Discovery of novel PROTACs based on multi-targeted angiogenesis inhibitors.

Anti-angiogenesis has been proved to be an effective strategy for the treatment of tumors. Anti-angiogenic drugs had achieved certain therapeutic effects. However, drug resistance also gradually emerged and limited the application of angiogenesis inhibitors. Proteolysis Targeting Chimeras (PROTACs) are bifunctional molecules capable of degrading proteins through the ubiquitin-proteasome system (UPS). Compared with traditional inhibitors, they displayed advantages of less dosage, lower toxicity and less resistance. In this study, we designed and synthesized a series of novel PROTACs based on our recently reported multi-targeted angiogenesis inhibitor S5. Preliminary biological evaluation of title PROTACs was carried out in various cell lines. The results indicated that these novel bifunctional PROTACs displayed potential in degrading BRAF protein. Their degradation mechanism showed that the degradation of BRAF by PROTAC-1 was dependent on binding to target proteins and E3 ubiquitin ligase. Our findings provided further evidence that these novel PROTACs could be considered in further application in overcome of clinical resistance of traditional angiogenesis inhibitors.

A bifunctional agent for efficient imaging of PD-L1 and antimelanoma activity.

Immune checkpoint inhibitors targeting PD-L1 lead to challenging patterns of efficacy and toxicity. Herein, by focusing on tracing the molecular biomarker of response to efficacy, we formulated a central hypothesis for the construction of theranostic functional monoclonal antibody incorporation with tracing ability based on fluorescence turn-on and controllable release strategies. Functional atezolizumab was constructed by in situ assembly of both biorthogonal group and controllable release group. The theranostic monoclonal antibodies achieved quantitative monitoring of PD-L1 on cells with different expression levels through biorthogonal light-up fluorescence, followed by the release of atezolizumab in combination with high tumor reduction conditions to promote immune activation. The combination of bio-orthogonal reaction-driven fluorescence turn-on and tumor microenvironment-responsive controllable release afforded theranostic bifunctional monoclonal antibodies for the detection of PD-L1 and combination therapy. Remarkably, these novel theranostics might be used as probes for fluorescent imaging and simultaneously achieving potent antitumor efficacy.

Discovery of novel antibody-drug conjugates bearing tissue protease specific linker with both anti-angiogenic and strong cytotoxic effects.

Bevacizumab is an FDA-approved class of monoclonal antibodies used to inhibit angiogenesis and promote normalization of blood vessels. It is usually combined with chemotherapeutic agents to treat a variety of solid tumors. However, the whole-body toxicities and toxicity associated with chemotherapy greatly limit the clinical use of this combination therapy. Antibody-drug conjugates (ADCs) couple monoclonal antibodies to cytotoxic molecules via a linker, utilizing the high specificity of monoclonal antibodies to tumor surface antigens to act as a "biological missile" to deliver chemotherapeutic drugs to the tumor site. Herein, we designed a bevacizumab-based ADC, Bevacizumab Vedotin, conjugating bevacizumab to the microtubulin inhibitor MMAE via a tissue protease-specific linker. Biological studies showed strong stability and good tumor cell targeting of our constructed ADCs; rapid drug release was achieved in the presence of exogenous histone protease B. In addition, Bevacizumab Vedotin exhibited good anti-proliferative, apoptosis-promoting and cell cycle-stalling effects on glioma (U87), hepatocellular carcinoma (HepG2), and breast cancer (MCF-7) cell lines. Further in vitro assays demonstrated the enhanced anti-migration activity against MCF-7, potent anti-angiogenic effects, and blockade of the VEGF/VEGFR pathway of Bevacizumab Vedotin.

DPPH Radical Scavenging Activity of New Phenolics from the Fermentation Broth of Mushroom Morehella importuna.

In recent years, wild morel mushroom species have begun to be widely cultivated in China due to their high edible and medicinal values. To parse the medicinal ingredients, we employed the technique of liquid-submerged fermentation to investigate the secondary metabolites of Morehella importuna. Two new natural isobenzofuranone derivatives (1-2) and one new orsellinaldehyde derivative (3), together with seven known compounds, including one o-orsellinaldehyde (4), phenylacetic acid (5), benzoic acid (6), 4-hydroxy-phenylacetic acid (7), 3,5-dihydroxybenzoic acid (8), N,N'-pentane-1,5-diyldiacetamide (9), and 1H-pyrrole-2-carboxylic acid (10), were obtained from the fermented broth of M. importuna. Their structures were determined according to the data of NMR, HR Q-TOF MS, IR, UV, optical activity, and single-crystal X-ray crystallography. TLC-bioautography displayed that these compounds possess significant antioxidant activity with the half DPPH free radical scavenging concentration of 1.79 (1), 4.10 (2), 4.28 (4), 2.45 (5), 4.40 (7), 1.73 (8), and 6.00 (10) mM. The experimental results would shed light on the medicinal value of M. importuna for its abundant antioxidants.

