RESUMO
UNLABELLED: Human noroviruses are a leading cause of gastroenteritis across the globe, but the pathogenic mechanisms responsible for disease are not well established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the open reading frame 2 (ORF2) gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence on an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effects on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome. IMPORTANCE: Human noroviruses are a major global cause of disease, yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells, and this directly correlates with virulence. Importantly, mutations impairing B cell infection had minimal effects on macrophage infection, revealing a potentially critical role for B cell infection in norovirus pathogenesis.
Assuntos
Linfócitos B/virologia , Proteínas do Capsídeo/metabolismo , Interações Hospedeiro-Patógeno , Norovirus/fisiologia , Norovirus/patogenicidade , Fatores de Virulência/metabolismo , Substituição de Aminoácidos , Estruturas Animais/virologia , Animais , Peso Corporal , Infecções por Caliciviridae/patologia , Infecções por Caliciviridae/virologia , Proteínas do Capsídeo/genética , Modelos Animais de Doenças , Feminino , Macrófagos/virologia , Masculino , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Análise de Sobrevida , Carga Viral , Virulência , Fatores de Virulência/genética , Replicação ViralRESUMO
The sweeteners saccharin (SAC) and acesulfame K (ACE) recently entered the topic of anticancer human carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, as they showed to selectively inhibit the tumor-associated CAs IX/XII over ubiquitous CAs. A drug design strategy is here reported, which took SAC and ACE as leads and produced a series of 2H-benzo[e][1,2,4]thiadiazin-3(4H)-one-1,1-dioxides (BTD). Many derivatives showed greater potency (KIs-CA IX 19.1-408.5 nM) and selectivity (II/IX SI 2-76) than the leads (KIs-CA IX 103, 2400 nM; II/IX-SI 56, >4) against CA IX/XII over off-target isoforms. A thorough X-ray crystallographic study depicted their binding mode to both CA II and IX-mimic. The most representative BTDs were characterized in vitro for their antitumor activity against A549, PC-3, and HCT-116 cancer cell lines both in normoxia and hypoxia. The two most effective compounds were assayed for their effect on several apoptosis markers, identifying promising leads for the development of new anticancer drugs.
Assuntos
Antineoplásicos/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Sacarina/análogos & derivados , Sacarina/farmacologia , Sulfonamidas/farmacologia , Tiazinas/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Anidrase Carbônica IX/antagonistas & inibidores , Inibidores da Anidrase Carbônica/síntese química , Inibidores da Anidrase Carbônica/química , Anidrases Carbônicas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Ensaios Enzimáticos , Humanos , Estrutura Molecular , Sacarina/síntese química , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Tiazinas/síntese química , Tiazinas/químicaRESUMO
Carbonic anhydrase IX (CA IX) has been identified as a biomarker and drug target for several malignant tumors due to its role in cancer cell growth and proliferation. Simple cyclic sulfonamides, like saccharin (SAC), have shown up to a 60-fold selectivity towards CA IX over other ubiquitous CA isoforms, with greater selectivity obtained applying the "tail-approach" to derivatize SAC with a methylene triazole linker that connected to a "tail" beta glucoside. These modifications of SAC led to an increased selectivity of more than 1000-fold towards CA IX, whereas clinically available CA inhibitors show little to no isoform selectivity. As part of our interest in the development of new CA inhibitors, we found the existing synthetic protocol, which relies on a N-tert-butyl saccharin intermediate, to be problematic in the final deprotection steps. We therefore describe an alternative approach to the synthesis of these compounds featuring a gentle "one pot" deprotection/cyclization as the final synthetic step, and report new galactosyl and glucosyl conjugates with low to mid nM inhibition of CA IX.
