ABSTRACT
Hitachimycin is a bicyclic macrolactam antibiotic with (S)-ß-phenylalanine (ß-Phe) at the starter position of the polyketide skeleton. While the enzymes that recognize ß-amino acids, modify the aminoacyl groups, and transfer the resultant dipeptide groups to the acyl carrier protein domains of polyketide synthases (PKSs) have been studied extensively, the post-PKS modification mechanism responsible for constructing the unique bicyclic structure of hitachimycin remains elusive. In this study, we first inactivated six genes encoding putative post-PKS modification enzymes, namely hitM1 to hitM6, in Streptomyces scabrisporus to determine their involvement in hitachimycin biosynthesis. The ΔhitM4 strain accumulated an all-trans-2,4,6,8,18-pentaene macrolactam, which was confirmed as a true intermediate in hitachimycin biosynthesis by cellular feeding experiments, and appears to be the initial intermediate in the post-PKS modification pathway. The ΔhitM1 strain accumulated 10-O-demethyl-10-oxohitachimycin (M1-A). In enzymatic experiments, M1-A was reduced by the NAD(P)H-dependent reductase HitM1 in the presence of NADPH. The product of the reaction catalyzed by HitM1 was converted to hitachimycin by the methyltransferase HitM6. We thus propose a plausible post-PKS modification mechanism for the biosynthesis of hitachimycin.
Subject(s)
Polyketide Synthases , Streptomyces , Streptomyces/enzymology , Streptomyces/metabolism , Streptomyces/genetics , Polyketide Synthases/metabolism , Polyketide Synthases/genetics , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/chemistry , Molecular StructureABSTRACT
Obtaining a microorganism strain with a broad-spectrum resistance property and highly efficient antifungal activity is important to the biocontrol strategy. Herein, a marine Streptomyces sp. HNBCa1 demonstrated a broad-spectrum resistance to 17 tested crop pathogenic fungi and exhibited a high biocontrol efficiency against mango anthracnose and banana fusarium wilt. To uncover the critical bioactive secondary metabolites basis, genome assembly and annotation, metabolomic analysis, and a semipreparative HPLC-based activity-guide method were employed. Finally, geldanamycin and ectoine involved in codifferential secondary metabolites were also found to be related to biosynthetic gene clusters in the genome of HNBCa1. Reblastatin and geldanamycin were uncovered in response to broad-spectrum resistance to the 17 crop pathogenic fungi. Our results suggested that reblastatin and geldanamycin were critical to maintaining the broad-spectrum resistance property and highly efficient antifungal activity of HNBCa1, which could be further developed as a biological control agent to control crop fungal diseases.
Subject(s)
Fusarium , Lactams, Macrocyclic , Plant Diseases , Secondary Metabolism , Streptomyces , Streptomyces/genetics , Streptomyces/metabolism , Streptomyces/chemistry , Plant Diseases/microbiology , Lactams, Macrocyclic/pharmacology , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/chemistry , Fusarium/drug effects , Benzoquinones/pharmacology , Benzoquinones/metabolism , Benzoquinones/chemistry , Fungi/genetics , Antifungal Agents/pharmacology , Antifungal Agents/metabolism , Antifungal Agents/chemistryABSTRACT
Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower (P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower (P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased (P < 0.05) the blastocysts rates. In the third experiment, the association of 2 µM 17AAG with HS for 12 h during IVM resulted in lower (P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.
