Engineered Imine Reductase for Asymmetric Synthesis of Dextromethorphan Key Intermediate.

(S)-1-(4-Methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-1-(4-methoxybenzyl)-OHIQ) is the key intermediate of the nonopioid antitussive dextromethorphan. In this study, (S)-IR61-V69Y/P123A/W179G/F182I/L212V (M4) was identified with a 766-fold improvement in catalytic efficiency compared with wide-type IR61 through enzyme engineering. M4 could completely convert 200 mM of 1-(4-methoxybenzyl)-3,4,5,6,7,8-hexahydroisoquinoline into (S)-1-(4-methoxybenzyl)-OHIQ in 77% isolated yield, with >99% enantiomeric excess and a high space-time yield of 542 g L-1 day-1, demonstrating a great potential for the synthesis of dextromethorphan intermediate in industrial applications.

TMEPAI promotes degradation of the NF-κB signaling pathway inhibitory protein IκBα and contributes to tumorigenesis.

The transmembrane prostate androgen-induced protein (TMEPAI) is known to be highly expressed in various types of cancer and promoted oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. Here we reported that expression of TMEPAI activated the NF-κB signaling. TMEPAI showed direct interaction with NF-κB pathway inhibitory protein IκBα. Though ubiquitin ligase Nedd4 (neural precursor cell expressed, developmentally down-regulated 4) did not interact with IκBα directly, TMEPAI recruited Nedd4 for ubiquitination of IκBα, leading to IκBα degradation through the proteasomal and lysosomal pathway, and promoted activation of NF-κB signaling. Further study indicated NF-κB signaling is involved in TMEPAI-induced cell proliferation and tumor growth in immune deficient mice. This finding helps to further understand the mechanism of TMEPAI on tumorigenesis and suggests TMEPAI is potential target for cancer treatment.

The novel prolyl hydroxylase-2 inhibitor caffeic acid upregulates hypoxia inducible factor and protects against hypoxia.

BACKGROUND & AIMS: Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors. METHODS: An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out. RESULTS: Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group. CONCLUSIONS: This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia.

Production of functional recombinant prolyl hydroxylase-2 enzyme in insect cells for small molecule inhibitor screening studies.

Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates the hypoxia inducible factor (HIF) which then targets it for degradation in oxygenated cells. Inhibition of the activity of the PHD2 enzyme under hypoxic environmental conditions acts to upregulate HIF. Thus, PHD2 inhibitors may serve as a promising treatment for HIF-dependent diseases. In this study, recombinant PHD2 protein was successfully expressed using a baculovirus-insect cell expression secretory system. PHD2 was purified and in combination with bacterially expressed functional von Hippel Lindau protein-elongin B-elongin C (VBC) protein complex was used to successfully develop a fluorescence-based PHD2 activity assay. Myricetin was identified as a novel potent PHD2 inhibitor by high-throughput screening of a natural compound library. Further studies showed that treatment of human neuroblastoma SH-SY5Y cells with Myricetin increased HIF-1α protein levels. These results indicate that the insect cell expression system is capable of producing highly active recombinant PHD2 protein from which a fluorescence-based activity assay can be developed for high-throughput screening applications.

Quercetin inhibiting the PD-1/PD-L1 interaction for immune-enhancing cancer chemopreventive agent.

Targeting the PD-1/PD-L1 immune checkpoints has achieved significant positive results in the treatment of multiple cancers. Quercetin is one of the most abundant dietary flavonoids found in various vegetables and fruits, and has a wide range of biological activities including immunomodulation. Here we report that quercetin dihydrate was screened and shown to inhibit the PD-1/PD-L1 interaction. Treatment with quercetin dihydrate promoted the killing activity of T cells on MDA-MB-231 and NCI-H460 cancer cells. Experiments using the xenograft mouse model showed that the growth rate of tumor volumes and masses in the quercetin dihydrate-treated mice were decreased. Immunohistochemistry of the tumors showed that CD8, GZMB, and IFN-γ were increased in the quercetin dihydrate-treated mice. These results suggest that quercetin dihydrate attenuates the inhibitory effect of PD-L1 on T cells by inhibiting the PD-1/PD-L1 interaction, which has an exciting potential to be used as a cancer chemopreventive agent.

