RESUMO
A phosphonate analog of N-acetyl neuraminic acid (PANA) has been designed as a potential neuraminidase (NA) inhibitor and synthesized as both the alpha (ePANA) and beta (aPANA) anomers. Inhibition of type A (N2) and type B NA activity by ePANA was approximately a 100-fold better than by sialic acid, but inhibition of type A (N9) NA was only ten-fold better than by sialic acid. The aPANA compound was not a strong inhibitor for any of the NA strains tested. The crystal structures at 2.4 A resolution of ePANA complexed to type A (N2) NA, type A (N9) NA and type B NA and aPANA complexed to type A (N2) NA showed that neither of the PANA compounds distorted the NA active site upon binding. No significant differences in the NA-ePANA complex structures were found to explain the anomalous inhibition of N9 neuraminidase by ePANA. We put forward the hypothesis that an increase in the ePANA inhibition compared to that caused by sialic acid is due to (1) a stronger electrostatic interaction between the inhibitor phosphonyl group and the active site arginine pocket and (2) a lower distortion energy requirement for binding of ePANA.
Assuntos
Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Neuraminidase/antagonistas & inibidores , Organofosfonatos/farmacologia , Ácidos Siálicos/farmacologia , Sítios de Ligação , Ácido N-Acetilneuramínico , Especificidade da EspécieRESUMO
Recently, the novel cyclooctylpyranone HIV protease inhibitor 1 was identified in our labs, and an X-ray structure of this inhibitor complexed with HIV-2 protease was obtained. This crystal structure was used to develop two strategies for creating derivatives of 1 with enhanced enzyme inhibitory activity. The first strategy, substitution on the cyclooctyl ring, met with limited success, but provided some interesting information about the conformationally-flexible cycloocytyl ring on the inhibitors. The second strategy, substitution at the meta position of the aryl ring, was far more successful and generated compounds, such as the carboxamide derivatives 41 (Ki = 3.0 +/- 0.4 nM) and 36 (Ki = 4.0 +/- 0.8 nM), which were significantly more active than the corresponding unsubstituted cycloocytlpyranone 2 (Ki = 11.7 +/- 4.7 nM). An X-ray crystal structure of 36 complexed with HIV-1 protease indicated the increase in binding affinity is most likely due to the additional interactions between the amide substituent and the S3 region of the protease.
Assuntos
Inibidores da Protease de HIV/síntese química , Pironas/síntese química , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Gráficos por Computador , Cristalografia por Raios X , Desenho de Fármacos , Protease de HIV/química , Protease de HIV/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Pironas/química , Pironas/farmacologiaRESUMO
The low oral bioavailability and rapid biliary excretion of peptide-derived HIV protease inhibitors have limited their utility as potential therapeutic agents. Our broad screening program to discover non-peptidic HIV protease inhibitors previously identified compound I (phenprocoumon, Ki = 1 microM) as a lead template. Structure-based design of potent non-peptidic inhibitors, utilizing crystal structures of HIV protease/inhibitor complexes, provided a rational basis for the previously reported carboxamide-containing 4-hydroxycoumarins and 4-hydroxy-2-pyrones. The amino acid containing compound V (Ki = 4 nM) provided an example of a promising new series of HIV protease inhibitors with significantly improved enzymatic binding affinity. In this report, further structure-activity relationship studies, in which the carboxamide is replaced by a sulfonamide functionality, led to the identification of another series of nonamino acid containing promising inhibitors with significantly enhanced enzyme binding affinity and in vitro antiviral activity. The most active diastereomer of the sulfonamide-containing pyrone XVIII (Ki = 0.5 nM) shows improved antiviral activity (IC50 = 0.6 nM) and represents an example of a new design direction for the discovery of more potent non-peptidic HIV protease inhibitors as potential therapeutic agents for the treatment of HIV infection.