Subject(s)
In Vitro Oocyte Maturation Techniques , Oocytes , Cattle , Animals , In Vitro Oocyte Maturation Techniques/methods , Oocytes/physiology , Lactams, Macrocyclic/pharmacology , Lactams, Macrocyclic/metabolism , Heat-Shock Response , Blastocyst/physiology , HSP90 Heat-Shock Proteins/geneticsABSTRACT
Mitochondria are key tumor drivers, but their suitability as a therapeutic target is unknown. Here, we report on the preclinical characterization of Gamitrinib (GA mitochondrial matrix inhibitor), a first-in-class anticancer agent that couples the Heat Shock Protein-90 (Hsp90) inhibitor 17-allylamino-geldanamycin (17-AAG) to the mitochondrial-targeting moiety, triphenylphosphonium. Formulated as a stable (≥24 weeks at -20°C) injectable suspension produced by microfluidization (<200 nm particle size), Gamitrinib (>99.5% purity) is heavily bound to plasma proteins (>99%), has intrinsic clearance from liver microsomes of 3.30 mL/min/g and minimally penetrates a Caco-2 intestinal monolayer. Compared to 17-AAG, Gamitrinib has slower clearance (85.6 ± 5.8 mL/min/kg), longer t1/2 (12.2 ± 1.55 h), mean AUC0-t of 783.1 ± 71.3 hâng/mL, and unique metabolism without generation of 17-AG. Concentrations of Gamitrinib that trigger tumor cell killing (IC50 ~1-4 µM) do not affect cytochrome P450 isoforms CYP1A2, CYP2A6, CYP2B6, CYP2C8 or ion channel conductance (Nav1.5, Kv4.3/KChIP2, Cav1.2, Kv1.5, KCNQ1/mink, HCN4, Kir2). Twice weekly IV administration of Gamitrinib to Sprague-Dawley rats or beagle dogs for up to 36 d is feasible. At dose levels of up to 5 (rats)- and 12 (dogs)-fold higher than therapeutically effective doses in mice (10 mg/kg), Gamitrinib treatment is unremarkable in dogs with no alterations in clinical-chemistry parameters, heart function, or tissue histology, and causes occasional inflammation at the infusion site and mild elevation of serum urea nitrogen in rats (≥10 mg/kg/dose). Therefore, targeting mitochondria for cancer therapy is feasible and well tolerated. A publicly funded, first-in-human phase I clinical trial of Gamitrinib in patients with advanced cancer is ongoing (ClinicalTrials.gov NCT04827810).
Subject(s)
Antineoplastic Agents , Mitochondria , Neoplasms , Animals , Antineoplastic Agents/adverse effects , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Benzoquinones/metabolism , Benzoquinones/pharmacology , Caco-2 Cells , Clinical Trials, Phase I as Topic , Dogs , Feasibility Studies , HSP90 Heat-Shock Proteins/metabolism , Humans , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/pharmacology , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Neoplasms/drug therapy , Rats , Rats, Sprague-DawleyABSTRACT
The effect of transporters and enzymes on drug pharmacokinetics is increasingly evaluated using genetically modified animals that have these proteins either knocked-out or their human orthologues transgenically expressed. Analysis of pharmacokinetic data obtained in such experiments is typically performed using non-compartmental analysis (NCA), which has limitations such as not being able to identify the PK parameter that is affected by the genetic modification of the enzymes or transporters and the requirement of intense and homogeneous sampling of all subjects. Here we used a compartmental population pharmacokinetic modeling approach using PK data from a series of genetically modified mouse experiments with lorlatinib to extend the results and conclusions from previously reported NCA analyses. A compartmental population pharmacokinetic model was built and physiologically plausible covariates were evaluated for the different mouse strains. With the model, similar effects of the strains on the area under the concentration-time curve (AUC) from 0 to 8 hours were found as for the NCA. Additionally, the differences in AUC between the strains were explained by specific effects on clearance and bioavailability for the strain with human expressing CYP3A4. Finally, effects of multidrug efflux transporters ATP-binding cassette (ABC) sub-family B member 1 (ABCB1) and G member 2 (ABCG2) on brain efflux were quantified. Use of compartmental population PK modeling yielded additional insight into the role of drug-metabolizing enzymes and drug transporters in mouse experiments compared to the NCA. Furthermore, these models allowed analysis of heterogeneous pooled datasets and the sparse organ concentration data in contrast to classical NCA analyses.
Subject(s)
Lactams , Pyrazoles , Aminopyridines , Animals , Humans , Lactams, Macrocyclic/metabolism , MiceABSTRACT
Cancer stem cells (CSCs) are nowadays one of the major focuses in tumor research since this subpopulation was revealed to be a great obstacle for successful treatment. The identification of CSCs in pediatric solid tumors harbors major challenges because of the immature character of these tumors. Here, we present CD34, CD90, OV-6 and cell-surface vimentin (csVimentin) as reliable markers to identify CSCs in hepatoblastoma cell lines. We were able to identify CSC characteristics for the subset of CD34+CD90+OV-6+csVimentin+-co-expressing cells, such as pluripotency, self-renewal, increased expression of EMT markers and migration. Treatment with Cisplatin as the standard chemotherapeutic drug in hepatoblastoma therapy further revealed the chemo-resistance of this subset, which is a main characteristic of CSCs. When we treated the cells with the Hsp90 inhibitor 17-AAG, we observed a significant reduction in the CSC subset. With our study, we identified CSCs of hepatoblastoma using CD34, CD90, OV-6 and csVimentin. This set of markers could be helpful to estimate the success of novel therapeutic approaches, as resistant CSCs are responsible for tumor relapses.
