RESUMO
The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4R,5R)-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus. Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4R,5R)-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.
Assuntos
Proteínas de Bactérias , Ácido Corísmico/metabolismo , Genes Bacterianos/fisiologia , Imunossupressores/metabolismo , Família Multigênica/fisiologia , Sirolimo/metabolismo , Streptomyces , Tacrolimo/análogos & derivados , Tacrolimo/metabolismo , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Ácido Corísmico/química , Imunossupressores/química , Sirolimo/química , Streptomyces/enzimologia , Streptomyces/genética , Tacrolimo/químicaRESUMO
The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.
Assuntos
Melhoramento Genético/métodos , Mutagênese Sítio-Dirigida/métodos , Recombinação Genética/genética , Sirolimo/metabolismo , Streptomyces/fisiologia , Sirolimo/isolamento & purificação , Especificidade da Espécie , Streptomyces/classificaçãoRESUMO
Potent 3,4-disubstituted benzofuran P1' MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1' moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.
Assuntos
Benzofuranos/química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/química , Animais , Benzofuranos/síntese química , Benzofuranos/farmacologia , Metaloproteinase 13 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Ligação Proteica , Coelhos , Albumina Sérica/química , Relação Estrutura-AtividadeRESUMO
Inhibitors of matrix metalloprotease (MMP)-13 and tumor necrosis factor-alpha converting enzyme (TACE) have been highly sought as potential therapeutic agents for the treatment of osteoarthritis and rheumatoid arthritis, respectively. This review focuses on the published literature on these inhibitors from 2001 to mid-2003. Significant advances have been reported in the design and synthesis of potent and selective inhibitors of MMP-13 using hydroxamic acid and non-hydroxamate zinc chelators on a variety of scaffolds. TACE inhibitors based on variations of known MMP inhibitors scaffolds and novel designs have been reported. Selectivity profiles for these inhibitors range from broad-spectrum to TACE-specific. Future clinical studies on these and other inhibitors will determine which MMP, or set of MMPs, must be inhibited for efficacy and long-term safety.
Assuntos
Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/síntese química , Proteínas ADAM , Proteína ADAM17 , Animais , Artrite Reumatoide/tratamento farmacológico , Quelantes , Desenho de Fármacos , Humanos , Ácidos Hidroxâmicos/síntese química , Metaloproteinase 13 da Matriz , Osteoartrite/tratamento farmacológico , Inibidores de Proteases/uso terapêutico , Relação Estrutura-Atividade , ZincoRESUMO
A series of 4-alkynyloxy phenyl sulfanyl, sulfinyl and sulfony alkyl and piperidine-4-carboxylic acid hydroxamides were synthesized. Their structure-activity relationships, against tumor necrosis factor-alpha (TACE) and matrix metalloproteinase (MMP) inhibitor activities, are presented by investigating the oxidation state on sulfur and altering the P1' substituent. The sulfonyl derivatives 20-24 carrying a 4-butynyloxy moiety were selective TACE inhibitors over the MMPs tested. The sulfinyl derivatives showed a preference for a specific oxidation on sulfur as in compounds 25-28. The selectivity over MMPs was also demonstrated in the sulfonyl series. The enhanced cellular activity was achieved upon incorporating a butynyloxy substituent in the piperidene series. Compounds 64 and 65 were potent inhibitors of TNF-alpha release in the mouse at 100 mg/kg po.
Assuntos
Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Sulfetos/síntese química , Sulfonas/síntese química , Sulfóxidos/síntese química , Proteínas ADAM , Proteína ADAM17 , Animais , Cristalografia por Raios X , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Camundongos , Modelos Moleculares , Estrutura Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Oxirredução , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacologia , Relação Estrutura-Atividade , Sulfetos/química , Sulfetos/farmacologia , Sulfonas/química , Sulfonas/farmacologia , Sulfóxidos/química , Sulfóxidos/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. These enzymes are strictly regulated by endogenous inhibitors such as tissue inhibitors of MMPs and alpha(2)-macroglobulins. Overexpression of these enzymes has been implicated in various pathological disorders such as arthritis, tumor metastasis, cardiovascular diseases, and multiple sclerosis. Developing effective small-molecule inhibitors to modulate MMP activity is one approach to treat these degenerative diseases. The present work focuses on the discovery and SAR of novel N-hydroxy-alpha-phenylsulfonylacetamide derivatives, which are potent, selective, and orally active MMP inhibitors.
Assuntos
Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/síntese química , Sulfonas/síntese química , Proteínas ADAM , Proteína ADAM17 , Administração Oral , Animais , Bioensaio , Cartilagem/efeitos dos fármacos , Cartilagem/enzimologia , Bovinos , Diálise , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Metaloproteinase 13 da Matriz , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Osteoartrite/tratamento farmacológico , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
The matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that play a key role in both physiological and pathological tissue degradation. In our preceding paper, we have reported on a series of novel and orally active N-hydroxy-alpha-phenylsulfonylacetamide derivatives. However, these compounds had two drawbacks (moderate selectivity and chirality issues). To circumvent these two problems, a series of novel and orally active N-substituted 4-benzenesulfonylpiperidine-4-carboxylic acid hydroxyamide derivatives have been synthesized. The present paper deals with the synthesis and SAR of these compounds. Among the several compounds synthesized, derivative 55 turned out to be a potent, selective, and an orally active MMP inhibitor in the clinically relevant advanced rabbit osteoarthritis model. Detailed pharmacokinetics and metabolism data are described.
