RESUMO
Amphetamines (AMPHs) are globally abused. With no effective treatment for AMPH addiction to date, there is urgent need for the identification of druggable targets that mediate the reinforcing action of this stimulant class. AMPH-stimulated dopamine efflux is modulated by protein kinase C (PKC) activation. Inhibition of PKC reduces AMPH-stimulated dopamine efflux and locomotor activity. The only known CNS-permeant PKC inhibitor is the selective estrogen receptor modulator tamoxifen. In this study, we demonstrate that a tamoxifen analog, 6c, which more potently inhibits PKC than tamoxifen but lacks affinity for the estrogen receptor, reduces AMPH-stimulated increases in extracellular dopamine and reinforcement-related behavior. In rat striatal synaptosomes, 6c was almost fivefold more potent at inhibiting AMPH-stimulated dopamine efflux than [3H]dopamine uptake through the dopamine transporter (DAT). The compound did not compete with [3H]WIN 35,428 binding or affect surface DAT levels. Using microdialysis, direct accumbal administration of 1 µM 6c reduced dopamine overflow in freely moving rats. Using LC-MS, we demonstrate that 6c is CNS-permeant. Systemic treatment of rats with 6 mg/kg 6c either simultaneously or 18 h prior to systemic AMPH administration reduced both AMPH-stimulated dopamine overflow and AMPH-induced locomotor effects. Finally, 18 h pretreatment of rats with 6 mg/kg 6c s.c. reduces AMPH-self administration but not food self-administration. These results demonstrate the utility of tamoxifen analogs in reducing AMPH effects on dopamine and reinforcement-related behaviors and suggest a new avenue of development for therapeutics to reduce AMPH abuse.
Assuntos
Anfetamina/farmacologia , Dopamina/metabolismo , Psicotrópicos/farmacologia , Reforço Psicológico , Tamoxifeno/análogos & derivados , Tamoxifeno/administração & dosagem , Animais , Estimulantes do Sistema Nervoso Central/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Dopaminérgicos/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/embriologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Autoadministração , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismo , TrítioRESUMO
We recently reported the development of a novel inhibitor of Rho-mediated gene transcription (1, CCG-203971) that is efficacious in multiple animal models of acute fibrosis, including scleroderma, when given intraperitoneally. The modest in vivo potency and poor pharmacokinetics (PK) of this lead, however, make it unsuitable for long term efficacy studies. We therefore undertook a systematic medicinal chemistry effort to improve both the metabolic stability and the solubility of 1, resulting in the identification of two analogs achieving over 10-fold increases in plasma exposures in mice. We subsequently showed that one of these analogs (8f, CCG-232601) could inhibit the development of bleomycin-induced dermal fibrosis in mice when administered orally at 50mg/kg, an effect that was comparable to what we had observed earlier with 1 at a 4-fold higher IP dose.
Assuntos
Ácidos Nipecóticos/farmacocinética , Ácidos Nipecóticos/uso terapêutico , Fator Rho/antagonistas & inibidores , Escleroderma Sistêmico/tratamento farmacológico , Pele/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Administração Oral , Animais , Modelos Animais de Doenças , Fibrose , Células HEK293 , Humanos , Camundongos , Ácidos Nipecóticos/administração & dosagem , Ácidos Nipecóticos/química , Fator Rho/metabolismo , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia , Elemento de Resposta Sérica/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Transativadores/antagonistas & inibidores , Transativadores/metabolismoRESUMO
The clinical selective estrogen receptor modulator tamoxifen is also a modest inhibitor of protein kinase C, a target implicated in several untreatable brain diseases such as amphetamine abuse. This inhibition and tamoxifen's ability to cross the blood brain barrier make it an attractive scaffold to conduct further SAR studies toward uncovering effective therapies for such diseases. Utilizing the known compound 6a as a starting template and guided by computational tools to derive physicochemical properties known to be important for CNS permeable drugs, the design and synthesis of a small series of novel triarylacrylonitrile analogues have been carried out providing compounds with enhanced potency and selectivity for PKC over the estrogen receptor relative to tamoxifen. Shortened synthetic routes compared to classical procedures have been developed for analogues incorporating a ß-phenyl ring, which involve installing dialkylaminoalkoxy side chains first off the α and/or α' rings of a precursor benzophenone and then condensing the resultant ketones with phenylacetonitrile anion. A second novel, efficient and versatile route utilizing Suzuki chemistry has also been developed, which will allow for the introduction of a wide range of ß-aryl or ß-heteroaryl moieties and side-chain substituents onto the acrylonitrile core. For analogues possessing a single side chain off the α- or α'-ring, novel 2D NMR experiments have been carried out that allow for unambiguous assignment of E- and Z-stereochemistry. From the SAR analysis, one compound, 6c, shows markedly increased potency and selectivity for inhibiting PKC with an IC50 of 80nM for inhibition of PKC protein substrate and >10µM for binding to the estrogen receptor α (tamoxifen IC50=20µM and 222nM, respectively). The data on 6c provide support for further exploration of PKC as a druggable target for the treatment of amphetamine abuse.
