ABSTRACT
Site-specific incorporation of unnatural amino acids (UAAs) with similar incorporation efficiency to that of natural amino acids (NAAs) and low background activity is extremely valuable for efficient synthesis of proteins with diverse new chemical functions and design of various synthetic auxotrophs. However, such efficient translation systems remain largely unknown in the literature. Here, we describe engineered chimeric phenylalanine systems that dramatically increase the yield of proteins bearing UAAs, through systematic engineering of the aminoacyl-tRNA synthetase and its respective cognate tRNA. These engineered synthetase/tRNA pairs allow single-site and multi-site incorporation of UAAs with efficiencies similar to those of NAAs and high fidelity. In addition, using the evolved chimeric phenylalanine system, we construct a series of E. coli strains whose growth is strictly dependent on exogenously supplied of UAAs. We further show that synthetic auxotrophic cells can grow robustly in living mice when UAAs are supplemented.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Directed Molecular Evolution/methods , Escherichia coli/genetics , Phenylalanine/metabolism , Protein Biosynthesis , RNA, Transfer/genetics , Amino Acids/metabolism , Amino Acids/pharmacology , Amino Acyl-tRNA Synthetases/metabolism , Animals , Base Pairing , Biomimetic Materials/metabolism , Biomimetic Materials/pharmacology , Cell Engineering , Escherichia coli/metabolism , Gene Expression , Genes, Reporter , Germ-Free Life , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Nucleic Acid Conformation , Phenylalanine/pharmacology , Plasmids/chemistry , Plasmids/metabolism , RNA, Transfer/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Two novel alkaloids compounds together with fifteen know metabolites were identified from Aspergillus ochraceus. The stereochemistry features of the new molecules were determined via HRESIMS, NMR, ECD, and XRD analyses. Amongst these, compounds two compounds exhibited potential efficacy as anti-Parkinson's disease with the EC50 values of 2.30 and 2.45â µM, respectively. ADMET prediction showed that these compounds owned favorable drug-like characteristics and safe toxicity scores towards CNS drugs. Virtual screening analyses manifested that the compounds exhibited not only robust and reliable interactions to adenosine receptors A2A , but also higher binding selectivity to A2A receptors than to A1 and A3 receptors. Molecular dynamics simulation demonstrated the reliability of molecular docking results and the stability of the complexes obtained with the novel compounds and A2A receptors in natural environments. It is the first time that anti-PD lead compounds have been identified from Aspergillus ochraceus and targeting adenosine A2A receptors.
Subject(s)
Adenosine A2 Receptor Antagonists/pharmacology , Antiparkinson Agents/pharmacology , Aspergillus ochraceus/chemistry , Receptor, Adenosine A2A/metabolism , Adenosine A2 Receptor Antagonists/chemistry , Adenosine A2 Receptor Antagonists/metabolism , Adenosine A2 Receptor Antagonists/pharmacokinetics , Animals , Antiparkinson Agents/chemistry , Antiparkinson Agents/metabolism , Antiparkinson Agents/pharmacokinetics , Cell Line, Tumor , Drug Evaluation, Preclinical , Female , Humans , Male , Mice , Molecular Docking Simulation , Molecular Dynamics Simulation , Neuroprotective Agents/chemistry , Neuroprotective Agents/metabolism , Neuroprotective Agents/pharmacokinetics , Neuroprotective Agents/pharmacology , Rats , StereoisomerismABSTRACT
Hyperjaponol H (1), a new filicinic acid-based meroterpenoid, with a 6/6/10 ring system trans-fused by hetero-Diels-Alder cycloaddition between a germacrane sesquiterpenoid and a filicinic acid moiety, was isolated from aerial parts of Hypericum japonicum. The elucidation of its structure and absolute configuration were accomplished by the analyses of extensive spectroscopic data and the comparison of Cotton effects of electron circular dichroism (ECD) with previously reported ones. The bioactivity assay showed that hyperjaponol H exhibited a moderate inhibitory efficacy on lytic Epstein-Barr virus (EBV) DNA replication in B95-8 cells.