Assuntos
4-Hidroxicumarinas/química , Inibidores da Protease de HIV/química , HIV-1/enzimologia , HIV-2/enzimologia , Pironas/química , Sulfonamidas/química , 4-Hidroxicumarinas/farmacologia , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Desenho de Fármacos , Inibidores da Protease de HIV/síntese química , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Femprocumona/análogos & derivados , Femprocumona/química , Pironas/síntese química , Pironas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/farmacologiaRESUMO
From a broad screening program, the 4-hydroxycoumarin phenprocoumon (I) was previously identified as a lead template with HIV protease inhibitory activity. The crystal structure of phenprocoumon/HIV protease complex initiated a structure-based design effort that initially identified the 4-hydroxy-2-pyrone U-96988 (II) as a first-generation clinical candidate for the potential treatment of HIV infection. Based upon the crystal structure of the 4-hydroxy-2-pyrone III/HIV protease complex, a series of analogues incorporating a 5,6-dihydro-4-hydroxy-2-pyrone template were studied. It was recognized that in addition to having the required pharmacophore (the 4-hydroxy group with hydrogen-bonding interaction with the two catalytic aspartic acid residues and the lactone moiety replacing the ubiquitous water molecule in the active site), these 5,6-dihydro-4-hydroxy-2-pyrones incorporated side chains at the C-6 position that appropriately extended into the S1' and S2' subsites of the enzyme active site. The crystal structures of a number of representative 5,6-dihydro-4-hydroxy-2-pyrones complexed with the HIV protease were also determined to provide better understanding of the interaction between the enzyme and these inhibitors to aid the structure-based drug design effort. The crystal structures of the ligands in the enzyme active site did not always agree with the conformations expected from experience with previous pyrone inhibitors. This is likely due to the increased flexibility of the dihydropyrone ring. From this study, compound XIX exhibited reasonably high enzyme inhibitory activity (Ki = 15 nM) and showed antiviral activity (IC50 = 5 microM) in the cell-culture assay. This result provided a research direction which led to the discovery of active 5,6-dihydro-4-hydroxy-2-pyrones as potential agents for the treatment of HIV infection.
Assuntos
Inibidores da Protease de HIV/síntese química , Pironas/síntese química , Linhagem Celular , Cristalografia por Raios X , Desenho de Fármacos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Pironas/química , Pironas/farmacologia , Espectrofotometria InfravermelhoRESUMO
A broad screening program previously identified phenprocoumon (1) as a small molecule template for inhibition of HIV protease. Subsequent modification of this lead through iterative cycles of structure-based design led to the activity enhancements of pyrone and dihydropyrone ring systems (II and V) and amide-based substitution (III). Incorporation of sulfonamide substitution within the dihydropyrone template provided a series of highly potent HIV protease inhibitors, with structure-activity relationships described in this paper. Crystallographic studies provided further information on important binding interactions responsible for high enzymatic binding. These studies culminated in compound VI, which inhibits HIV protease with a Ki value of 8 pM and shows an IC90 value of 100 nM in antiviral cell culture. Clinical trials of this compound (PNU-140690, Tipranavir) for treatment of HIV infection are currently underway.
Assuntos
Fármacos Anti-HIV , Inibidores da Protease de HIV , Protease de HIV/metabolismo , Piridinas , Pironas , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Humanos , Ligação de Hidrogênio , Camundongos , Modelos Moleculares , Ligação Proteica , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Pironas/química , Pironas/metabolismo , Pironas/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , SulfonamidasRESUMO
Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.
Assuntos
Inibidores da Protease de HIV/síntese química , Pironas/síntese química , Animais , Linhagem Celular , Cristalografia por Raios X , Cães , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacocinética , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Modelos Moleculares , Pironas/química , Pironas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
In extensive burns it becomes difficult for fibroblasts to migrate from the periphery of the healthy tissue and colonize the injured area. Even under such circumstances healing takes place, and this is attributed to the differentiation of circulating fibrocytes which enter the wound site. This normal cell type is identified in keloid fibroblasts: it expresses fibrocyte markers and secretes extra cellular matrix proteins. In-vitro collagen contraction assay reveals that fibrocytes contract collagen gels with an efficacy similar to normal fibroblasts. The contribution of fibrocytes to the formation of keloid fibroblasts in post-burn healing is discussed.