Caffeine promotes the expression of telomerase reverse transcriptase to regulate cellular senescence and aging.

Telomere shortening is one of the main causes of cellular senescence. Caffeine is a natural stimulant most commonly found in coffee and tea. In this study, caffeine was found to promote the expression of telomerase reverse transcriptase (TERT) at both mRNA and protein levels, and consequently extended the telomere length and prevented cellular senescence. Knockdown of TERT eliminated the effect of caffeine on telomere elongation. Moreover, animal studies indicated that caffeine promoted the expression of TERT and extended the telomere length in the thymus and spleen of mice treated with caffeine for a long period of eight months. In addition, caffeine restored the decline of organ index and improved the histological structural change of the thymus, spleen and liver of mice due to aging. These results suggest that caffeine promotes the expression of TERT to delay cellular senescence and aging, which help to understand the mechanism for the beneficial effects of caffeine containing foods on health.

Cetrimonium bromide promotes the clearance of lipids by activating the TFEB-mediated autophagosome-lysosome pathway in hepatic cells.

Autophagy plays a key role in the metabolism of macromolecules via the degradative abilities of the lysosome. Transcription factor EB (TFEB) regulates autophagosome biogenesis and lysosome function, and promoting TFEB activity has emerged as a potential strategy for the treatment of metabolic disorders. Herein, we report that cetrimonium bromide (CTAB; a quaternary ammonium compound) promotes autophagy and lysosomal biogenesis by inducing the nuclear translocation of TFEB in hepatic cells. Knockdown of TFEB mediated by short hairpin RNA inhibits CTAB-induced autophagy and lysosomal biogenesis. Mechanistically, CTAB treatment inhibits the Akt-mTORC1 signaling pathway. Moreover, CTAB treatment significantly increases lipid metabolism in both palmitate- and oleate-treated HepG2 cells, and this increase was attenuated by knockdown of TFEB. Collectively, our results indicate that CTAB activates the autophagosome-lysosome pathway via inducing the nuclear translocation of TFEB by inhibiting the mTORC1 signaling pathway. These results add to the collective understanding of TFEB function and provide new insights into CTAB-mediated lipid metabolism.

Esomeprazole inhibits the lysosomal cysteine protease legumain to prevent cancer metastasis.

Legumain is a newly discovered lysosomal cysteine protease that can cleave asparagine bonds and plays crucial roles in regulating immunity and cancer metastasis. Legumain has been shown to be highly expressed in various solid tumors, within the tumor microenvironment and its levels are directly related to tumor metastasis and poor prognosis. Therefore, legumain presents as a potential cancer therapeutic drug target. In this study, we have identified esomeprazole and omeprazole as novel legumain small molecule inhibitors by screening an FDA approved-drug library. These compounds inhibited enzyme activity of both recombinant and endogenous legumain proteins with esomeprazole displaying the highest inhibitory effect. Further molecular docking analysis also indicated that esomeprazole, the S- form of omeprazole had the most stable binding to legumain protein compared to R-omeprazole. Transwell assay data showed that esomeprazole and omeprazole reduced MDA-MB-231 breast cancer cell invasion without effecting cell viability. Moreover, an in vivo orthotopic transplantation nude mouse model study showed that esomeprazole reduced lung metastasis of MDA-MB-231 breast cancer cells. These results indicated that esomeprazole has the exciting potential to be used in anti-cancer therapy by preventing cancer metastasis via the inhibition of legumain enzyme activity. Graphical abstract.

Selection and characterization of a novel affibody peptide and its application in a two-site ELISA for the detection of cancer biomarker alpha-fetoprotein.