Subject(s)
Butyrophenones/pharmacology , Cyclohexenes/pharmacology , Hypericum/chemistry , Sesquiterpenes, Germacrane/pharmacology , Terpenes/pharmacology , Animals , Antiviral Agents/pharmacology , Butyrophenones/chemistry , Carbon-13 Magnetic Resonance Spectroscopy , Cell Line , Cell Survival/drug effects , Circular Dichroism , Cyclohexenes/chemistry , Ganciclovir/pharmacology , Herpesvirus 4, Human/drug effects , Humans , Proton Magnetic Resonance Spectroscopy , Sesquiterpenes, Germacrane/chemistry , Terpenes/chemistry , Virus Replication/drug effectsABSTRACT
Kaposi's sarcoma associated herpesvirus (KSHV) has gained considerable attention as a type of carcinogenic pathogen. Recent research suggests that KSHV has participated in the pathogenesis of Kaposi's sarcoma-related malignant neoplastic diseases. Viral lytic infection might be pivotal for the etiopathogenesis of KSHV-induced diseases; however, most clinical KSHV lytic replication inhibitors like ganciclovir, nelfinavir, or cidofovir do not restrain virus replication effectively enough to achieve clinical efficacy. In our continued pharmaceutical studies on Chinese herbal medicines, new acylphloroglucinol-based meroterpenoid enantiomers have been discovered from Hypericum japonicum. Most of these metabolites have potential inhibitory activities that target KSHV lytic replication. Amongst these analogues, compounds 1a and 1b possess an unreported ring system cyclopenta[b]chromene. Compounds 1a with 4a exhibit stronger inhibitory activities towards the lytic replication of KSHV in Vero cells. In addition, 1a and 4a have IC50 values of 8.30 and 4.90 µM and selectivity indexes of 23.49 and 25.70, respectively. Qualitative and quantitative SAR and molecular docking studies for acylphloroglucinol-based meroterpenoids with regard to anti-KSHV activity were conducted. An explanation for the variation in the activity and selectivity indexes was proposed in accordance with the predicted binding pose found with molecular docking to a putative target, thymidylate synthase (kTS). Compounds 1a and 4a have potential for further development and optimization of their anti-KSHV activities which could lead to new candidate drugs.
ABSTRACT
Two new glycosides, cinnacassides F (1) and G (2), with a rare geranylphenylacetate carbon skeleton, were isolated from the barks of Cinnamomum cassia, along with three known analogues, cinnacassides A (3), B (4) and C (5). The structures of the new compounds were elucidated on the basis of extensive NMR spectroscopic analyses and chemical method. Compounds 1-5 were investigated for their immunomodulatory activities, and compounds 1, 3 and 4 showed differential immunosuppressive activities against murine lymphocytes.
Subject(s)
Cinnamomum aromaticum/chemistry , Glycosides/chemistry , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Phenylacetates/chemistry , Animals , Drug Evaluation, Preclinical/methods , Glycosides/pharmacology , Lymphocytes/drug effects , Magnetic Resonance Spectroscopy , Mice , Molecular Structure , Phenylacetates/pharmacologyABSTRACT
Two pairs of new enantiomers with unusual 5,5-spiroketal cores, termed (±)-japonones A and B [(±)-1 and (±)-2], were obtained from Hypericum japonicum Thunb. The absolute configurations of (±)-1 and (±)-2 were characterized by extensive analyses of spectroscopic data and calculated electronic circular dichroism (ECD) spectra, the application of modified Mosher's methods, and the assistance of quantum chemical predictions (QCP) of (13)C NMR chemical shifts. Among these metabolites, (+)-1 exhibited some inhibitory activity on Kaposi's sarcoma associated herpesvirus (KSHV). Virtual screening of (±)-1 and (±)-2 were conducted using the Surflex-Dock module in the Sybyl software, and (+)-1 exhibited ability to bind with ERK to form key interactions with residues Lys52, Pro56, Ile101, Asp165, Gly167 and Val99.
