Herein, we report the discovery and characterization of a novel class of PAK4 inhibitors with a quinazoline scaffold. Based on the shape and chemical composition of the ATP-binding pocket of PAKs, we chose a 2,4-diaminoquinazoline series of inhibitors as a starting point. Guided by X-ray crystallography and a structure-based drug design (SBDD) approach, a series of novel 4-aminoquinazoline-2-carboxamide PAK4 inhibitors were designed and synthesized. The inhibitors' selectivity, therapeutic potency, and pharmaceutical properties were optimized. One of the best compounds, 31 (CZh226), showed remarkable PAK4 selectivity (346-fold vs PAK1) and favorable kinase selectivity profile. Moreover, this compound potently inhibited the migration and invasion of A549 tumor cells by regulating the PAK4-directed downstream signaling pathways in vitro. Taken together, these data support the further development of 31 as a lead compound for PAK4-targeted anticancer drug discovery and as a valuable research probe for the further biological investigation of group II PAKs.
Despite recent great advances in microbial culture, most microbes have not yet been cultured, and the impact of medium composition on the isolation of microbes from natural systems has not been elucidated. To optimize media for culturing marine microbes, microbial communities in three sediment samples were described using high-throughput sequencing (HTS) and culture-dependent techniques. HTS revealed communities dominated by Gammaproteobacteria, and culture-based methods revealed communities dominated by Actinobacteria. Among the total operational taxonomic units (OTUs) from the HTS dataset, 6% were recovered in the culture collection. Four potentially novel bacterial strains belonging to Oceaniovalibus, Psychrobacter and Salegentibacter were isolated. The combination of media cultured more taxa than any single medium. Nutrient-rich and single-carbon/nitrogen-source media supported the growth of relatively few taxa, and the quality of nitrogen strongly influenced the types of bacteria isolated.
The bacterial prepeptidase C-terminal (PPC) domain can be found in the C termini of a wide variety of proteases that are secreted by marine bacteria. However, the functions of these PPC domains remain unknown due to a lack of systematic research. Here, the binding and swelling abilities of eight PPC domains from six different proteases were compared systematically via scanning electron microscopy (SEM), enzyme assays, and fluorescence spectroscopy. These PPC domains all possess the ability to bind and swell insoluble collagen. PPC domains can expose collagen monomers but cannot disrupt the pyridinoline cross-links or unwind the collagen triple helix. This ability can play a synergistic role alongside collagenase in collagen hydrolysis. Site-directed mutagenesis of the PPC domain from Vibrio anguillarum showed that the conserved polar and aromatic residues Y6, D26, D28, Y30, W42, E53, C55, and Y65 and the hydrophobic residues V10, V18, and I57 played key roles in substrate binding. Molecular dynamic simulations were conducted to investigate the interactions between PPC domains and collagen. Most PPC domains have a similar mechanism for binding collagen, and the hydrophobic binding pocket of PPC domains may play an important role in collagen binding. This study sheds light on the substrate binding mechanisms of PPC domains and reveals a new function for the PPC domains of bacterial proteases in substrate degradation. IMPORTANCE Prepeptidase C-terminal (PPC) domains commonly exist in the C termini of marine bacterial proteases. Reports examining PPC have been limited, and its functions remain unclear. In this study, eight PPCs from six different bacteria were examined. Most of the PPCs possessed the ability to bind collagen, feathers, and chitin, and all PPCs could significantly swell insoluble collagen. PPCs can expose collagen monomers but cannot disrupt pyridinoline cross-links or unwind the collagen triple helix. This swelling ability may also play synergistic roles in collagen hydrolysis. Comparative structural analyses and the examination of PPC mutants revealed that the hydrophobic binding pockets of PPCs may play important roles in collagen binding. This study provides new insights into the functions and ecological significance of PPCs, and the molecular mechanism of the collagen binding of PPCs was clarified, which is beneficial for the protein engineering of highly active PPCs and collagenase in the pharmaceutical industry and of artificial biological materials.
Bile stress tolerance is a crucial characteristic of probiotics for surviving in the human gastrointestinal tract. The mechanism underlying the effect of L-malic acid on enhancing the glycodeoxycholic acid (GDCA) tolerance of Lacticaseibacillus paracasei L9 was investigated herein. Bile tolerance specificity assays revealed that Lc. paracasei L9 was more sensitive to GDCA than to taurocholic acid, glycocholic acid, and taurodeoxycholic acid. Notably, L-malic acid significantly enhanced the GDCA tolerance of Lc. paracasei L9 by increasing the pH of the medium. The role of the malolactic enzyme pathway in enhancing GDCA resistance was investigated using molecular techniques. Confocal laser scanning and scanning electron microscopy revealed that L-malic acid preserved membrane permeability and cellular morphology, thereby protecting bacterial cells from GDCA stress-induced damage. The study also demonstrated that L-malic acid enhanced bile tolerance in different species of lactobacilli. These findings provide a novel protective mechanism for coping with bile stress in lactobacilli.
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