Alpha-fetoprotein (AFP) is one of the most important biomarkers associated with primary liver cancer, and the main approaches for diagnosis are based on immunoassay. Affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in imaging diagnosis, clinical therapeutics and biotechnology research. The aim of this study was therefore to develop a novel affibody-based ELISA for detection of AFP. After three rounds of biopanning, six AFP-binding affibody peptides were selected using phage display technology, among them affibody ZAFPD2 showed high and specific binding affinity to AFP. An affibody dimer of ZAFPD2 was created, named (ZAFP D2)2, expressed in E.coli and the purified (ZAFP D2)2 recombinant protein showed higher binding affinity to AFP, as well as high thermal stability. A novel affibody-based two-site ELISA method using ZAFPD2 or (ZAFP D2)2 and polyclonal antibody to detect AFP was developed, the detection limit of the immunoassay using (ZAFP D2)2 was 2 ng mL-1 that was 4 times lower than ZAFPD2, which meets the requirements for practical application. Therefore, this concept of affibody-based ELISA may provide a new method for the detection of various cancer biomarkers.

Production of a novel bispecific protein ULBP1×CD19-scFv targeting the NKG2D receptor and CD19 to promote the activation of NK cells.

Natural killer (NK) cells are potent cytotoxic effector cells of the innate immune system and play an important role in tumor immunosurveillance and control. NKG2D is an activating receptor of NK cells. The NKG2D receptor-ligand system has contributed to immune cells recognizing tumor cells and the tumor microenvironment. In order to stretch the application of NK cells on adoptive immunotherapy for B-cell malignancies, we designed and produced a novel bispecific ULBP1×CD19-scFv fusion protein, in which the extracellular domain of NKG2D ligand ULBP1 was fused to a single chain variable fragment (scFv) of anti-CD19. The vector expressing ULBP1×CD19-scFv protein was constructed and expressed in Pichia pastoris. Effects of medium composition, concentration of methanol as the inducer, induction time and broth content in shake flask on the expression of the recombinant protein were investigated. The results showed that the optimized conditions for ULBP1×CD19-scFv expression were 1% methanol induction for 96 h with 15% broth content. The secreted recombinant protein was purified using ammonium sulfate fractionation and Ni-NTA affinity chromatography and the purity is about 93%. The cytotoxicity of NK92-MI cells against CD19+ Raji cells was enhanced in the presence of purified ULBP1×CD19-scFv protein. These results indicated that ULBP1 could be used as an activating element of bispecific killer engagers (BiKEs) and Pichia pastoris yeast might be an alternative expression host for BiKEs production.

[A high efficiency cloning approach of multi-points combinational mutagenesis].

The combination of high-quality mutagenesis and effective screening can improve the efficiency of enzyme directed evolution. In this study, a high efficiency cloning construction method of Multi-points Combinational Mutagenesis (MCM) was developed. Efficient multi-point combination mutations were performed in this MCM method by introducing DNA assembly, fusion PCR and hybridization techniques. After optimization, the efficiency of MCM was tested by directed evolution of benzoylformate decarboxylase. The obtained number of Colony Forming Units (CFUs) by electroporation to competent cells E. coli Trelief™ 5α exceeded 106 CFUs/µg DNA. Test results show that 90/100 clones were precisely assembled. The efficiency of simultaneous mutation at 5 sites (L109, L110, H281, Q282 and A460) was up to 88%. Finally, a mutant enzyme (L109Y, L110D, H281G, Q282V and A460M) with a 10-fold increase in kcat/Km was obtained. Therefore, this method can effectively create diverse mutant libraries and promote the rapid development of enzyme directed evolution.

Screening and production of an affibody inhibiting the interaction of the PD-1/PD-L1 immune checkpoint.