Assuntos
Acrilonitrila/farmacologia , Desenho de Fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Tamoxifeno/farmacologia , Acrilonitrila/síntese química , Acrilonitrila/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Ligação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tamoxifeno/químicaRESUMO
RecA is central to maintaining genome integrity in bacterial cells. Despite the near-ubiquitous conservation of RecA in eubacteria, the pathways that facilitate RecA loading and repair center assembly have remained poorly understood in Bacillus subtilis. Here, we show that RecA rapidly colocalizes with the DNA polymerase complex (replisome) immediately following DNA damage or damage-independent replication fork arrest. In Escherichia coli, the RecFOR and RecBCD pathways serve to load RecA and the choice between these two pathways depends on the type of damage under repair. We found in B. subtilis that the rapid localization of RecA to repair centers is strictly dependent on RecO and RecR in response to all types of damage examined, including a site-specific double-stranded break and damage-independent replication fork arrest. Furthermore, we provide evidence that, although RecF is not required for RecA repair center formation in vivo, RecF does increase the efficiency of repair center assembly, suggesting that RecF may influence the initial stages of RecA nucleation or filament extension. We further identify single-stranded DNA binding protein (SSB) as an additional component important for RecA repair center assembly. Truncation of the SSB C terminus impairs the ability of B. subtilis to form repair centers in response to damage and damage-independent fork arrest. With these results, we conclude that the SSB-dependent recruitment of RecOR to the replisome is necessary for loading and organizing RecA into repair centers in response to DNA damage and replication fork arrest.
Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/genética , Replicação do DNA/efeitos dos fármacos , DNA Bacteriano/genética , Uracila/análogos & derivados , Bacillus subtilis/citologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Dano ao DNA , Reparo do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Mutação , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes de Fusão , Uracila/farmacologiaRESUMO
Resistance to antibiotics is an increasingly dire threat to human health that warrants the development of new modes of treating infection. We recently identified 1 (CCG-2979) as an inhibitor of the expression of streptokinase, a critical virulence factor in Group A Streptococcus that endows blood-borne bacteria with fibrinolytic capabilities. In this report, we describe the synthesis and biological evaluation of a series of novel 5,6-dihydrobenzo[h]quinazolin-4(3H)-one analogs of 1 undertaken with the goal of improving the modest potency of the lead. In addition to achieving an over 35-fold increase in potency, we identified structural modifications that improve the solubility and metabolic stability of the scaffold. The efficacy of two new compounds 12c (CCG-203592) and 12k (CCG-205363) against biofilm formation in Staphylococcus aureus represents a promising additional mode of action for this novel class of compounds.
Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Quinazolinas/química , Quinazolinas/farmacologia , Streptococcus/enzimologia , Estreptoquinase/antagonistas & inibidores , Animais , Antibacterianos/metabolismo , Antibacterianos/toxicidade , Biofilmes/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Quinazolinas/metabolismo , Quinazolinas/toxicidade , Solubilidade , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus/efeitos dos fármacos , Streptococcus/genética , Streptococcus/fisiologia , Estreptoquinase/genética , Estreptoquinase/metabolismo , Relação Estrutura-AtividadeRESUMO
Staphylococcus aureus is a major human pathogen and one of the more prominent pathogens causing biofilm related infections in clinic. Antibiotic resistance in S. aureus such as methicillin resistance is approaching an epidemic level. Antibiotic resistance is widespread among major human pathogens and poses a serious problem for public health. Conventional antibiotics are either bacteriostatic or bacteriocidal, leading to strong selection for antibiotic resistant pathogens. An alternative approach of inhibiting pathogen virulence without inhibiting bacterial growth may minimize the selection pressure for resistance. In previous studies, we identified a chemical series of low molecular weight compounds capable of inhibiting group A streptococcus virulence following this alternative anti-microbial approach. In the current study, we demonstrated that two analogs of this class of novel anti-virulence compounds also inhibited virulence gene expression of S. aureus and exhibited an inhibitory effect on S. aureus biofilm formation. This class of anti-virulence compounds could be a starting point for development of novel anti-microbial agents against S. aureus.
Assuntos
Biofilmes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Quinazolinas/farmacologia , Estreptoquinase/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Humanos , Resistência a Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Testes de Sensibilidade Microbiana , Quinazolinas/síntese química , Bibliotecas de Moléculas Pequenas , Estreptoquinase/genéticaRESUMO
Translocations involving the mixed lineage leukemia (MLL) gene result in human acute leukemias with very poor prognosis. The leukemogenic activity of MLL fusion proteins is critically dependent on their direct interaction with menin, a product of the multiple endocrine neoplasia (MEN1) gene. Here we present what are to our knowledge the first small-molecule inhibitors of the menin-MLL fusion protein interaction that specifically bind menin with nanomolar affinities. These compounds effectively reverse MLL fusion protein-mediated leukemic transformation by downregulating the expression of target genes required for MLL fusion protein oncogenic activity. They also selectively block proliferation and induce both apoptosis and differentiation of leukemia cells harboring MLL translocations. Identification of these compounds provides a new tool for better understanding MLL-mediated leukemogenesis and represents a new approach for studying the role of menin as an oncogenic cofactor of MLL fusion proteins. Our findings also highlight a new therapeutic strategy for aggressive leukemias with MLL rearrangements.
Assuntos
Antineoplásicos/farmacologia , Leucemia/tratamento farmacológico , Proteína de Leucina Linfoide-Mieloide/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células HEK293 , Histona-Lisina N-Metiltransferase , Humanos , Leucemia/genética , Leucemia/metabolismo , Leucemia/patologia , Camundongos , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Relação Estrutura-AtividadeRESUMO
AG490 is a tyrosine kinase inhibitor with activity against Jak2 and apoptotic activity in specific leukemias. Due to its weak kinase inhibitory activity and poor pharmacology, we conducted a cell-based screen for derivatives with improved Jak2 inhibition and activity in animals. Two hits emerged from an initial small chemical library screen, and more detailed structure-activity relationship studies led to the development of WP1130 with 50-fold greater activity in suppressing Jak2-dependent cytokine signaling than AG490. However, WP1130 did not directly suppress Jak2 kinase activity, but mediated Jak2 ubiquitination resulting in its trafficking through HDAC6 to perinuclear aggresomes without cytokine stimulation or SOCS-1 induction. Jak2 primarily contained K63-linked ubiquitin polymers, and mutation of this lysine blocked Jak2 ubiquitination and mobilization in WP1130-treated cells. Further analysis demonstrated that WP1130, but not AG490, acts as a deubiquitinating enzyme (DUB) inhibitor, possibly through a Michael addition reaction. We conclude that chemical modification of AG490 resulted in development of a DUB inhibitor with activity against a DUB capable of modulating Jak2 ubiquitination, trafficking and signal transduction.