New Sesquiterpenoids from the Fermented Broth of Termitomyces albuminosus and their Anti-Acetylcholinesterase Activity.

Termitomyces albuminosus is the symbiotic edible mushroom of termites and cannot be artificially cultivated at present. In the project of exploring its pharmaceutical metabolites by microbial fermentation, four new selinane type sesquiterpenoids-teucdiol C (1), D (2), E (3), and F (4), together with two known sesquiterpenoids teucdiol B (5) and epi-guaidiol A (6)-were obtained from its fermented broth of T. albuminosus. Their structures were elucidated by the analysis of NMR data, HR Q-TOF MS spectral data, CD, IR, UV, and single crystal X-ray diffraction. Epi-guaidiol A showed obvious anti-acetylcholinesterase activity in a dose-dependent manner. The experimental results displayed that T. albuminosus possess the pharmaceutical potential for Alzheimer's disease, and it was an effective way to dig new pharmaceutical agent of T. albuminosus with the microbial fermentation technique.

Gunnilactams A-C, Macrocyclic Tetralactams from the Mycelial Culture of the Entomogenous Fungus Paecilomyces gunnii.

Three novel macrocyclic tetralactams, gunnilactam A (1), gunnilactam B (2), and gunnilactam C (3), were isolated from the submerged fermentation broth of Paecilomyces gunnii, an entomogenous fungus identified as the anamorph of Cordyceps gunnii. Their structures were determined using NMR data, HREIMS, and single-crystal X-ray crystallography. Gunnilactam A exhibited selective cytotoxic activity against human prostate cancer C42B cells with an IC50 value of 5.4 µM.

Inhibitory activities against rice pathogens of 8-hydroxy-2,4,6-octatriynamide from Agrocybe sp.

8-Hydroxy-2,4,6-octatriynamide, a natural polyacetylene with inhibitory activities against rice pathogens, was isolated from the liquid fermentation broth of strain Agrocybe sp. YB2005 during screening for new natural chemical agents to control rice pathogens. 8-hydroxy-2,4,6-octatriynamide was purified by consecutive chromatography over a Cl8 reversed phase silica gel, sephadex LH-20 and silica gel. The chemical structure of 8-hydroxy-2,4,6-octatriynamide was elucidated through spectroscopic analyses, including 1D- and 2D-NMR, ESI mass spectrometry and X-ray single crystal diffraction. Bioassays showed that 8-hydroxy-2,4,6-octatriynamide could significantly inhibit growth of Xanthomonas oryzae with an MIC of 53.1 µM in a 96-well plate and the growth of Rhizoctonia solani at 1.02 mM in a 24-well plate. When rice leaves were inoculated with Magnaporthe grisea and cultured in artificial nutrition liquid containing 0.34 mM 8-hydroxy-2,4,6-octatriynamide, no rice blast was observed. The present study implied that 8-hydroxy-2,4,6-octatriynamide could be a candidate agent against rice pathogens.

Production of the siderophore lysochelin in rich media through maltose-promoted high-density growth of Lysobacter sp. 3655.

Siderophores are produced by bacteria in iron-restricted conditions. However, we found maltose could induce the biosynthesis of the siderophore lysochelin in Lysobacter sp. 3655 in rich media that are not compatible with siderophore production. Maltose markedly promoted cell growth, with over 300% increase in cell density (OD600) when LB medium was added with maltose (LBM). While lysochelin was not detectable when OD600 in LBM was below 5.0, the siderophore was clearly produced when OD600 reached 7.5 and dramatically increased when OD600 was 15.0. Coincidently, the transcription of lysochelin biosynthesis genes was remarkably enhanced following the increase of OD600. Conversely, the iron concentration in the cell culture dropped to 1.2 µM when OD600 reached 15.0, which was 6-fold lower than that in the starting medium. Moreover, mutants of the maltose-utilizing genes (orf2677 and orf2678) or quorum-sensing related gene orf644 significantly lowered the lysochelin yield. Transcriptomics analysis showed that the iron-utilizing/up-taking genes were up-regulated under high cell density. Accordingly, the transcription of lysochelin biosynthetic genes and the yield of lysochelin were stimulated when the iron-utilizing/up-taking genes were deleted. Finally, lysochelin biosynthesis was positively regulated by a TetR regulator (ORF3043). The lysochelin yield in orf3043 mutant decreased to 50% of that in the wild type and then restored in the complementary strain. Together, this study revealed a previously unrecognized mechanism for lysochelin biosynthetic regulation, by which the siderophore could still be massively produced in Lysobacter even grown in a rich culture medium. This finding could find new applications in large-scale production of siderophores in bacteria.

