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Biochemistry Jan 2022The structural diversification of natural products is instrumental to their versatile bioactivities. In this context, redox tailoring enzymes are commonly involved in...
The structural diversification of natural products is instrumental to their versatile bioactivities. In this context, redox tailoring enzymes are commonly involved in the modification and functionalization of advanced pathway intermediates en route to the mature natural products. In recent years, flavoprotein monooxygenases have been shown to mediate numerous redox tailoring reactions that include not only (aromatic) hydroxylation, Baeyer-Villiger oxidation, or epoxidation reactions but also oxygenations that are coupled to extensive remodeling of the carbon backbone, which are often central to the installment of the respective pharmacophores. In this Perspective, we will highlight recent developments and discoveries in the field of flavoenzyme catalysis in bacterial natural product biosynthesis and illustrate how the flavin cofactor can be fine-tuned to enable chemo-, regio-, and stereospecific oxygenations via distinct flavin-C4a-peroxide and flavin-N5-(per)oxide species. Open questions remain, e.g., regarding the breadth of chemical reactions enabled particularly by the newly discovered flavin-N5-oxygen adducts and the role of the protein environment in steering such cascade-like reactions. Outstanding cases involving different flavin oxygenating species will be exemplified by the tailoring of bacterial aromatic polyketides, including enterocin, rubromycins, rishirilides, mithramycin, anthracyclins, chartreusin, jadomycin, and xantholipin. In addition, the biosynthesis of tropone natural products, including tropolone and tropodithietic acid, will be presented, which features a recently described prototypical flavoprotein dioxygenase that may combine flavin-N5-peroxide and flavin-N5-oxide chemistry. Finally, structural and mechanistic features of selected enzymes will be discussed as well as hurdles for their application in the formation of natural product derivatives via bioengineering.
Topics: Bacteria; Bacterial Proteins; Biological Products; Biosynthetic Pathways; Flavins; Flavoproteins; Oxidation-Reduction; Polyketides; Substrate Specificity
PubMed: 34962769
DOI: 10.1021/acs.biochem.1c00763 -
Doklady. Biochemistry and Biophysics Sep 2021GC-rich stretches in the DNA minor groove are the established intracellular targets for the aureolic acid group of antibiotics such as olivomycin A and its semisynthetic...
GC-rich stretches in the DNA minor groove are the established intracellular targets for the aureolic acid group of antibiotics such as olivomycin A and its semisynthetic analogue olivamide. We demonstrated here that both antibiotics at nanomolar concentrations inhibited transcription of the c-Myc oncogene in cultured human tumor cells. The mechanism of transcriptional inhibition did not require the full-length binding site for Sp1, a GC-dependent transcriptional factor. GC quartets with the nucleotide sequences optimal for drug binding are sufficient for c-Myc transcriptional block by the aureolic acid derivatives.
Topics: Plicamycin
PubMed: 34697733
DOI: 10.1134/S1607672921050094 -
Cell Death & Disease Oct 2021Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the...
Colorectal cancers (CRC) can be classified into four consensus molecular subtypes (CMS), among which CMS1 has the best prognosis, contrasting with CMS4 that has the worst outcome. CMS4 CRC is notoriously resistant against therapeutic interventions, as demonstrated by preclinical studies and retrospective clinical observations. Here, we report the finding that two clinically employed agents, everolimus (EVE) and plicamycin (PLI), efficiently target the prototypic CMS4 cell line MDST8. As compared to the prototypic CMS1 cell line LoVo, MDST8 cells treated with EVE or PLI demonstrated stronger cytostatic and cytotoxic effects, increased signs of apoptosis and autophagy, as well as a more pronounced inhibition of DNA-to-RNA transcription and RNA-to-protein translation. Moreover, nontoxic doses of EVE and PLI induced the shrinkage of MDST8 tumors in mice, yet had only minor tumor growth-reducing effects on LoVo tumors. Altogether, these results suggest that EVE and PLI should be evaluated for their clinical activity against CMS4 CRC.
Topics: Adaptor Proteins, Signal Transducing; Animals; Antineoplastic Combined Chemotherapy Protocols; Cell Proliferation; Colorectal Neoplasms; Cytoskeletal Proteins; Everolimus; Humans; Mice; Plicamycin
PubMed: 34675191
DOI: 10.1038/s41419-021-04270-x -
Journal of Nanobiotechnology Sep 2021Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an...
BACKGROUND
Sarcomas comprise a group of aggressive malignancies with very little treatment options beyond standard chemotherapy. Reposition of approved drugs represents an attractive approach to identify effective therapeutic compounds. One example is mithramycin (MTM), a natural antibiotic which has demonstrated a strong antitumour activity in several tumour types, including sarcomas. However, its widespread use in the clinic was limited by its poor toxicity profile.