Assuntos
Anidrase Carbônica IX/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicoconjugados/síntese química , Glicoconjugados/farmacologia , Sacarina/química , Biocatálise , Anidrase Carbônica IX/antagonistas & inibidores , Técnicas de Química Sintética , Ciclização , Inibidores Enzimáticos/química , Glicoconjugados/química , Concentração Inibidora 50RESUMO
Calcification of soft tissue leads to serious diseases and has been associated with bacterial chronic infections. However, the origin and the molecular mechanisms of calcification remain unclear. Here we hypothesized that a human pathogen Pseudomonas aeruginosa deposits extracellular calcium, a process requiring carbonic anhydrases (CAs). Transmission electron microscopy confirmed the formation of 0.1-0.2⯵m deposits by P. aeruginosa PAO1 growing at 5â¯mM CaCl2, and X-ray elemental analysis confirmed they contain calcium. Quantitative analysis of deposited calcium showed that PAO1 deposits 0.35 and 0.75â¯mM calcium/mg protein when grown at 5â¯mM and 10â¯mM CaCl2, correspondingly. Fluorescent microscopy indicated that deposition initiates at the cell surface. We have previously characterized three PAO1 ß-class CAs: psCA1, psCA2, and psCA3 that hydrate CO2 to HCO3-, among which psCA1 showed the highest catalytic activity (Lotlikar et. al. 2013). According to immunoblot and RT-qPCR, growth at elevated calcium levels increases the expression of psCA1. Analyses of the deletion mutants lacking one, two or all three psCA genes, determined that psCA1 plays a major role in calcium deposition and contributes to the pathogen's virulence. In-silico modeling of the PAO1 ß-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3 (T59, A61A, A101, and A108), and these differences may play a role in catalytic rate and thus calcium deposition. A series of inhibitors were tested against the recombinant psCA1, among which aminobenzene sulfonamide (ABS) and acetazolamide (AAZ), which inhibited psCA1 catalytic activity with KIs of 19â¯nM and 37â¯nM, correspondingly. The addition of ABS and AAZ to growing PAO1 reduced calcium deposition by 41 and 78, respectively. Hence, for the first time, we showed that the ß-CA psCA1 in P. aeruginosa contributes to virulence likely by enabling calcium salt deposition, which can be partially controlled by inhibiting its catalytic activity.
Assuntos
Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Anidrase Carbônica I/metabolismo , Membrana Celular/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Acetazolamida/farmacologia , Proteínas de Bactérias/genética , Calcinose , Anidrase Carbônica I/genética , Membrana Celular/ultraestrutura , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Humanos , Microscopia Eletrônica de Transmissão , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Deleção de Sequência/genética , Sulfonamidas/farmacologia , Virulência/genéticaRESUMO
Carbonic anhydrase IX (CA IX) expression is important for the regulation of pH in hypoxic tumors and is emerging as a therapeutic target for the treatment of various cancers. Recent studies have demonstrated the selectivity of sucrose, saccharin, and acesulfame potassium for CA IX over other CA isoforms. Reported here is the X-ray crystal structure of CA IX-mimic in complex with sucralose determined to â¼1.5 Å resolution. Furthermore, this structure is compared to the aforementioned sweetener/carbohydrate structural studies in order to determine active site properties of CA IX that promote selective binding. This structural analysis provides a further understanding of CA IX isoform specific inhibition to facilitate the design of new inhibitors and anticancer drugs.
RESUMO
Human carbonic anhydrase IX (CA IX) is upregulated in neoplastic tissues; as such, it is studied as a drug target for anticancer chemotherapy. Inhibition of CA IX has been shown to be therapeutically favorable in terms of reducing tumor growth. Previously, saccharin, a commonly used artificial sweetener, has been observed to selectively inhibit CA IX over other CA isoforms. In this study, X-ray crystallography showed acesulfame potassium (Ace K) binding directly to the catalytic zinc in CA IX (mimic) and through a bridging water in CA II. This modulation in binding is reflected in the binding constants, with Ace K inhibiting CA IX but not other CA isoforms. Hence, this study establishes the potential of Ace K (an FDA approved food additive) as a lead compound in the design and development of CA IX specific inhibitors.
Assuntos
Anidrase Carbônica II/antagonistas & inibidores , Anidrase Carbônica II/metabolismo , Anidrase Carbônica IX/antagonistas & inibidores , Anidrase Carbônica IX/metabolismo , Tiazinas/metabolismo , Tiazinas/farmacologia , Anidrase Carbônica II/química , Anidrase Carbônica IX/química , Inibidores da Anidrase Carbônica/metabolismo , Inibidores da Anidrase Carbônica/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Pseudomonas aeruginosa is a Gram-negative facultative anaerobe belonging to the Pseudomonadaceae family. It is a multidrug-resistant opportunistic human pathogen, a common cause of life-threatening nosocomial infections, and a key bacterial agent in cystic fibrosis and endocarditis. The bacterium exhibits intrinsic resistance to most antibacterial agents, including aminoglycosides and quinolones. Hence, the identification of new drug targets for P.â aeruginosa is ongoing. PsCA3 is a ß-class carbonic anhydrase (ß-CA) that catalyzes the reversible hydration of carbon dioxide to bicarbonate and represents a new class of antimicrobial target. Previously, inhibitor screening studies of psCA3 have shown that a series of small anions including sulfamide (SFN), imidazole (IMD), and 4-methylimidazole (4MI), and thiocyanate (SCN) inhibit the enzyme with efficiencies in the micro- to millimolar range. Herein the X-ray crystal structures of these inhibitors in complex with psCA3 are presented and compared with human CAâ II. This structural survey into the binding modes of small anions forms the foundation for the development of inhibitors against ß-CAs and more selective inhibitors against P.â aeruginosa.