An affibody is a 58 amino acids peptide derived from the Z domain of staphylococcal protein A and generally applied in areas such as imaging diagnosis, clinical therapeutics and biotechnology research. To screen for an affibody targeting the immune checkpoint PD-L1, a combinatorial affibody library was generated in yeast using degenerate overlap PCR primers and In-fusion technology. Z-j1 and Z-j2 affibodies targeting the Ig-like V domain of PD-L1 were screened and identified from this combinatorial library using the yeast two hybrid system. The Z-j1 and Z-j2 recombinant affibody proteins were over produced in E.coli and purified. ELISA and GST pull-down assays showed that recombinant Z-j1 and Z-j2 affibody proteins bound with high affinity to PD-L1 and inhibited the interaction of PD-1/PD-L1. Thus, novel affibodies targeting the immune checkpoint PD-1/PD-L1 were identified and produced in this study and have the potential to be used in cancer immunotherapy.

Piperlongumine-induced nuclear translocation of the FOXO3A transcription factor triggers BIM-mediated apoptosis in cancer cells.

The transcription factor forkhead box O 3A (FOXO3A) is a tumor suppressor that promotes cell cycle arrest and apoptosis. Piperlongumine (PL), a plant alkaloid, is known to selectively kill tumor cells while sparing normal cells. However, the mechanism of PL-induced cancer cell death is not fully understood. We report here that an association of FOXO3A with the pro-apoptotic protein BIM (also known as BCL2-like 11, BCL2L11) has a direct and specific function in PL-induced cancer cell death. Using HeLa cells stably expressing a FOXO3A-GFP fusion protein and several other cancer cell lines, we found that PL treatment induces FOXO3A dephosphorylation and nuclear translocation and promotes its binding to the BIM gene promoter, resulting in the up-regulation of BIM in the cancer cell lines. Accordingly, PL inhibited cell viability and caused intrinsic apoptosis in a FOXO3A-dependent manner. Of note, siRNA-mediated FOXO3A knockdown rescued the cells from PL-induced cell death. In vivo, the PL treatment markedly inhibited xenograft tumor growth, and this inhibition was accompanied by the activation of the FOXO3A-BIM axis. Moreover, PL promoted FOXO3A dephosphorylation by inhibiting phosphorylation and activation of Akt, a kinase that phosphorylates FOXO3A. In summary, our findings indicate that PL activates the FOXO3A-BIM apoptotic axis by promoting dephosphorylation and nuclear translocation of FOXO3A via Akt signaling inhibition. These findings uncover a critical mechanism underlying the effects of PL on cancer cells.

Discovery of Myricetin as a Potent Inhibitor of Human Flap Endonuclease 1, Which Potentially Can Be Used as Sensitizing Agent against HT-29 Human Colon Cancer Cells.

Human flap endonuclease 1 (hFEN1) is instrumental in DNA replication and repair. It is able to cleave the 5' single-stranded protrusion (also known as 5' flap) resulting from strand displacement reactions. In light of its crucial functions, hFEN1 is now deemed as a nontrivial target in the DNA damage response system for anticancer drug development. Herein, we report that myricetin and some natural flavonoids are able to inhibit hFEN1. Structure-activity relationship, inhibitory mechanisms, molecular docking, and cancer cell-based assays have been performed. Our original findings expand the activity of flavonoids and may pave the way for flavonoid-assisted targeted cancer therapy.

Clomiphene citrate induces nuclear translocation of the TFEB transcription factor and triggers apoptosis by enhancing lysosomal membrane permeabilization.