Subject(s)
Antigens, CD34/metabolism , Antigens, Differentiation/metabolism , Benzoquinones/metabolism , Hepatoblastoma/genetics , Lactams, Macrocyclic/metabolism , Liver Neoplasms/genetics , Neoplastic Stem Cells/metabolism , Thy-1 Antigens/metabolism , Vimentin/metabolism , Hepatoblastoma/pathology , Humans , Liver Neoplasms/pathologyABSTRACT
We have previously shown deep-sea-derived Streptomyces koyangensis SCSIO 5802 to produce two types of active secondary metabolites, abyssomicins and candicidins. Here, we report the complete genome sequence of S. koyangensis SCSIO 5802 employing bioinformatics to highlight its potential to produce at least 21 categories of natural products. In order to mine novel natural products, the production of two polycyclic tetramate macrolactams (PTMs), the known 10-epi-HSAF (1) and a new compound, koyanamide A (2), was stimulated via inactivation of the abyssomicin and candicidin biosynthetic machineries. Detailed bioinformatics analyses revealed a PKS/NRPS gene cluster, containing 6 open reading frames (ORFs) and spanning ~16 kb of contiguous genomic DNA, as the putative PTM biosynthetic gene cluster (BGC) (termed herein sko). We furthermore demonstrate, via gene disruption experiments, that the sko cluster encodes the biosynthesis of 10-epi-HSAF and koyanamide A. Finally, we propose a plausible biosynthetic pathway to 10-epi-HSAF and koyanamide A. In total, this study demonstrates an effective approach to cryptic BGC activation enabling the discovery of new bioactive metabolites; genome mining and metabolic profiling methods play key roles in this strategy.
Subject(s)
Lactams, Macrocyclic/metabolism , Streptomyces , Aquatic Organisms , Genome , Humans , Multigene Family , Phytotherapy , Whole Genome SequencingABSTRACT
Polycyclic tetramate macrolactams (PoTeMs) are a fast-growing family of antibiotic natural products found in phylogenetically diverse microorganisms. Surprisingly, none of the PoTeMs have been investigated for potential physiological functions in their producers. Here, we used heat-stable antifungal factor (HSAF), an antifungal PoTeM from Lysobacter enzymogenes, as a model to show that PoTeMs form complexes with iron ions, with an association constant (Ka ) of 2.71 × 106 M-1 The in vivo and in vitro data showed formation of 2:1 and 3:1 complexes between HSAF and iron ions, which were confirmed by molecular mechanical and quantum mechanical calculations. HSAF protected DNA from degradation in high concentrations of iron and H2O2 or under UV radiation. HSAF mutants of L. enzymogenes barely survived under oxidative stress and exhibited markedly increased production of reactive oxygen species (ROS). Exogenous addition of HSAF into the mutants significantly prevented ROS production and restored normal growth in the mutants under the oxidative stress. The results reveal that the function of HSAF is to protect the producer microorganism from oxidative damage rather than as an iron-acquisition siderophore. The characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. The study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells.IMPORTANCE PoTeMs are a family of structurally distinct metabolites that have been found in a large number of bacteria. Although PoTeMs exhibit diverse therapeutic properties, the physiological function of PoTeMs in the producer microorganisms had not been investigated. HSAF from Lysobacter enzymogenes is an antifungal PoTeM that has been subjected to extensive studies for mechanisms of biosynthesis, regulation, and antifungal activity. Using HSAF as a model system, we here showed that the characteristic structure of PoTeMs, a 2,4-pyrrolidinedione-embedded macrolactam, may represent a new iron-chelating scaffold of microbial metabolites. In L. enzymogenes, HSAF functions as a small-molecule modulator for oxidative damage caused by iron, H2O2, and UV light. Together, the study demonstrated a previously unrecognized strategy for microorganisms to modulate oxidative damage to the cells. HSAF represents the first member of the fast-growing PoTeM family of microbial metabolites whose potential biological function has been studied.
