RESUMO
Deregulation of the phosphoinositide-3-OH kinase (PI(3)K) pathway has been implicated in numerous pathologies including cancer, diabetes, thrombosis, rheumatoid arthritis and asthma. Recently, small-molecule and ATP-competitive PI(3)K inhibitors with a wide range of selectivities have entered clinical development. In order to understand the mechanisms underlying the isoform selectivity of these inhibitors, we developed a new expression strategy that enabled us to determine to our knowledge the first crystal structure of the catalytic subunit of the class IA PI(3)K p110 delta. Structures of this enzyme in complex with a broad panel of isoform- and pan-selective class I PI(3)K inhibitors reveal that selectivity toward p110 delta can be achieved by exploiting its conformational flexibility and the sequence diversity of active site residues that do not contact ATP. We have used these observations to rationalize and synthesize highly selective inhibitors for p110 delta with greatly improved potencies.
Assuntos
Domínio Catalítico , Fosfatidilinositol 3-Quinases/química , Inibidores de Proteínas Quinases/química , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Simulação por Computador , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Fosfatidilinositol 3-Quinases/metabolismo , Domínios e Motivos de Interação entre Proteínas , Spodoptera , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Malaria remains a major killer in many parts of the world. Recently, there has been an increase in the role of public-private partnerships inciting academic and industrial scientists to merge their expertise in drug-target validation and in the early stage of drug discovery to identify potential new medicines. There is a need to identify and characterize new molecules showing high efficacy, low toxicity with low propensity to induce resistance in the parasite. In this context, we have studied the structural requirements of the inhibition of PfCDPK1. This is a calcium-dependent protein kinase expressed in Plasmodium falciparum, which has been genetically confirmed as essential for survival. A primary screening assay has been developed. A total of 54000 compounds were tested, yielding two distinct chemical series of nanomolar small molecule inhibitors. The most potent members of each series were further characterized through enzymatic and biophysical analyses. Dissociation rates of the inhibitor-kinase complexes were shown to be key parameters to differentiate both series. Finally, a homology-based model of the kinase core domain has been built which allows rational design of the next generation of inhibitors.
Assuntos
Plasmodium falciparum/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Animais , Luminescência , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Proteínas Quinases/isolamento & purificação , Proteínas Quinases/metabolismo , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Especificidade por SubstratoRESUMO
Chemokines are a family of small chemoattractant cytokines that have an important role in controlling leukocyte migration. The finding that some chemokines and their receptors are upregulated in both acute and chronic inflammatory diseases, and that they are key players in the development of AIDS, has provided the pharmaceutical industry with new targets for therapeutic intervention in these diseases. Although the chemokine system shows apparent redundancy in vitro, target validation is possible largely through expression studies in human disease tissues and the use of transgenic and knockout mice as disease models. Several approaches are being developed to block the effects of chemokines, including small-molecule antagonists of chemokine receptors, modified chemokines and antibodies directed against chemokine receptors. Here, we describe the rationale behind these different approaches, the pitfalls that have been encountered and future perspectives.
Assuntos
Quimiocinas/antagonistas & inibidores , Animais , Antagonistas dos Receptores CCR5 , Eosinófilos/fisiologia , Glicosaminoglicanos/metabolismo , Humanos , Psoríase/tratamento farmacológico , Receptores CCR3 , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/fisiologia , Transdução de SinaisRESUMO
Class I phosphoinositide 3-kinases (PI3Ks), in particular PI3Kgamma, have become attractive drug targets for inflammatory and autoimmune diseases. Here, we disclose a novel series of furan-2-ylmethylene thiazolidinediones as selective, ATP-competitive PI3Kgamma inhibitors. Structure-based design and X-ray crystallography of complexes formed by inhibitors bound to PI3Kgamma identified key pharmacophore features for potency and selectivity. An acidic NH group on the thiazolidinedione moiety and a hydroxy group on the furan-2-yl-phenyl part of the molecule play crucial roles in binding to PI3K and contribute to class IB PI3K selectivity. Compound 26 (AS-252424), a potent and selective small-molecule PI3Kgamma inhibitor emerging from these efforts, was further profiled in three different cellular PI3K assays and shown to be selective for class IB PI3K-mediated cellular effects. Oral administration of 26 in a mouse model of acute peritonitis led to a significant reduction of leukocyte recruitment.