Assuntos
Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz , Osteoartrite/tratamento farmacológico , Piperidinas/síntese química , Inibidores de Proteases/síntese química , Sulfonas/síntese química , Proteínas ADAM , Proteína ADAM17 , Administração Oral , Animais , Sítios de Ligação , Bioensaio , Cartilagem/efeitos dos fármacos , Cartilagem/enzimologia , Bovinos , Cristalografia por Raios X , Diálise , Cães , Haplorrinos , Humanos , Ácidos Hidroxâmicos/farmacocinética , Ácidos Hidroxâmicos/farmacologia , Masculino , Metaloproteinase 13 da Matriz , Metaloproteinases da Matriz/química , Metaloendopeptidases/antagonistas & inibidores , Camundongos , Modelos Moleculares , Piperidinas/farmacocinética , Piperidinas/farmacologia , Inibidores de Proteases/farmacocinética , Inibidores de Proteases/farmacologia , Coelhos , Ratos , Relação Estrutura-Atividade , Sulfonas/farmacocinética , Sulfonas/farmacologiaRESUMO
Uterine leiomyomata, or fibroids, are benign tumors of the uterine myometrium that significantly affect up to 30% of reproductive-age women. Despite being the primary cause of hysterectomy in the United States, accounting for up to 200,000 procedures annually, the etiology of leiomyoma remains largely unknown. As a basis for understanding leiomyoma pathogenesis and identifying targets for pharmacotherapy, we conducted transcriptional profiling of leiomyoma and unaffected myometrium from humans and Eker rats, the best characterized preclinical model of leiomyomata. A global comparison of mRNA from leiomyoma versus myometrium in human and rat identified a highly significant overlap of dysregulated gene expression in leiomyomata. An unbiased pathway analysis using a method of gene-set enrichment based on the sigPathway algorithm detected the mammalian target of rapamycin (mTOR) pathway as one of the most highly up-regulated pathways in both human and rat tumors. To validate this pathway as a therapeutic target for uterine leiomyomata, preclinical studies were conducted in Eker rats. These rats develop uterine leiomyomata as a consequence of loss of Tsc2 function and up-regulation of mTOR signaling. Inhibition of mTOR in female Eker rats with the rapamycin analogue WAY-129327 for 2 weeks decreased mTOR signaling and cell proliferation in tumors, and treatment for 4 months significantly decreased tumor incidence, multiplicity, and size. These results identify dysregulated mTOR signaling as a component of leiomyoma etiology across species and directly show the dependence of uterine leiomyomata with activated mTOR on this signaling pathway for growth.
Assuntos
Leiomioma/metabolismo , Proteínas Quinases/metabolismo , Neoplasias Uterinas/metabolismo , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leiomioma/genética , Miométrio/metabolismo , Miométrio/fisiologia , Análise Serial de Proteínas , Proteínas Quinases/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR , Neoplasias Uterinas/genéticaRESUMO
Modification of alpha-biphenylsulfonamidocarboxylic acids led to potent and selective MMP-13 inhibitors. Compound 16 showed 100% oral bioavailability in rats and demonstrated >50% inhibition of bovine cartilage degradation at 10 ng/mL.
Assuntos
Colagenases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/farmacocinética , Inibidores de Metaloproteinases de Matriz , Osteoartrite/tratamento farmacológico , Administração Oral , Animais , Benzofuranos/síntese química , Benzofuranos/química , Colagenases/química , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Metaloproteinase 13 da Matriz , Modelos Moleculares , Estrutura Molecular , Ratos , Especificidade por SubstratoRESUMO
The structure-based design and synthesis of a series of novel biphenyl sulfonamide carboxylic acids as potent MMP-13 inhibitors with selectivity over MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-14, Aggrecanase 1, and TACE are described.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores de Metaloproteinases de Matriz , Aminação , Benzofuranos/química , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacologia , Colagenases/metabolismo , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Metaloproteinase 13 da Matriz , Estrutura Molecular , Relação Estrutura-Atividade , Enxofre/químicaRESUMO
A series of benzodiazepine inhibitors of the MMPs and TACE has been developed. These compounds display an interesting selectivity profile and should be useful tools for exploring the biological relevance of such selectivity.
Assuntos
Benzodiazepinas/farmacologia , Ácidos Hidroxâmicos , Inibidores de Metaloproteinases de Matriz , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Pirazinas , Proteínas ADAM , Proteína ADAM17 , Benzodiazepinas/síntese química , Cartilagem/efeitos dos fármacos , Cartilagem/enzimologia , Indicadores e Reagentes , Inibidores de Proteases/síntese química , Relação Estrutura-Atividade , SulfonamidasRESUMO
Twenty-five 2-phenyl-5,6-dihydro-2H-thieno[3,2-c]pyrazol-3-ol derivatives were synthesized for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good inhibitory activity against Staphylococcus aureus MurB, MurC and MurD enzymes in vitro and antimicrobial activity against gram-positive bacteria including MRSA, VRE and PRSP. However, when they were tested in the presence of 4% bovine serum albumin, the MIC values increased to greater than 128 microg/mL against PRSP. None of the compounds demonstrated activity against gram-negative bacteria at MIC <32 microg/mL.