Subject(s)
Lactams, Macrocyclic/metabolism , Lysobacter/metabolism , Ferrous Compounds/pharmacology , Hydrogen Peroxide/pharmacology , Lysobacter/drug effects , Lysobacter/radiation effects , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , Reactive Oxygen Species/metabolism , Ultraviolet RaysABSTRACT
Covering from 1992 to the end of 2020-11-20.Genetically-encoded polyenic macrolactams, which are constructed by Nature using hybrid polyketide synthase/nonribosomal peptide synthase (PKSs/NRPSs) assembly lines, are part of the large collection of natural products isolated from bacteria. Activation of cryptic (i.e., silent) gene clusters in these microorganisms has more recently allowed to generate and eventually isolate additional members of the family. Having two unsaturated fragments separated by short saturated chains, the primary macrolactam is posited to undergo transannular reactions and further rearrangements thus leading to the generation of a structurally diverse collection of polycyclic (natural) products and oxidized derivatives. The review will cover the challenges that scientists face on the isolation of these unstable compounds from the cultures of the producing microorganisms, their structural characterization, biological activities, optimized biogenetic routes, as well as the skeletal rearrangements of the primary structures of the natural macrolactams derived from pericyclic reactions of the polyenic fragments. The efforts of the synthetic chemists to emulate Nature on the successful generation and structural confirmation of these natural products will also be reported.
Subject(s)
Actinobacteria , Biological Products/metabolism , Lactams, Macrocyclic/metabolism , Actinobacteria/enzymology , Actinobacteria/genetics , Molecular Structure , Peptide Synthases/metabolism , Polyketide Synthases/metabolism , Secondary MetabolismABSTRACT
Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.
Subject(s)
Biological Products/metabolism , Lactams, Macrocyclic/metabolism , Multigene Family , Streptomyces/genetics , Organisms, Genetically Modified , Streptomyces/metabolismABSTRACT
A novel 17-allylamino-17-demethoxygeldanamycin (17-AAG) glucoside (1) was obtained from in vitro enzymatic glycosylation using a UDP-glycosyltransferase (YjiC). The water-solubility of compound 1 was approximately 10.5 times higher than that of the substrate, 17-AAG. Compound 1 showed potential anti-proliferative activities against five human cancer cell lines, with IC50 values ranging from 5.26 to 28.52 µM. Further studies also indicated that compound 1 could inhibit the growth of CNE-2Z cells by inducing the degradation of Hsp90 client proteins (Akt, c-Raf, Bcl-2, and HIF-1α). In addition, compound 1 showed greater potential anti-tumor efficacy than 17-AAG in nude mice xenografted with CNE-2Z cells. Therefore, we suggest that in vitro enzymatic glycosylation is a powerful approach for the structural optimization of 17-AAG.
Subject(s)
Antineoplastic Agents/pharmacology , Benzoquinones/pharmacology , Glucosides/pharmacology , Glycosyltransferases/metabolism , Lactams, Macrocyclic/pharmacology , Uridine Diphosphate/metabolism , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Benzoquinones/chemistry , Benzoquinones/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Glucosides/biosynthesis , Glucosides/chemistry , Glycosylation , Humans , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/metabolism , Mice , Mice, Nude , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Structure-Activity RelationshipABSTRACT
17-N-allylamino-17-demethoxygeldanamycin (17-AAG, tanespimycin) is an inhibitor of heat shock protein 90 (Hsp90), which has been studied in the treatment of cancer such as leukemia or solid tumors. Alternatively, 17-AAG may represent a promising therapeutic agent against leishmaniasis. However, the delivery of 17-AAG is difficult due to its poor aqueous solubility. For exploring the therapeutic value of 17-AAG, we developed solid lipid nanoparticles (SLN) by double emulsion method. SLN exhibited ~100 nm, PDI < 0.2 and zeta potential ~20 mV. In addition, SLN were morphologically spherical with negligible aggregation. The entrapment efficiency of 17-AAG into the lipid matrix reached at nearly 80%. In a separate set of experiments, fluorescent SLN (FITC-labeled) showed a remarkable macrophage uptake, peaking within 2 h of incubation by confocal microscopy. Regarding the drug internalization as critical step for elimination of intracellular Leishmania, this finding demonstrates an important feature of the developed SLN. Collectively, these data indicate the feasibility of developing SLN as potential delivery systems for 17-AAG in leishmaniasis chemotherapy.