Subject(s)
Antiviral Agents/chemistry , Drug Discovery , Herpesvirus 8, Human/drug effects , Hypericum/chemistry , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Circular Dichroism , Herpesvirus 8, Human/pathogenicity , Humans , Molecular Structure , Sarcoma, Kaposi/drug therapy , Sarcoma, Kaposi/virology , StereoisomerismABSTRACT
Hypericum japonicum (Guttiferae), a type of annual or perennial herb, has been historically applied to cure infectious hepatitis, acute and chronic hepatitis, gastrointestinal disorder, and internal hemorrhage. In our successive studies on the genus Hypericum, two new α-pyrones termed japopyrones A and B (1 and 2) were isolated from H. japonicum. Their structures and absolute configurations were established by the comprehensive analyses of spectroscopic data, the application of the Single-crystal X-ray diffraction structural analysis, and the experimental electronic circular dichroism (ECD) spectra. Bioactivity screenings suggested that compound 2 possessed the potential inhibition efficacy on lytic replication of Kaposi's sarcoma associated herpesvirus (KSHV) with an IC50 29.46 µM and a selective index of higher than 6.79, respectively.
Subject(s)
Hypericum/chemistry , Lactones/chemistry , Lactones/isolation & purification , Pyrones/chemistry , Pyrones/isolation & purification , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Cell Line , Circular Dichroism , Herpesvirus 8, Human/drug effects , Humans , Lactones/pharmacology , Molecular Structure , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Pyrones/pharmacology , Virus Replication/drug effects , X-Ray DiffractionABSTRACT
(±)-Japonicols A-D (1a/1b-4a/4b), four pairs of new phloroglucinol-based terpenoid enantiomers, were isolated from Hypericum japonicum. Their absolute configurations were confirmed through comparison of their experimental and calculated electronic circular dichroism spectra and single-crystal X-ray diffraction analyses. Compounds 1a/1b, 2a/2b, and 3a/3b possess 2-oxabicyclo[3.3.1]nonane, pyrano[3,2-b]pyran, and benzo[b]cyclopenta[e]oxepine ring systems, respectively. The effects of the phloroglucinols on anti-Kaposi's sarcoma-associated herpesvirus were assessed, and 2a exhibited a moderate inhibitory effect, with an EC50 value of 8.75 µM and a selectivity index of 16.06.
Subject(s)
Antiviral Agents , Drugs, Chinese Herbal , Herpesvirus 8, Human/drug effects , Hypericum/chemistry , Phloroglucinol , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Crystallography, X-Ray , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Drugs, Chinese Herbal/pharmacology , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Phloroglucinol/analogs & derivatives , Phloroglucinol/chemistry , Phloroglucinol/isolation & purification , Phloroglucinol/pharmacology , StereoisomerismABSTRACT
Seven filicinic acid-based meroterpenoids (1-7), possessing 6/6/11, 6/6/7/5, or 6/6/10 ring systems, were isolated from Hypericum japonicum. All of them have novel skeletons with the incorporation of sesquiterpenoid moieties to an acylated filicinic acid. Compounds 2a and 4 exhibited significant efficacy on anti-Epstein-Barr virus, with EC50 values of 0.57 and 0.49 µM, respectively. Furthermore, compounds 2a and 4 were well accommodated to the binding pocket of 2GV9 predicted by the molecular docking.
Subject(s)
Antiviral Agents/pharmacology , Cyclohexenes/pharmacology , Herpesvirus 4, Human/drug effects , Hypericum/chemistry , Terpenes/pharmacology , Animals , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Cell Line, Tumor , Cyclohexenes/chemistry , Cyclohexenes/isolation & purification , Dose-Response Relationship, Drug , Microbial Sensitivity Tests , Molecular Conformation , Molecular Docking Simulation , Structure-Activity Relationship , Terpenes/chemistry , Terpenes/isolation & purificationABSTRACT
Zanthoxylum schinifolium Sieb. et Zucc. (Rutaceae), a dioecious shrub with hooked prickly branches, has been used as folk medicine for the treatment of the common cold, stomach ache, diarrhea, and jaundice in China, Korea, and Japan. In our phytochemical investigations on this genus, a new megastigmane sesquiterpenoid, which is referred to as schinifolenol A (1), was isolated from Z. schinifolium. The stereochemistry was characterized via the analyses of extensive spectra. The absolute configuration was established by the application of a modified Mosher's experiment and assisted by a time-dependent density functional theory (TD-DFT) on calculated electronic circular dichroism (ECD). Bioactivity screenings showed that compound 1 exhibited a safe hypotoxicity and a better selectivity on anti-Kaposi's sarcoma associated herpes virus (KSHV).