In this study, we established a new methodology for preparing 5-nucleotidase (5-NT) with the aim of enhancing our understanding of its enzyme activity and laying a basis for regulating the content of umami-enhancing nucleotides in pork. 5-NT was prepared with Sephadex gel filtration and reverse-phase high-performance liquid chromatography, and its enzymatic properties and catalytic activity were evaluated. The results show that the molecular weight of the prepared 5-NT was 57 kDa, the optimal catalytic temperature was 40 °C, and the optimal pH was 8. Zn2+, and sucrose showed inhibitory effects on the activity of 5-NT, while K+, Na+, Ca2+, Mg2+, glucose, fructose, and trehalose promoted the activity of the studied compound. The prepared 5-NT exhibited higher catalytic activity and selectivity against IMP compared with its commercial counterpart, while its catalytic activity against XMP was not significant (p > 0.05). In brief, we established a new methodology for preparing 5-NT, enhancing our understanding of its enzyme activity and providing a solid basis for regulating the content of umami-enhancing nucleotides in pork through the control of endogenous 5-NT activity.

Zika virus (ZIKV), one of the flaviviruses, has attracted worldwide attention since its large epidemics around Brazil. Association of ZIKV infection with microcephaly and neurological problems such as Guillain-Barré syndrome has prompted intensive pathological investigations. However, there is still a long way to go on the discovery of effective anti-ZIKV therapeutics. In this study, an in silico screening of the National Cancer Institute (NCI) diversity set based on ZIKV NS3 helicase was performed using a molecular docking approach. Selected compounds with drug-like properties were subjected to cell-based antiviral assays resulting in the identification of two novel lead compounds (named Compounds 1 and 2). They inhibited ZIKV infection with IC50 values at the micro-molar level (8.5 μM and 15.2 μM, respectively). Binding mode analysis, absolute binding free energy calculation, and structure-activity relationship studies of these two compounds revealed their possible interactions with ZIKV NS3 helicase, suggesting a mechanistic basis for further optimization. These two novel small molecules may represent new leads for the development of inhibitory drugs against ZIKV.

G-protein coupled receptors (GPCRs) are implicated in many diseases and attractive targets for drug discovery. Peptide fragments derived from protein ligands of GPCRs are commonly used as probes of GPCR function and as leads for drug development. However, these peptide fragments lack the structural integrity of their parent full-length protein ligands and often show low receptor affinity, which limits their research and therapeutic values. It remains a challenge to efficiently generate high affinity peptide inhibitors of GPCRs. We have investigated a combinational approach involving the synthetic covalent linkage of two low affinity peptide fragments to determine if the strategy can yield high affinity GPCR inhibitors. We examined this design approach using the chemokine receptor CXCR4 as a model of GPCR system. Here, we provide a proof of concept demonstration by designing and synthesizing two peptides, AR5 and AR6, that combine a peptide fragment derived from two viral ligands of CXCR4, vMIP-II and HIV-1 envelope glycoprotein gp120. AR5 and AR6 display nanomolar binding affinity, in contrast to the weak micromolar CXCR4 binding of each peptide fragment alone, and inhibit HIV-1 entry via CXCR4. Further studies were carried out for the representative peptide AR6 using western blotting and site-directed mutagenesis in conjunction with molecular dynamic simulation and binding free energy calculation to determine how the peptide interacts with CXCR4 and inhibits its downstream signaling. These results demonstrate that this combinational approach is effective for generating nanomolar active inhibitors of CXCR4 and may be applicable to other GPCRs.