Subject(s)
Benzoquinones/metabolism , Benzoquinones/pharmacology , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/pharmacology , Lipids/chemistry , Macrophages/metabolism , Nanoparticles/chemistry , Animals , Benzoquinones/administration & dosage , Benzoquinones/chemistry , Drug Carriers/therapeutic use , HSP90 Heat-Shock Proteins/therapeutic use , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/chemistry , Leishmania , Leishmaniasis/drug therapy , Molecular Structure , SolubilityABSTRACT
While an initial clinical absorption, distribution, metabolism, and excretion (ADME) study (Study 1; N = 6) with 100 mg/100 µCi [14 C]lorlatinib, radiolabeled on the carbonyl carbon, confirmed that the primary metabolic pathways for lorlatinib are oxidation (N-demethylation, N-oxidation) and N-glucuronidation, it also revealed an unanticipated, intramolecular cleavage metabolic pathway of lorlatinib, yielding a major circulating benzoic acid metabolite (M8), and an unlabeled pyrido-pyrazole substructure. Concerns regarding the fate of unknown metabolites associated with this intramolecular cleavage pathway led to conduct of a second ADME study (Study 2; N = 6) of identical design but with the radiolabel positioned on the pyrazole ring. Results were similar with respect to the overall mass balance, lorlatinib plasma exposures, and metabolic profiles in excreta for the metabolites that retained the radiolabel in both studies. Differences were observed in plasma total radioactivity exposures (2-fold area under the plasma concentration-time curve from time 0 to infinity difference) and relative ratios of the percentage of dose recovered in urine vs feces (48% vs 41% in Study 1; 28% vs 64% in Study 2). In addition, an approximately 3-fold difference in the mean molar exposure ratio of M8 to lorlatinib was observed for values derived from metabolic profiling data relative to those derived from specific bioanalytical methods (0.5 vs 1.4 for Studies 1 and 2, respectively). These interstudy differences were attributed to a combination of factors, including alteration of radiolabel position, orthogonal analytical methodologies, and intersubject variability, and illustrate that results from clinical ADME studies are not unambiguous and should be interpreted within the context of the specific study design considerations.
Subject(s)
Carbon Radioisotopes/metabolism , Carbon Radioisotopes/pharmacokinetics , Lactams, Macrocyclic/metabolism , Lactams, Macrocyclic/pharmacokinetics , Administration, Oral , Adolescent , Adult , Aminopyridines , Biotransformation , Carbon Radioisotopes/administration & dosage , Carbon Radioisotopes/chemistry , Drug Elimination Routes , Feces/chemistry , Healthy Volunteers , Humans , Lactams , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/chemistry , Male , Middle Aged , Models, Biological , Pyrazoles , Solutions , Young AdultABSTRACT
Hepatitis C virus, causative agent of chronic viral hepatitis, infects 71 million people worldwide and is divided into seven genotypes and multiple subtypes with sequence identities between 68 to 82%. While older generation direct-acting antivirals had varying effectiveness against different genotypes, the newest NS3/4A protease inhibitors including glecaprevir (GLE) have pan-genotypic activity. The structural basis for pan-genotypic inhibition and effects of polymorphisms on inhibitor potency were not well-known due to lack of crystal structures of GLE-bound NS3/4A or genotypes other than 1. In this study, we determined the crystal structures of NS3/4A from genotypes 1a, 3a, 4a, and 5a in complex with GLE. Comparison with the highly similar grazoprevir indicated the mechanism of GLE's drastic improvement in potency. We found that, while GLE is highly potent against wild-type NS3/4A of all genotypes, specific resistance-associated substitutions (RASs) confer orders of magnitude loss in inhibition. Our crystal structures reveal molecular mechanisms behind pan-genotypic activity of GLE, including potency loss due to RASs at D168. Our structures permit for the first time analysis of changes due to polymorphisms among genotypes, providing insights into design principles that can aid future drug development and potentially can be extended to other proteins.