Assuntos
Furanos/síntese química , Inibidores de Fosfoinositídeo-3 Quinase , Tiazolidinedionas/síntese química , Doença Aguda , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/fisiologia , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Classe Ib de Fosfatidilinositol 3-Quinase , Cristalografia por Raios X , Furanos/química , Furanos/farmacologia , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/química , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Monócitos/efeitos dos fármacos , Monócitos/fisiologia , Neutrófilos/imunologia , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Peritonite/imunologia , Fosfatidilinositol 3-Quinases/química , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Estrutura-Atividade , Tiazolidinedionas/química , Tiazolidinedionas/farmacologia , TioglicolatosRESUMO
The biological activity of chemokines requires interactions with cell surface proteoglycans. We have determined the structure of the chemokine RANTES (regulated on activation normal T cell expressed) in the presence of heparin-derived disaccharide analogs by X-ray crystallography. These structures confirm the essential role of the BBXB motif in the interaction between the chemokine and the disaccharide. Unexpected interactions were observed in the 30s loop and at the amino terminus. Mutant RANTES molecules were designed to abrogate these interactions and their biological activity examined in vivo. The K45E mutant within the BBXB motif lost the capacity to bind heparin and the ability to elicit cellular recruitment. The Y3A mutant maintained its capacity to bind heparin but was unable to elicit cellular recruitment. Finally, a tetrasaccharide is the smallest oligosaccharide which effectively abolishes the ability of RANTES to recruit cells in vivo. These crystallographic structures provide a description of the molecular interaction of a chemokine with glycosaminoglycans.
Assuntos
Quimiocina CCL5/química , Quimiocinas/antagonistas & inibidores , Sequência de Carboidratos , Quimiocina CCL5/genética , Cristalografia por Raios X , Heparina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação ProteicaRESUMO
BACKGROUND: Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins. METHODOLOGY/PRINCIPAL FINDINGS: We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1:1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent pi-pi interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine. CONCLUSIONS/SIGNIFICANCE: However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the "address" whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.
Assuntos
Quimiocina CCL3/química , Quimiocina CCL3/metabolismo , Receptores de Quimiocinas/química , Receptores de Quimiocinas/metabolismo , Rhipicephalus/metabolismo , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Glicosilação , Humanos , Modelos Moleculares , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126-1136), and may be therapeutically useful as novel antiinflammatory agents in the future.
Assuntos
Anti-Inflamatórios/metabolismo , Quimiocinas/metabolismo , Receptores de Quimiocinas/metabolismo , Animais , Borrelia burgdorferi , Clonagem Molecular , DNA Complementar/metabolismo , Humanos , Inflamação , Concentração Inibidora 50 , Conformação Molecular , Ligação Proteica , Rhipicephalus sanguineus , Células Th1/metabolismo , Células Th2/metabolismoRESUMO
Protein therapeutics represent a rapidly growing proportion of marketed drugs and have an undisputed place alongside chemistry-based oral therapies; indeed, for certain indications they are the only effective therapy. Therapeutic proteins can be mined from diverse sources to target interactions that are not accessible to small molecules, and can be engineered to have optimal pharmacological properties. Nevertheless, the development of such therapeutics is hampered by several issues, such as cost of production, patient compliance, immunogenicity and reticence of reimbursement agencies to pay for their use in chronic treatment. Herein we review some of these issues in detail. The application of technological advancements will address some of these issues and enable the development of a growing number of biological therapies that will continue to improve patient quality of life for decades to come.