All-in-one nanotheranostic platform based on tumor microenvironment: new strategies in multimodal imaging and therapeutic protocol.

Conventional tumor diagnosis and treatment approaches have significant limitations in clinical application, whereas personalized theranostistic nanoplatforms can ensure advanced diagnosis, precise treatment, and even a good prognosis in cancer. Tumor microenvironment (TME)-targeted therapeutic strategies offer absolute advantages in all aspects compared to tumor cell-targeted therapeutic strategies. It is essential to create a TME-responsive all-in-one nanotheranostic platform to facilitate individualized tumor treatment. Based on the TME-responsive multifunctional nanotheranostic platform, we focus on the combined use of multimodal imaging and therapeutic protocols and summary and outlooks on the latest advanced nanomaterials and structures for creating the integrated nanotheranostic system based on material science, which provide insights and reflections on the development of innovative TME-targeting tools for cancer theranostics.

Recent Advances in Signaling Pathways and Kinase Inhibitors for Leukemia Chemotherapy.

Leukemia is a malignant clonal disease of hematopoietic stem cells, which accounts for about 3% of the total incidence of tumors and is particularly prevalent among children and adolescents. It mainly includes four types of leukemia, namely ALL, AML, CLL, and CML, which are often aggressive and challenging diseases to treat. Several signaling pathways are dysregulated in almost all types of leukemia, such as JAK, PI3K, and MAPK, and others are dysregulated in specific types of leukemia, like Wnt/ß-catenin, Hedgehog, FLT3, Bcr-Abl, and so on. Many efforts have been devoted to developing small molecule inhibitors targeting protein kinases involved in leukemia-related signaling pathways. In this review, we focus on the study of signaling pathways and protein kinases that developed as targets of anti-leukemia drug therapy and report the research progress of relevant small molecule kinase inhibitors over the last five years.

Identification and Biosynthetic Study of the Siderophore Lysochelin in the Biocontrol Agent Lysobacter enzymogenes.

Lysobacter is a genus of bacteria emerging as new biocontrol agents in agriculture. Although iron acquisition is essential for the bacteria, no siderophore has been identified from any Lysobacter. Here, we report the identification of the first siderophore, N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (lysochelin), and its biosynthetic gene cluster from Lysobacter enzymogenes. Intriguingly, the deletion of the spermidine biosynthetic gene encoding arginine decarboxylase or SAM decarboxylase eliminated lysochelin and the antifungals, HSAF and its analogues, which are key to the disease control activity and to the survival of Lysobacter under oxidative stresses caused by excess iron. The production of lysochelin and the antifungals is greatly affected by iron concentration. Together, the results revealed a previously unrecognized system, in which L. enzymogenes produces a group of small molecules, lysochelin, spermidine, and HSAF and its analogues, that are affected by iron concentration and critical to the growth and survival of the biocontrol agent.

Discovery of intracellular self-assembly protein degraders driven by tumor-specific activatable bioorthogonal reaction.

Proteolysis Targeting Chimera (PROTAC) is a type of bifunctional chimeric molecule that can directly degrade the binding proteins through the ubiquitin-proteasome pathway. PROTAC has shown great potential in overcoming drug resistance and targeting undruggable targets. However, there are still many shortcomings that need to be solved urgently, including worse membrane permeability and bioavailability induced by their large molecular weight. Herein, we used intracellular self-assembly strategy to construct tumor-specific PROTACs via small molecular precursors. We developed two types of precursors incorporated with azide and alkyne as biorthogonal groups, respectively. These small precursors with improved membrane permeability could react facilely with each other under the catalysis of copper ions with high concentration in tumor tissues, affording novel PROTACs. These novel intracellular self-assembled PROTACs could effectly induce degradation of VEGFR-2 and EphB4 in U87 cells. Meanwhile, they could also promote apoptosis and block cells in S phase. These tumor-specific intracellular self-assembled PROTACs exhibited high selectivity due to the high concentration of copper content in tumor tissue. Moreover, this new strategy could reduce the molecular weight of PROTACs, as well as improve the membrane permeability. These results will greatly expand the applications of bioorthogonal reaction in discovery of novel PROTACs.