Assuntos
Endopeptidases/metabolismo , Janus Quinase 2/metabolismo , Inibidores de Proteases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Tirfostinas/farmacologia , Ubiquitinação , Substituição de Aminoácidos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cianoacrilatos , Avaliação Pré-Clínica de Medicamentos , Endopeptidases/química , Ensaios Enzimáticos , Proteínas de Choque Térmico HSP90/metabolismo , Desacetilase 6 de Histona , Histona Desacetilases/metabolismo , Humanos , Concentração Inibidora 50 , Interleucina-6/farmacologia , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nitrilas/farmacologia , Fosforilação , Inibidores de Proteassoma , Transporte Proteico/efeitos dos fármacos , Proteólise , Piridinas/farmacologia , Fator de Transcrição STAT3/metabolismo , Fator de Transcrição STAT5/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
We explored the D-arabinose 5-phosphate isomerase (KdsD, E.C. 5.3.1.13) from Francisella tularensis, a highly infectious gram-negative pathogen that has raised concern as a potential bioweapon, as a target for the development of novel chemotherapeutics. F. tularensis KdsD was expressed in Escherichia coli from a synthetic gene, purified, and characterized. A group of hydroxamates designed to be mimics of the putative enediol intermediate in the enzyme's catalytic mechanism were prepared and tested as inhibitors of F. tularensis KdsD. The best inhibitor, which has an IC(50) of 7 µM, is the most potent KdsD inhibitor reported to date.
Assuntos
Álcoois/farmacologia , Aldose-Cetose Isomerases/antagonistas & inibidores , Antibacterianos/farmacologia , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Francisella tularensis/efeitos dos fármacos , Álcoois/síntese química , Álcoois/química , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Alcenos/síntese química , Alcenos/química , Alcenos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Escherichia coli/genética , Francisella tularensis/enzimologia , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Concentração Inibidora 50 , Estrutura MolecularRESUMO
An extraordinarily potent and hepatoselective class of HMG-CoA reductase inhibitors containing a pyrazole core was recently reported; however, its development was hampered by a long and difficult synthetic route. We attempted to circumvent this obstacle by preparing closely related analogs wherein the key dihydroxyheptanoic acid sidechain was tethered to the pyrazole core via an oxygen linker ('oxypyrazoles'). This minor change reduced the total number of synthetic steps from 14 to 7. Although the resulting analogs maintained much of the in vitro and cell activity of the pyrazoles, inferior in vivo activity precluded further development. Caco-2 cell permeability data suggest that enhanced cellular efflux of the oxypyrazoles relative to the pyrazoles may be responsible for the poor in vivo activity.
Assuntos
Desenho de Fármacos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Animais , Linhagem Celular , Cricetinae , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Fígado/efeitos dos fármacos , Fígado/enzimologia , Estrutura Molecular , Células Musculares/efeitos dos fármacos , Células Musculares/enzimologia , Pirazóis/síntese química , Ratos , Relação Estrutura-AtividadeRESUMO
This manuscript describes the design and synthesis of a series of pyrrole-based inhibitors of HMG-CoA reductase for the treatment of hypercholesterolemia. Analogs were optimized using structure-based design and physical property considerations resulting in the identification of 44, a hepatoselective HMG-CoA reductase inhibitor with excellent acute and chronic efficacy in a pre-clinical animal models.
Assuntos
Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Pirróis/química , Pirróis/farmacologia , Animais , Cricetinae , Relação Dose-Resposta a Droga , Desenho de Fármacos , Fluorbenzenos , Hiperlipidemias/tratamento farmacológico , Fígado/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Pirimidinas , Rosuvastatina Cálcica , Relação Estrutura-Atividade , SulfonamidasRESUMO
Recent literature has suggested the benefit of selective PPARdelta agonists for the treatment of atherosclerosis and other disease states associated with the metabolic syndrome. Herein we report the synthesis and structure-activity relationships of a series of novel and selective PPARdelta agonists. Our search began with identification of a novel benzothiophene template which was modified by the addition of various thiazolyl, isoxazolyl, and benzyloxy-benzyl moieties. Further elucidation of the SAR led to the identification of benzofuran and indole based templates. During the course of our research, we discovered three new chemical templates with varying degrees of affinity and potency for PPARdelta versus the PPARalpha and PPARgamma subtypes.