemBridge database using the most promising lead as the search target produced four additional LPA(3) antagonists and a potent dual LPA(1&2) antagonist.
Assuntos
Simulação por Computador , Lisofosfolipídeos/antagonistas & inibidores , Modelos Químicos , Modelos Moleculares , Animais , Células CHO , Cálcio/química , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Bovinos , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Diglicerídeos/química , Diglicerídeos/farmacologia , Isoxazóis/química , Isoxazóis/farmacologia , Lisofosfolipídeos/agonistas , Lisofosfolipídeos/metabolismo , Propionatos/química , Propionatos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Ratos , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/biossíntese , Receptores de Ácidos Lisofosfatídicos/genética , Rodopsina/química , Homologia Estrutural de ProteínaRESUMO
Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.
Assuntos
Aminoácidos/metabolismo , Diferenciação Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Biologia Computacional , Sequência Conservada , Citometria de Fluxo , Humanos , Ligantes , Lisofosfolipídeos/metabolismo , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/metabolismo , Mutação Puntual/genética , Ratos , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Computational modeling and its application in ligand screening and ligand receptor interaction studies play important roles in structure-based drug design. A series of sphingosine 1-phosphate (S1P) receptor ligands with varying potencies and receptor selectivities were docked into homology models of the S1P(1-5) receptors. These studies provided molecular insights into pharmacological trends both across the receptor family as well as at single receptors. This study identifies ligand recognition features that generalize across the S1P receptor family, features unique to the S1P(4) and S1P(5) receptors, and suggests significant structural differences of the S1P(2) receptor. Docking results reveal a previously unknown sulfur-aromatic interaction between the S1P(4) C5.44 sulfur atom and the phenyl ring of benzimidazole as well as pi-pi interaction between F3.33 of S1P(1,4,5) and aromatic ligands. The findings not only confirm the importance of a cation-pi interaction between W4.64 and the ammonium of S1P at S1P(4) but also predict the same interaction at S1P(5). S1P receptor models are validated for pharmacophore development including database mining and new ligand discovery and serve as tools for ligand optimization to improve potency and selectivity.
Assuntos
Modelos Moleculares , Receptores de Ácidos Lisofosfatídicos/química , Receptores de Lisoesfingolipídeo/química , Sequência de Aminoácidos , Sítios de Ligação , Ligantes , Dados de Sequência Molecular , Estrutura Molecular , Ligação Proteica , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P1 from S1P to 18:1 LPA. RESULTS: We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P4. This mutation severely affected the response of S1P4 to S1P in a [35S]GTP gamma S binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P4 receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors. CONCLUSIONS: Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P1 receptor.