New abscisic acid-related metabolites from Phellinus vaninii.

Two new and three known abscisic acid-related metabolites were obtained from the potato dextrose agar culture of Phellinus vaninii YB2005. Their structures were established on the basis of detailed spectroscopic analyses, including 1D NMR, 2D NMR, and HR-Q-TOF-MS techniques. The putative biosynthesis pathway of these secondary metabolites would decipher the mechanism of the symbiosis between plant and fungi from the view of chemistry.

Identification of the Biosynthetic Gene Cluster for the anti-MRSA Lysocins through Gene Cluster Activation Using Strong Promoters of Housekeeping Genes and Production of New Analogs in Lysobacter sp. 3655.

The Gram-negative gliding bacteria Lysobacter represent a new and rich source for bioactive natural products. In an effort to discover new antibiotics, we found a cryptic biosynthetic gene cluster (BGC) in Lysobacter sp. 3655 that shared a high similarity with the putative lysocin BGC identified in silico previously from Lysobacter sp. RH2180-5. Lysocins are cyclic lipodepsipeptides with potent activity against MRSA (methicillin-resistant Staphylococcus aureus) using a novel mode of action, but the lysocin BGC had not been experimentally verified so far. Using an activity-guided screening, we isolated the main antibiotic compound and confirmed it to be lysocin E. However, the putative lysocin BGC was barely transcribed in the wild type, in which lysocins were produced only in specific conditions and in a negligible amount. To activate the putative lysocin BGC, we screened for strongly transcribed housekeeping genes in strain 3655 and found several powerful promoters. Upon engineering the promoters into the BGC, the lysocin gene transcription was significantly enhanced and the lysocin yield was markedly increased. With readily detectable lysocins production in the engineered strains, we showed that lysocin production was abolished in the gene deletion mutant and then restored in the complementary strain, even when grown in conditions that did not support the wild type for lysocin production. Moreover, the engineered strain produced multiple new lysocin congeners. The determination of the lysocin BGC and the Lysobacter promoters will facilitate the ongoing efforts for yield improvement and new antibiotic biosynthesis using synthetic biology strategies.

A Sphingosine-type cerebroside in Clavicorona pyxidata induce fruit body formation.

Clavicorona pyxidata is a wild edible and medicinal mushroom that is rich in bioactive natural products and has thus been extensively used as traditional medicine in China. The present study has determined that the organic crude extract prepared from a fermented culture of C. pyxidata imparted auto-inhibitory effects on mycelial growth and then induced the formation of fruiting bodies. By monitoring bioactivity, one compound was isolated via successive chromatography over silica gel, Sephadex LH-20, and Cl8-reversed phase silica gel and was identified as a known sphingosine-type cerebroside by nuclear magnetic resonance (NMR) and physicochemical data, namely, (4E, 8E)-N-D-2'-hydroxypalmitoyl-1-O-ß-D-glucopyranosyl-9-methyl-4,8-sphingadienine. The application of this cerebroside at a concentration of 200 µg/disc paper resulted in the inhibition of aerial hyphal growth of C. pyxidata. The findings of the present study indicated that this C. pyxidata cerebroside is a fruiting body-inducing substance (FIS).

Anti-Inflammatory Effect of a Polyphenol-Enriched Fraction from Acalypha wilkesiana on Lipopolysaccharide-Stimulated RAW 264.7 Macrophages and Acetaminophen-Induced Liver Injury in Mice.

A polyphenol-enriched fraction (PEF) from Acalypha wilkesiana, whose leaves have been traditionally utilized for the treatment of diverse medical ailments, was investigated for the anti-inflammatory effect and molecular mechanisms by using lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages and acetaminophen- (APAP-) induced liver injury mouse model. Results showed that PEF significantly attenuated LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2) in RAW 264.7 macrophages. PEF also reduced the secretion of proinflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin- (IL-) 1ß, and IL-6 in LPS-stimulated RAW 264.7 macrophages. Moreover, PEF potently inhibited LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) as well as the activation of nuclear factor-κB (NF-κB) by preventing the degradation of inhibitor κB-α (IκB-α). In vivo, PEF pretreatment ameliorated APAP-induced liver injury and hepatic inflammation, as presented by decreased hepatic damage indicators and proinflammatory factors at both plasma and gene levels. Additionally, PEF pretreatment remarkably diminished Toll-like receptor 3 (TLR3) and TLR4 expression and the subsequent MAPKs and NF-κB activation. HPLC analysis revealed that two predominantly polyphenolic compounds present in PEF were geraniin and corilagin. These results indicated that PEF has an anti-inflammatory effect, and its molecular mechanisms may be involved in the inactivation of the TLR/MAPK/NF-κB signaling pathway, suggesting the therapeutic potential of PEF for inflammatory diseases.