RESULTS
In order to improve the therapeutic index of MTM, we have loaded MTM into newly developed nanocarrier formulations. First, polylactide (PLA) polymeric nanoparticles (NPs) were generated by nanoprecipitation. Also, liposomes (LIP) were prepared by ethanol injection and evaporation solvent method. Finally, MTM-loaded hydrogels (HG) were obtained by passive loading using a urea derivative non-peptidic hydrogelator. MTM-loaded NPs and LIP display optimal hydrodynamic radii between 80 and 105 nm with a very low polydispersity index (PdI) and encapsulation efficiencies (EE) of 92 and 30%, respectively. All formulations show a high stability and different release rates ranging from a fast release in HG (100% after 30 min) to more sustained release from NPs (100% after 24 h) and LIP (40% after 48 h). In vitro assays confirmed that all assayed MTM formulations retain the cytotoxic, anti-invasive and anti-stemness potential of free MTM in models of myxoid liposarcoma, undifferentiated pleomorphic sarcoma and chondrosarcoma. In addition, whole genome transcriptomic analysis evidenced the ability of MTM, both free and encapsulated, to act as a multi-repressor of several tumour-promoting pathways at once. Importantly, the treatment of mice bearing sarcoma xenografts showed that encapsulated MTM exhibited enhanced therapeutic effects and was better tolerated than free MTM.
CONCLUSIONS
Overall, these novel formulations may represent an efficient and safer MTM-delivering alternative for sarcoma treatment.
Topics: Animals; Anti-Bacterial Agents; Antineoplastic Agents; Chondrosarcoma; Drug Compounding; Female; Humans; Hydrogels; Liposomes; Mice; Mice, Nude; Nanoparticles; Plicamycin; Polyesters; Sarcoma
PubMed: 34488783
DOI: 10.1186/s12951-021-01008-x -
Frontiers in Immunology 2021The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and...
The axis of Programmed cell death-1 receptor (PD-1) with its ligand (PD-L1) plays a critical role in colorectal cancer (CRC) in escaping immune surveillance, and blocking this axis has been found to be effective in a subset of patients. Although blocking PD-L1 has been shown to be effective in 5-10% of patients, the majority of the cohorts show resistance to this checkpoint blockade (CB) therapy. Multiple factors assist in the growth of resistance to CB, among which T cell exhaustion and immunosuppressive effects of immune cells in the tumor microenvironment (TME) play a critical role along with other tumor intrinsic factors. We have previously shown the polyketide antibiotic, Mithramycin-A (Mit-A), an effective agent in killing cancer stem cells (CSCs) and in a subcutaneous murine model. Since TME plays a pivotal role in CB therapy, we tested the immunomodulatory efficacy of Mit-A with anti-PD-L1 mAb (αPD-L1) combination therapy in an immunocompetent MC38 syngeneic orthotopic CRC mouse model. Tumors and spleens were analyzed by flow cytometry for the distinct immune cell populations affected by the treatment, in addition to RT-PCR for tumor samples. We demonstrated the combination treatment decreases tumor growth, thus increasing the effectiveness of the CB. Mit-A in the presence of αPD-L1 significantly increased CD8 T cell infiltration and decreased immunosuppressive granulocytic myeloid-derived suppressor cells and anti-inflammatory macrophages in the TME. Our results revealed Mit-A in combination with αPD-L1 has the potential for augmented CB therapy by turning an immunologically "cold" into "hot" TME in CRC.
Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Disease Models, Animal; Female; Immune Checkpoint Inhibitors; Mice; Mice, Inbred C57BL; Plicamycin
PubMed: 34381456
DOI: 10.3389/fimmu.2021.706133 -
Cancer Science Sep 2021Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo...
Heterogeneous nuclear ribonucleoprotein L-like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial-mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from -273 to -10 base pairs upstream of the transcription start site identified by 5'-RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB-downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.
Topics: Binding Sites; Cell Proliferation; Colorectal Neoplasms; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Gene Knockout Techniques; HT29 Cells; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Neoplasm Metastasis; Plicamycin; Promoter Regions, Genetic; Proto-Oncogene Proteins c-myb; Sp1 Transcription Factor; Transcription, Genetic; Transfection
PubMed: 34286904
DOI: 10.1111/cas.15069 -
Journal of Virology Aug 2021Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The...
