RESUMO
Mammalian lethal with SEC13 protein 8 (mLST8), is an indispensable protein subunit of mammalian target of rapamycin (mTOR) signaling pathway that interacts with the kinase domain of mTOR protein, thereby stabilizing its active site. Experimental studies reported the over expression of mLST8 in human colon and prostate cancers by activation of both mTORC1/2 complexes and subsequent downstream substrates leading to tumor progression. Considering its role, targeting mLST8 protein would be a therapeutic approach against tumor progression in colon and prostate cancers. Hence, using in silico structure based drug design approach, the comparative binding patterns of 1,1'-binapthyl-2,2'diol (BINOL), 1-(2-carboxynaphth-1yl)-2-naphthoic acid (SCF-12) and their analogs in the cavity of mLST8 were explored. ADME and binding energy calculations led to the identification of five compounds with favorable Glide (G) scores and implicated the importance of Asn132 and Gln225 as key binding residues. Molecular dynamics (MD) simulations and free energy landscape (FEL) approaches helped in elucidating the binding mechanism and suggested the possibility of ligands 1-3 namely, ZINC01765622, ZINC62723702 and ZINC02576980 to be promising antagonists for mLST8. Thus, this study substantiates the prospect of targeting mLST8 protein using potent hits which could hinder tumor progression in colon and prostate cancers.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Naftóis/química , Neoplasias da Próstata/tratamento farmacológico , Homólogo LST8 da Proteína Associada a mTOR/química , Ácidos Carboxílicos/química , Domínio Catalítico/efeitos dos fármacos , Simulação por Computador , Desenho de Fármacos , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/química , Simulação de Dinâmica Molecular , Terapia de Alvo Molecular , Naftalenos/química , Naftóis/farmacologia , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Homólogo LST8 da Proteína Associada a mTOR/antagonistas & inibidoresRESUMO
Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.
Assuntos
Oligopeptídeos/genética , Poliaminas/metabolismo , Priapismo/metabolismo , Proteínas e Peptídeos Salivares/genética , Anemia Falciforme/metabolismo , Animais , Arginase/metabolismo , Diaminas/farmacologia , Modelos Animais de Doenças , Masculino , Camundongos , Oligopeptídeos/metabolismo , Ornitina Descarboxilase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Putrescina/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas e Peptídeos Salivares/metabolismo , Regulação para Cima , Poliamina OxidaseRESUMO
TetraPEGylated canine Hb, [SP (succinimidophenyl)-PEG5K]4-canine-Hb, with PEGylation at its four reactive cysteine residues (a111 and b93) has been prepared and characterized. The hydrodynamic volume and the molecular radius of (SP-PEG5K)4-canine-Hb are intermediate to those of di- and hexaPEGylated human Hb as expected. However, the COP (colloidal osmotic pressure) of tetraPEGylated canine Hb is closer to that of hexaPEGylated human Hb than to that of diPEGylated human Hb. The O2 affinity of tetraPEGylated canine Hb is higher than that of canine Hb and comparable with that of hexaPEGylated Hb. The O2 affinity of tetraPEGylated canine Hb is not responsive to the presence of DPG (diphosphoglycerate) or chloride, but it retains almost full response to L-35, an allosteric effector that interacts at the aa-end of the central cavity. The tetraPEGylated canine Hb is vasoinactive in hamster in 10% top load infusion studies. It is also essentially non-hypertensive in an extreme exchange haemodilution protocol in hamster just as di- and hexaPEGylated human Hb. The O2 delivery by tetraPEGylated canine Hb is comparable with that of hexaPEGylated Hb but not as efficient as diPEGylated Hb. These results demonstrate that PEGylation-induced solution properties of PEG [poly(ethylene glycol)]-Hb conjugates are dictated by the level and chemistry of PEGylation and the interplay of these plays a critical role in tissue oxygenation. The studies imply the need to establish the right level (and/or pattern) of PEGylation and O2 affinity of Hb-PEG adducts in designing O2-carrying plasma volume expanders, and this remains the primary challenge in the design of PEGylated Hb as blood substitutes.
Assuntos
Hemoglobinas/química , Hemoglobinas/metabolismo , Oxigênio/metabolismo , Polietilenoglicóis/química , Animais , Gatos , Galinhas , Cricetinae , Cães , Humanos , Masculino , Maleimidas/química , Mesocricetus , Camundongos , Ligação Proteica , OvinosRESUMO
The oxygen transport capacity of nonhypertensive polyethylene glycol (PEG)-conjugated hemoglobin solutions were investigated in the hamster chamber window model. Microvascular measurements were made to determine oxygen delivery in conditions of extreme hemodilution [hematocrit (Hct) 11%]. Two isovolemic hemodilution steps were performed with a 6% Dextran 70 (70-kDa molecular mass) plasma expander until Hct was 35% of control. Isovolemic blood volume exchange was continued using two surface-modified PEGylated hemoglobins (P5K2, P(50) = 8.6, and P10K2, P(50) = 8.3; P(50) is the hemoglobin Po(2) corresponding to its 50% oxygen saturation) until Hct was 11%. P5K2 and P10K2 are PEG-conjugated hemoglobins that maintain most of the hemoglobin allosteric properties and have a cooperativity index of n = 2.2. The effects of these molecular solutions were compared with those obtained in a previous study using MP4, a PEG-modified hemoglobin whose P(50) was 5.4 and cooperativity was 1.2 (Tsai et al., Am J Physiol Heart Circ Physiol 285: H1411-H1419, 2003). Tissue oxygen levels were higher after P5K2 (7.0 +/- 2.5 mmHg) and P10K2 (6.3 +/- 2.3 mmHg) versus MP4 (1.7 +/- 0.5 mmHg) or the nonoxygen carrier Dextran 70 (1.3 +/- 1.2 mmHg). Microvascular oxygen delivery was higher after P5K2 and P10K2 (2.22 and 2.34 ml O(2)/dl blood) compared with MP4 (1.41 ml O(2)/dl blood) or Dextran 70 (0.90 ml O(2)/dl blood); however, all these values were lower than control (7.42 ml O(2)/dl blood). The total hemoglobin in blood was similar in all cases; therefore, the improvement in tissue Po(2) and oxygen delivery appears to be due to the increased cooperativity of the new molecules.