RESUMO
The type VII secretion system (T7SS) of Staphylococcus aureus is a multiprotein complex dedicated to the export of several virulence factors during host infection. This virulence pathway plays a key role in promoting bacterial survival and the long-term persistence of staphylococcal abscess communities. The expression of the T7SS is activated by bacterial interaction with host tissues including blood serum, nasal secretions, and pulmonary surfactant. In this work we identify the major stimulatory factors as host-specific cis-unsaturated fatty acids. Increased T7SS expression requires host fatty acid incorporation into bacterial biosynthetic pathways by the Saureus fatty acid kinase (FAK) complex, and FakA is required for virulence. The incorporated cis-unsaturated fatty acids decrease Saureus membrane fluidity, and these altered membrane dynamics are partially responsible for T7SS activation. These data define a molecular mechanism by which Saureus cells sense the host environment and implement appropriate virulence pathways.
Assuntos
Interações Hospedeiro-Patógeno , Ácido Linoleico/metabolismo , Staphylococcus aureus/fisiologia , Sistemas de Secreção Tipo VII/fisiologia , Animais , Humanos , Camundongos , Fatores de Virulência/metabolismoRESUMO
The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral ß-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.
Assuntos
Bactérias Gram-Negativas/metabolismo , Lipoproteínas/metabolismo , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Lipoproteínas/genética , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genéticaRESUMO
Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis. These cells are classically defined as a subset of morphologically defined A single (As) spermatogonia, which can produce more SSCs or they can give rise to nonstem As cells that, upon replication, generate A paired (Apr) and then A aligned (Aal) spermatogonia. These latter two cell types, along with the nonstem As cells, function as transit-amplifying progenitor cells. It is known that glial cell line-derived neurotrophic factor (GDNF) is essential for maintaining all of these cells, but it is unknown if or how the responses of these cells change as they progress down the pathway to differentiated type A1 spermatogonia. We address this issue by using a chemical-genetic approach to inhibit GDNF signaling in vivo and an in vitro approach to increase GDNF stimulation. We show that inhibition for 2 days suppresses replication of As, Apr, and Aal spermatogonia to an equal extent, whereas stimulation by GDNF preferentially increases replication of As and Apr spermatogonia. We also test if inhibiting GDNF signaling causes As, Apr, and Aal spermatogonia to express Kit, an essential step in their differentiation into type A1 spermatogonia. Inhibition for 3 or 7 days produces a progressive increase in the percentages of As, Apr, and Aal undergoing differentiation, with the largest increase observed in Aal spermatogonia. Finally, we demonstrate that numbers of SSCs decrease more slowly than numbers of progenitor spermatogonia when GDNF signaling is inhibited. Taken together, these data suggest that there are significant changes in the responses to GDNF as SSCs give rise to progenitor spermatogonia, which replicate and gradually differentiate into type A1 spermatogonia.
Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Células-Tronco Adultas/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica/fisiologia , Masculino , Camundongos , Túbulos Seminíferos/citologia , Transdução de Sinais , Técnicas de Cultura de TecidosRESUMO
Analog-sensitive (AS) kinase technology is a powerful approach for studying phospho-signaling pathways in diverse organisms and physiological processes. The key feature of this technique is that a kinase-of-interest can be mutated to sensitize it to inhibitor analogs that do not target wild-type (WT) kinases. In theory, this enables specific inhibition of any kinase in cells and in mouse models of human disease. Typically, these inhibitors are identified from a small library of molecules based on the pyrazolopyrimidine (PP) scaffold. However, we recently identified a subset of native human kinases, including the Ephrin A kinase family, that are sensitive to commonly used PP inhibitors. In an effort to develop a bioorthogonal AS-kinase inhibitor and to extend this technique to PP-sensitive kinases, we sought an alternative inhibitor scaffold. Here we report the structure-based design of synthetically tractable, potent, and extremely selective AS-kinase inhibitors based on the natural product staurosporine. We demonstrate that these molecules, termed staralogs, potently target AS kinases in cells, and we employ X-ray crystallography to elucidate their mechanism of efficacy. Finally, we demonstrate that staralogs target AS mutants of PP-sensitive kinases at concentrations where there is little to no inhibition of native human kinases. Thus, staralogs represent a new class of AS-kinase inhibitors and a core component of the chemical genetic tool kit for probing kinase-signaling pathways.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Animais , Sítios de Ligação , Carbazóis/química , Carbazóis/farmacologia , Humanos , Camundongos , Modelos Moleculares , Mutação , Proteínas Quinases/genética , Relação Estrutura-AtividadeRESUMO
The cancer drug, Imatinib, is a selective Abl kinase inhibitor that does not inhibit the closely related kinase c-Src. This one drug and its ability to selectively inhibit Abl over c-Src has been a guiding principle in virtually all kinase drug discovery efforts in the last 15 years. A prominent hypothesis explaining the selectivity of Imatinib is that Abl has an intrinsic ability to adopt an inactive conformation (termed DFG-out), whereas c-Src appears to pay a high intrinsic energetic penalty for adopting this conformation, effectively excluding Imatinib from its ATP pocket. This explanation of the difference in binding affinity of Imatinib for Abl versus c-Src makes the striking prediction that it would not be possible to design an inhibitor that binds to the DFG-out conformation of c-Src with high affinity. We report the discovery of a series of such inhibitors. We use structure-activity relationships and X-ray crystallography to confirm our findings. These studies suggest that small molecules are capable of inducing the generally unfavorable DFG-out conformation in c-Src. Structural comparison between c-Src in complex with these inhibitors allows us to speculate on the differential selectivity of Imatinib for c-Src and Abl.
Assuntos
Preparações Farmacêuticas/química , Piperazinas/química , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Pirimidinas/química , Benzamidas , Cristalografia por Raios X , Mesilato de Imatinib , Modelos Moleculares , Estrutura Molecular , Ligação Proteica , Proteínas Proto-Oncogênicas pp60(c-src)/química , Proteínas Proto-Oncogênicas pp60(c-src)/genéticaRESUMO
Calcium dependent protein kinase 1 (CDPK1) is an essential enzyme in the opportunistic pathogen Toxoplasma gondii. CDPK1 controls multiple processes that are critical to the intracellular replicative cycle of T. gondii including secretion of adhesins, motility, invasion, and egress. Remarkably, CDPK1 contains a small glycine gatekeeper residue in the ATP binding pocket making it sensitive to ATP-competitive inhibitors with bulky substituents that complement this expanded binding pocket. Here we explored structure-activity relationships of a series of pyrazolopyrimidine inhibitors of CDPK1 with the goal of increasing selectivity over host enzymes, improving antiparasite potency, and improving metabolic stability. The resulting lead compound 24 exhibited excellent enzyme inhibition and selectivity for CDPK1 and potently inhibited parasite growth in vitro. Compound 24 was also effective at treating acute toxoplasmosis in the mouse, reducing dissemination to the central nervous system, and decreasing reactivation of chronic infection in severely immunocompromised mice. These findings provide proof of concept for the development of small molecule inhibitors of CDPK1 for treatment of CNS toxoplasmosis.
Assuntos
Antiprotozoários/química , Antiprotozoários/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Toxoplasmose Cerebral/tratamento farmacológico , Animais , Antiprotozoários/farmacocinética , Feminino , Humanos , Masculino , Camundongos , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Pirazóis/química , Pirimidinas/química , Relação Estrutura-Atividade , Toxoplasma/efeitos dos fármacos , Toxoplasma/enzimologia , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose Cerebral/prevenção & controleRESUMO
Analog-sensitive AS Kinase technology allows for rapid, reversible, and highly specific inhibition of individual engineered kinases in cells and in mouse models of human diseases. The technique consists of two parts: a kinase containing a space-creating mutation in the ATP-binding pocket and a bulky ATP-competitive small molecule inhibitor that complements the shape of the mutant ATP pocket. This strategy enables dissection of phospho-signaling pathways, elucidation of the physiological function of individual kinases, and characterization of the pharmacology of clinical-kinase inhibitors. Here, we present an overview of AS technology and describe a stepwise approach for generating AS Kinase mutants and identifying appropriate small molecule inhibitors. We also describe commonly encountered technical obstacles and provide strategies to overcome them.