Porcine epidemic diarrhea virus (PEDV) is an enteric coronavirus causing acute intestinal infection in pigs, with high mortality often seen in neonatal pigs. The newborns rely on innate immune responses against invading pathogens because of lacking adaptive immunity. However, how PEDV disables the innate immunity of newborns toward severe infection remains unknown. We found that PEDV infection led to reduced expression of histone deacetylases (HDACs), especially HDAC1, in porcine IPEC-J2 cells. HDACs are considered important regulators of innate immunity. We hypothesized that PEDV interacts with certain host factors to regulate HDAC1 expression in favor of its replication. We show that HDAC1 acted as a negative regulator of PEDV replication in IPEC-J2 cells, as shown by chemical inhibition, gene knockout, and overexpression. A GC-box (GCCCCACCCCC) within the promoter region was identified for Sp1 binding in IPEC-J2 cells. Treatment of the cells with Sp1 inhibitor mithramycin A inhibited HDAC1 expression, indicating direct regulation of HDAC1 expression by Sp1. Of the viral proteins that were overexpressed in IPEC-J2 cells, the N protein was found to be present in the nuclei and more inhibitory to transcription. The putative nuclear localization sequence PKKNKSR contributed to its nuclear localization. The N protein interacted with Sp1 and interfered with its binding to the promoter region, thereby inhibiting its transcriptional activity for expression. Our findings reveal a novel mechanism of PEDV evasion of the host responses, offering implications for studying the infection processes of other coronaviruses. The enteric coronavirus porcine epidemic diarrhea virus (PEDV) causes fatal acute intestinal infection in neonatal pigs that rely on innate immune responses. Histone deacetylases (HDACs) play important roles in innate immune regulation. Our study found PEDV suppresses HDAC1 expression via the interaction of its N protein and porcine Sp1, which identified a novel mechanism of PEDV evasion of the host responses to benefit its replication. This study suggests that other coronaviruses, including SARS-CoV and SARS-CoV-2, also make use of their N proteins to intercept the host immune responses in favor of their infection.
Topics: Animals; Cells, Cultured; Coronavirus Infections; Epithelial Cells; Histone Deacetylase 1; Intestinal Mucosa; Porcine epidemic diarrhea virus; Sp1 Transcription Factor; Swine; Swine Diseases; Viral Nonstructural Proteins; Virus Replication
PubMed: 34232065
DOI: 10.1128/JVI.00853-21 -
Life Sciences Sep 2021Colorectal cancer (CRC) with high metastasis rates has been known as a major cause of death worldwide. Lack of the specificity and insufficient concentrations of...
Colorectal cancer (CRC) with high metastasis rates has been known as a major cause of death worldwide. Lack of the specificity and insufficient concentrations of traditional chemotherapeutics at tumor site and their severe adverse effects necessitate development of new treatment strategies such as designing suitable nanocarriers for delivery of drugs, improving their pharmacological profiles and reducing adverse effects. We have developed a platform based on the poly-ursolic acid (poly-UA), a polymeric system with potential anticancer effect. Following the self-assembly of poly-UA into the nanoparticles (NPs), they were applied for delivery of mithramycin A (Mith-A), a promising candidate for CRC therapy, however, with some limitations such as rapid clearance and serious side effects. Mith-A-loaded poly-UA NPs with suitable physicochemical properties and efficient drug entrapment, released Mith-A in a controlled manner and provided suitable toxicity against the CT-26 colorectal cancer cells, increased accumulation in tumor, and protection against the detrimental features of the disease. Poly-UA NPs demonstrated therapeutic efficiency (in vivo and in vitro) by themselves. The prepared NPs induced no remarkable alteration of body weights or damages to the major organs in animals bearing tumor indicating the safety of NPs. The bioactive nanoformulation along with improving the pharmacological profile of Mith-A could provide a synergistic toxicity against the CRC.
Topics: Animals; Antineoplastic Agents; Cell Line; Colorectal Neoplasms; Drug Compounding; Humans; Male; Mice; Mice, Inbred BALB C
PubMed: 34186049
DOI: 10.1016/j.lfs.2021.119772 -
Cancer Immunology, Immunotherapy : CII Feb 2022Recent studies have shown that tumor-derived exosomes participate in the communication between tumor cells and their microenvironment and mediate malignant biological...
Recent studies have shown that tumor-derived exosomes participate in the communication between tumor cells and their microenvironment and mediate malignant biological behaviors including immune escape. In this study, we found that gastric cancer (GC) cell-derived exosomes could be effectively uptaken by Vγ9Vδ2 T cells, decrease the cell viability of Vγ9Vδ2 T cells, induce apoptosis, and reduce the production of cytotoxic cytokines IFN-γ and TNF-α. Furthermore, we demonstrated that exosomal miR-135b-5p was delivered into Vγ9Vδ2 T cells. Exosomal miR-135b-5p impaired the function of Vγ9Vδ2 T cells by targeting specificity protein 1 (SP1). More importantly, blocking the SP1 function by Plicamycin, an SP1 inhibitor, abolished the effect of stable miR-135b-5p knockdown GC cell-derived exosomes on Vγ9Vδ2 T cell function. Collectively, our results suggest that GC cell-derived exosomes impair the function of Vγ9Vδ2 T cells via miR-135b-5p/SP1 pathway, and targeting exosomal miR-135b-5p/SP1 axis may improve the efficiency of GC immunotherapy based on Vγ9Vδ2 T cells.
Topics: Apoptosis; Cell Proliferation; Exosomes; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Receptors, Antigen, T-Cell, gamma-delta; Sp1 Transcription Factor; Stomach Neoplasms; T-Lymphocytes; Tumor Cells, Cultured; Tumor Microenvironment
PubMed: 34159436
DOI: 10.1007/s00262-021-02991-8