Quand le patient est atteint de brûlures de grande extension, pour les fibroblastes il devient difficile de migrer de la périphérie du tissu sain pour coloniser la zone lésée. Même dans ces conditions la guérison a lieu, ce qui est attribué à la différenciation des fibrocytes en circulation qui entrent dans la plaie. Ce type normal de cellule a été identifié dans les fibroblastes chéloïdaux: il exprime des marqueurs des fibrocytes et sécrète des protéines de la matrice extracellulaire. Les épreuves in vitro sur la contraction du collagène révèlent que les fibrocytes contractent le gel de collagène avec une efficacité similaire à celle des fibroblastes normaux. Les Auteurs concluent avec une discussion sur la contribution des fibrocytes à la formation des fibroblastes chéloïdaux dans la phase finale de la guérison des brûlures.
RESUMO
Identifying the spectrum of genetic alterations that cooperate with critical oncogenes to promote transformation provides a foundation for understanding the diversity of clinical phenotypes observed in human cancers. Here, we performed integrated analyses to identify genomic alterations that co-occur with oncogenic BRAF in melanoma and abrogate cellular dependence upon this oncogene. We identified concurrent mutational inactivation of the PTEN and RB1 tumor suppressors as a mechanism for loss of BRAF/MEK dependence in melanomas harboring (V600E)BRAF mutations. RB1 alterations were mutually exclusive with loss of p16(INK4A), suggesting that whereas p16(INK4A) and RB1 may have overlapping roles in preventing tumor formation, tumors with loss of RB1 exhibit diminished dependence upon BRAF signaling for cell proliferation. These findings provide a genetic basis for the heterogeneity of clinical outcomes in patients treated with targeted inhibitors of the mitogen-activated protein kinase pathway. Our results also suggest a need for comprehensive screening for RB1 and PTEN inactivation in patients treated with RAF and MEK-selective inhibitors to determine whether these alterations are associated with diminished clinical benefit in patients whose cancers harbor mutant BRAF.
Assuntos
Melanoma/genética , Mutação , PTEN Fosfo-Hidrolase/fisiologia , Proteínas Proto-Oncogênicas B-raf/genética , Proteína do Retinoblastoma/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Quinases raf/fisiologia , Animais , Quinase 4 Dependente de Ciclina/genética , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologiaAssuntos
Desenho de Fármacos , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/farmacologia , HIV/efeitos dos fármacos , Pironas/química , Pironas/farmacologia , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Antivirais/química , Antivirais/farmacocinética , Antivirais/farmacologia , Cães , Protease de HIV/metabolismo , Inibidores da Protease de HIV/farmacocinética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , HIV-2/efeitos dos fármacos , HIV-2/enzimologia , Ligação de Hidrogênio , Modelos Moleculares , Método de Monte Carlo , Pironas/metabolismo , Pironas/farmacocinética , Ratos , Sulfonamidas/metabolismo , Sulfonamidas/farmacocinéticaRESUMO
Neuraminidase is one of the two glycoprotein spikes protruding from the influenza virus membrane. We have determined by X-ray crystallography the native structure of B/Lee/40 neuraminidase (NA) and the structures of its crystals soaked with a substrate, N-acetylneuraminyllactose (NANL), and an inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) at 1.8-A resolution. NANL was hydrolyzed by the crystalline NA to generate the product N-acetylneuraminic acid (NANA, also known as sialic acid), which is still able to bind to NA. In the difference Fourier map of the presumed NA-NANA complex, the moiety bound in the active site had a distorted boat conformation of NANA, but there is no significant electron density for O2. The structure of the bound moiety is not identical to that of chemically synthesized DANA soaked into NA crystals. Prolonged incubation of NANA with NA in solution at room temperature produced only a trace amount of DANA as detected by NMR. On the basis of our studies, a mechanism is proposed for the enzymatic hydrolysis by influenza virus neuraminidase.
Assuntos
Vírus da Influenza B/enzimologia , Neuraminidase/química , Neuraminidase/metabolismo , Ácidos Siálicos/metabolismo , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Lactose/análogos & derivados , Lactose/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Estrutura Molecular , Ácido N-Acetilneuramínico , Estrutura Secundária de Proteína , Ácidos Siálicos/químicaRESUMO
Potent, non-peptidic, dihydropyrone sulfonamide HIV protease inhibitors have been previously described. Crystallographic analysis of dihydropyrone sulfonamide inhibitor/HIV protease complexes suggested incorporation of a second, C2 symmetry-related sulfonamide group. Selected bis-sulfonamide dihydropyrone analogues display high HIV protease inhibitory activity.