The autophagy-lysosome pathway plays a central role in cellular homeostasis by regulating the cellular degradative machinery. The transcription factor EB (TFEB) regulates the biogenesis and function of both lysosomes and autophagosomes, and enhancement of TFEB function has emerged as an attractive therapeutic strategy for lysosome-related disorders. However, little is known about the role of TFEB activation in regulating the cellular fate. Here, we describe that clomiphene citrate (CC), a selective estrogen receptor modulator, promotes nuclear translocation of TFEB and increases lysosomal biogenesis in HeLa and MDA-MB-231 cells. Treatment with CC inhibits cell viability and causes apoptosis by increasing the release of proteases cathepsin B (CatB) and cathepsin D (CatD) from lysosomes into the cytosol. In contrast, knockdown of TFEB rescues the cells from CC-induced cell death. Furthermore, CC-induced TFEB activation also enhances the autophagy flux in HeLa cells. Knockdown of autophagy-related gene 7 (ATG7) significantly decreases the CC-induced CatB and CatD release and cell death, suggesting that autophagy contributes to the lysosomal membrane permeabilization (LMP) caused by CC. Altogether, these findings have broad implications for our understanding of TFEB function and provide new insights into CC pharmacological therapy.

[Screening efficient constitutive promoters in Corynebacterium glutamicum based on time-series transcriptome analysis].

Promoter, an essential regulatory element, is widely used for metabolic engineering of industrial strains. Corynebacterium glutamicum is an important industrial workhorse to produce various amino acids. However, strong constitutive promoters that are applicable to C. glutamicum are rarely reported. In this study, we first performed a time-series transcriptome analysis of a glutamate hyper-producing strain C. glutamicum SL4 by using RNA-Seq. Overall, we picked 10 samples at different time during the fermentation process. By analyzing the time-series transcriptome data, we selected 10 candidate genes with the highest transcriptional level. These genes were all transcribed stably during the fermentation process. We subsequently cloned the promoter sequences and evaluated the promoters' strength in strain SL4 using a red fluorescent protein reporter system. To evaluate the universality of the promoters in different C. glutamicum strains, we further tested the performance of some promoters in wild type C. glutamicum strains, including ATCC 13869 and ATCC 13032. The strongest promoter was further characterized using LacZ as a reporter in all the three C. glutamicum strains. Finally, we successfully obtained three constitutive promoters with universality, PcysK, PgapA and PfumC. PcysK is the most efficient promoter among the three C. glutamicum strains. In strains SL4 and ATCC 13869, the strength of PcysK is 2-fold of the strong inducible promoter Ptac using the red fluorescent protein as a reporter and 4-fold of Ptac using LacZ as a reporter. Moreover, the strength of PcysK reaches 30%-40% of Ptac in strain ATCC 13032. The promoter PcysK is identified as a strong promoter for the first time, which can be used as an efficient biobrick for metabolic engineering of synthesis pathways in C. glutamicum.

Harnessing the affinity of magnetic nanoparticles toward dye-labeled DNA and developing it as an universal aptasensor revealed by lipopolysaccharide detection.

In current study, we have found that several magnetic nanoparticles (MNPs) are able to absorb DNA molecules, and surface engineering would be beneficial to tune such interaction. We then have focused on the assembly of polyethylenimine (PEI) coated MNPs (PEI-MNPs) with ssDNA (single-stranded DNA) and found this assembly is mediated by two forces, namely the electrostatic interactions of surface charges of MNPs and the phosphate backbones of DNA; as well as the coordination of exterior iron ions (especially Fe3+) of MNPs and DNA phosphate backbones. The fluorescence of dye-labeled DNA is significantly quenched when being complexed with PEI-MNPs, which is proved to be caused by static quenching. This PEI-MNPs interact with DNA, which could be harnessed for devising a novel type of aptasensor. This has been examplified by the selective and sensitive detection of lipopolysaccharide (LPS). The LOD (limit of detection) is ∼35 ng/mL and the linear range from 50 ng/mL to 10 µg/mL. Compared with widely used graphene oxide (GO)‒ssDNA aptamer sensors, we also have demonstrated that the PEI-MNPs based sensor is able to better avoid non-specific DNA displacement by interfering proteins, generating more satisfactory signal-to-background ratio. Our proposed sensor could be a supplement to classic GO‒DNA sensors. In summary, our work provides fundamental understanding of MNPs‒DNA interactions and also paves the way for developing novel MNPs based sensing approaches, which would contribute to nano‒bio interface and DNA-assisted bio-analysis, DNA-coordinated nano-materials and DNA-directed assembly.