New apoptosis-inducing sesquiterpenoids from the mycelial culture of Chinese edible fungus Pleurotus cystidiosus.

Two new bisabolane-type sesquiterpenoids, pleuroton A (1) and pleuroton B (2), and three clitocybulol derivatives, clitocybulol D (3), clitocybulol E (4), and clitocybulol F (5), were obtained from the mycelial culture of edible fungus Pleurotus cystidiosus O. K. Mill by repeated column chromatography over RP-18, Sephadex LH-20, and silica gel. Their structures were determined according to nuclear magnetic resonance data, high-resolution electron impact mass spectrometry, and circular dichroism spectra. These new sesquiterpenoids exhibited significant cytotoxicity against two human prostate cancer DU-145 and C42B cells in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The median inhibitory concentration (IC50) of compounds 1, 2, 3, 4, and 5 was 174, 28, 233, 162, and 179 nM, respectively, against the DU-145 cell and was 104, 52, 163, 120, and 119 nM, respectively, against the C42B cell. Especially, pleuroton B (2) exhibited the strongest cytotoxity among these sesquiterpenoids, which was confirmed by the colony formation assay. Furthermore, pleuroton B (2) could trigger the apoptosis of DU-145 cells through the detection of apoptosis cells using annexin V-FITC staining by flow cytometry, the observation of condensed nuclei in the apoptosis cells, and the western blot analysis for the expression of apoptosis-related proteins Bcl-2, Bak, and Bax. Analysis of structure-activity relationships of these sesquiterpenoids revealed that the unusual functional moiety of pleuroton B should contribute to its significant bioactivity. These results display the pharmacological potential of P. cystidiosus.

[Construction of Saccharomyces cerevisiae mutant with knockout of SNF4 gene].

Construction and ethanol production effects of SNF4 gene knockout in Saccharomyces cerevisiae were described in this paper. For knockout of SNF4 gene in S. cerevisiae YS2, a PCR-amplified disruption cassette was used, encoding the short flanking homologous regions to the SNF4 gene and Kan(r) as selectable marker. The SNF4 gene disruption cassette was transformed into S. cerevisiae YS2 through LiAc/SS Carrier DNA/PEG. The positive transformants were grown on G418 plates and verified by PCR. The Kan(r) marker was rescued by transforming plasmid pSH65 into positive transformants and inducing expression of Cre recombinase in galactose-containing medium. Lastly, the YS2-deltaSNF4 strain, in which SNF4 allele gene were completely knocked out, was obtained by repeating the same procedure. The result of anaerobic fermentation showed that ethanol production of the SNF4 gene knockout strain had increased by 7.57 percent as compared with the original strain YS2. The experiment indicated ethanol production could be improved significantly with the approach ofSNF4 gene knockout by Cre-LoxP system.

Clavicorolides A and B, sesquiterpenoids from the fermentation products of edible fungus Clavicorona pyxidata.

Clavicorolides A (1) and B (2), two sesquiterpenoids possessing a novel backbone named as Clavicoronane-type, together with one new and two known Protoilludane-type sesquiterpenoids, namely, Tsugicolines M (3), A (4), and C (5), and Sterpurane (6), and Lactarane-type sesquiterpenoid Lactarorufin A (7), were isolated from the fermentation products of Clavicorona pyxidata YB2005. Their structures including relative and absolute configurations were elucidated on the basis of NMR data and analysis of X-ray single crystal diffraction.

Isolation, structure elucidation and apoptosis-inducing activity of new compounds from the edible fungus Lentinus striguellus.

Three new natural compounds, striguellolide A (1), striguellol A (2) and striguellone A (6), together with three known ones, 3,4-trans-dihydroxy-6-methoxy-2,2-dimethylchroman (3), 3-hydroxy-6- methoxy-2,2-dimethylchroman-4-one (4) and 3-hydroxy-6- methoxy-2,2-dimethylchroman (5), were isolated from the agar cultures of the edible fungal strain Lentinus striguellus. Their structures were elucidated by spectroscopic and mass spectrometric analyses, including 1D-, 2D-NMR and HR-ESI-Q-TOF-MS, and chemical reactions. Striguellone A showed cytotoxicity against HeLa cells by MTT assay and was found to be an activator of apoptosis, assessed by morphological observation and cell cycle analysis using the flow cytometer.