Assuntos
Desenho de Fármacos , Modelos Moleculares , Engenharia de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/química , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/antagonistas & inibidores , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Humanos , Ligantes , Conformação Molecular , Mutação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Propriedades de SuperfícieRESUMO
Activating mutations in FLT3 confer poor prognosis for individuals with acute myeloid leukemia (AML). Clinically active investigational FLT3 inhibitors can achieve complete remissions but their utility has been hampered by acquired resistance and myelosuppression attributed to a 'synthetic lethal toxicity' arising from simultaneous inhibition of FLT3 and KIT. We report a novel chemical strategy for selective FLT3 inhibition while avoiding KIT inhibition with the staurosporine analog, Star 27. Star 27 maintains potency against FLT3 in proliferation assays of FLT3-transformed cells compared with KIT-transformed cells, shows no toxicity towards normal human hematopoiesis at concentrations that inhibit primary FLT3-mutant AML blast growth, and is active against mutations that confer resistance to clinical inhibitors. As a more complete understanding of kinase networks emerges, it may be possible to define anti-targets such as KIT in the case of AML to allow improved kinase inhibitor design of clinical agents with enhanced efficacy and reduced toxicity.
Assuntos
Antineoplásicos/toxicidade , Doenças da Medula Óssea/induzido quimicamente , Leucemia Mieloide Aguda/tratamento farmacológico , Tirosina Quinase 3 Semelhante a fms/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Modelos Biológicos , Mutação , Relação Estrutura-Atividade , Peixe-Zebra , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
Engineered analog-sensitive (AS) protein kinases have emerged as powerful tools for dissecting phospho-signaling pathways, for elucidating the cellular function of individual kinases, and for deciphering unanticipated effects of clinical therapeutics. A crucial and necessary feature of this technology is a bioorthogonal small molecule that is innocuous toward native cellular systems but potently inhibits the engineered kinase. In order to generalize this method, we sought a molecule capable of targeting divergent AS-kinases. Here we employ X-ray crystallography and medicinal chemistry to unravel the mechanism of current inhibitors and use these insights to design the most potent, selective, and general AS-kinase inhibitors reported to date. We use large-scale kinase inhibitor profiling to characterize the selectivity of these molecules as well as examine the consequences of potential off-target effects in chemical genetic experiments. The molecules reported here will serve as powerful tools in efforts to extend AS-kinase technology to the entire kinome and the principles discovered may help in the design of other engineered enzyme/ligand pairs.
Assuntos
Desenho de Fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Quinases/química , Proteínas Quinases/metabolismoRESUMO
Toxoplasma gondii is sensitive to bulky pyrazolo [3,4-d] pyrimidine (PP) inhibitors due to the presence of a Gly gatekeeper in the essential calcium dependent protein kinase 1 (CDPK1). Here we synthesized a number of new derivatives of 3-methyl-benzyl-PP (3-MB-PP, or 1). The potency of PP analogues in inhibiting CDPK1 enzyme activity in vitro (low nM IC(50) values) and blocking parasite growth in host cell monolayers in vivo (low µM EC(50) values) were highly correlated and occurred in a CDPK1-specific manner. Chemical modification of the PP scaffold to increase half-life in the presence of microsomes in vitro led to identification of compounds with enhanced stability while retaining activity. Several of these more potent compounds were able to prevent lethal infection with T. gondii in the mouse model. Collectively, the strategies outlined here provide a route for development of more effective compounds for treatment of toxoplasmosis and perhaps related parasitic diseases.