2-(2-nitrobenzylidene) indolin-3-one compound inhibits transmembrane prostate androgen-induced protein (TMEPAI) expression and cancer cell proliferation.

OBJECTIVES: The transmembrane prostate androgen-induced protein (TMEPAI) is aberrantly expressed in many cancer and plays a crucial role in tumourigenesis, which makes it a potential cancer therapeutic target for drug discovery. MATERIALS AND METHODS: Here, we employed a firefly luciferase reporter driven by the TMEPAI gene promoter to screen for compound capable of inhibiting the expression of TMEPAI, and the effects of TMEPAI inhibitor on cancer cell proliferation were evaluated using the colony formation assay, cell cycle analysis, Ki-67 immunofluorescence assay and EdU incorporation assay. RESULTS: 2-(2-nitrobenzylidene) indolin-3-one (JHY-A007-50) was identified and shown to effectively inhibit the TMEPAI promoter activity. Further studies revealed that JHY-A007-50 specifically inhibited the expression of TMEPAI at both the mRNA and protein levels. Moreover, we found that JHY-A007-50 could inhibit cell proliferation and induce cell cycle arrest at the G1 phase. Our results showed that overexpression of TMEPAI decreased the inhibitory effects of JHY-A007-50 on cancer cell proliferation, and JHY-A007-50 did not affect the cell viability of HeLa cells knocked down of TMEPAI. CONCLUSIONS: Taken together, these results suggest that compound JHY-A007-50 mediates the downregulation of TMEPAI expression and inhibits cell proliferation in cancer cells.

Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors.

Legumain (LGMN) is a lysosomal protease that can specifically hydrolyze proteins after carboxyl-terminal asparagine residues. It has been reported that Legumain is highly expressed in many human tumors and promotes the migratory and invasive activity of cancer cells. Due to the limitation of an abundant and affordable source of endogenous active Legumain for further function studies, we produced the recombinant protein in Pichia pastoris. The pPICZα-LGMN expression plasmid was constructed and transformed into Pichia pastoris strain and positive recombinants were identified. Fermentation conditions were optimized and it was found that Legumain was most highly expressed under pH 6 culture conditions. In addition, the enzyme activity of the purified Legumain was tested using a fluorogenic substrate (Z-Ala-Ala-Asn-AMC) assay and the optimum pH for the autocatalytic activation of recombinant Legumain was very acidic at a pH value of 3. The recombinant protein was then used to screen a library of compounds and small molecule 1773 (Terramycin) was shown to effectively inhibit Legumain enzyme activity. These results indicate that the Pichia pastoris expression system can produce highly active recombinant Legumain protein allowing it to be used for High-throughput screening (HTS) applications.

Inhibitor of the human telomerase reverse trancriptase (hTERT) gene promoter induces cell apoptosis via a mitochondrial-dependent pathway.

Telomerase is aberrantly expressed in many cancers and plays an important role in the development of cellular immortality and oncogenesis, which makes it a potential cancer therapeutic target for drug discovery. Here, we constructed a firefly luciferase reporter driven by the human telomerase reverse trancriptase (hTERT) gene promoter to screen for inhibitory compounds. Compound 5c was discovered and shown to significantly inhibit the promoter activity of hTERT gene. Furthermore, five analogs of compound 5c were synthesized, and compound 8b was shown to be a more potent inhibitor of hTERT gene promoter activity and subsequent expression of hTERT mRNA and protein. The viability of HeLa cells was inhibited by a knockdown of hTERT gene expression, and the same effect was also observed by treating with compound 8b. Moreover, our results indicated that compound 8b induced apoptosis of HeLa cells, and activated caspase-9 and caspase-3 enzymes. Taken together, these results suggested that compound 8b down-regulates the expression of hTERT and induces mitochondrial-dependent apoptosis.