Subject(s)
Aminoisobutyric Acids/metabolism , Antiviral Agents/metabolism , Cyclopropanes/metabolism , Hepacivirus/enzymology , Lactams, Macrocyclic/metabolism , Leucine/analogs & derivatives , Proline/analogs & derivatives , Quinoxalines/metabolism , Serine Proteases/metabolism , Serine Proteinase Inhibitors/metabolism , Sulfonamides/metabolism , Viral Nonstructural Proteins/metabolism , Amides/chemistry , Amides/metabolism , Aminoisobutyric Acids/chemistry , Antiviral Agents/chemistry , Carbamates/chemistry , Carbamates/metabolism , Catalytic Domain , Crystallography, X-Ray , Cyclopropanes/chemistry , Lactams, Macrocyclic/chemistry , Leucine/chemistry , Leucine/metabolism , Mutation , Proline/chemistry , Proline/metabolism , Protein Binding , Quinoxalines/chemistry , Serine Proteases/chemistry , Serine Proteases/genetics , Serine Proteinase Inhibitors/chemistry , Sulfonamides/chemistry , Viral Nonstructural Proteins/chemistry , Viral Nonstructural Proteins/geneticsABSTRACT
More than two-third of known antibiotics are produced by actinomycetes of the genus Streptomyces. Unfortunately, the production rate from Streptomyces natural antibiotic is extremely slow and thus cannot satisfy industrial demand. In this study, the production of antibiotics by Streptomyces is enhanced by a "superplasmid" which including global regulatory factors afsR, cyclic adenosine receptor protein (CRP), RNA polymerase beta subunits (rpoB) with point mutation and acetyl coenzyme A carboxylase gene (accA2BE), these elements are controlled by the PermE* promoter and then transfer into Streptomyces coelicolor M145, Streptomyces mutabilis TRM45540, Streptomyces hygroscopicus XM201, and Streptomyces hygroscopicus ATCC29253 by conjugation to generate exconjugants. NMR, HPLC, and LC-MS analyses revealed that the superplasmid led to the overproduction of actinorhodin (101.90%), undecylprodigiosin (181.60%) in S. coelicolor M145:: pLQ003, of rapamycin (110%), hygrocin A (163.4%) in S. hygroscopicus ATCC29253:: pLQ003, and of actinomycin D (11.78%) in S. mutabilis TRM45540:: pLQ003, and also to the downregulation of geldanamycin in S. hygroscopicus XM201, but we found that mutant strains in mutant strains of S. hygroscopicus XM201 with regulatory factors inserted showed several peaks that were not found in wild-type strains. The results of the present work indicated that the regulator net working in Streptomyces was not uniform, the superplasmid we constructed possibly caused this overproduction and downregulation in different Streptomyces.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Genetic Engineering/methods , Plasmids/genetics , Streptomyces/genetics , Acetyl-CoA Carboxylase/genetics , Bacteria/drug effects , Benzoquinones/metabolism , Dactinomycin/biosynthesis , Lactams, Macrocyclic/metabolism , Point Mutation , Promoter Regions, GeneticABSTRACT
Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.
Subject(s)
Biological Products/metabolism , Lactams, Macrocyclic/metabolism , Multigene Family , Organisms, Genetically Modified , Streptomyces/metabolismABSTRACT
Three new polycyclic macrolactams, cyclamenols B-D (1-3), together with a known macrolactam, cyclamenol A (4), were isolated from the Streptomyces sp. OUCMDZ-4348. Their structures including absolute configurations were determined on the basis of spectroscopic analysis, chemical methods, and ECD calculations. The biosynthetic pathways involving intramolecular Diels-Alder reactions were proposed. Compound 1 exhibited selective inhibition against the gastric carcinoma cell line N87 with an IC50 value of 10.8 µM.
Subject(s)
Lactams, Macrocyclic/metabolism , Lactams/metabolism , Streptomyces/chemistry , Antarctic Regions , Cycloaddition Reaction , Lactams/chemistry , Lactams/isolation & purification , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/isolation & purification , Models, Molecular , Molecular Conformation , Species SpecificityABSTRACT
Genome mining of the marine Streptomyces seoulensis A01 enabled the identification of a giant type I polyketide synthase gene cluster ( asm). Heterologous expression of the cryptic asm cluster using a bacterial artificial chromosome vector in heterologous host led to the production of ansaseomycins A (1) and B (2). A plausible biosynthetic pathway was also proposed. Additionally, compounds 1 and 2 are active against K562 cell lines with IC50 values of 13.3 and 18.1 µM, respectively.
Subject(s)
Lactams, Macrocyclic/metabolism , Polyketide Synthases/metabolism , Streptomyces/metabolism , Biosynthetic Pathways , Chromosomes, Artificial, Bacterial , Lactams, Macrocyclic/chemistry , Molecular Structure , Multigene Family , Polyketide Synthases/chemistry , Polyketide Synthases/genetics , Streptomyces/chemistry , Streptomyces/geneticsABSTRACT
Bombyxamycins A and B (1 and 2) were discovered from a silkworm gut Streptomyces bacterium. Spectroscopic analysis and multiple-step chemical derivatization identified them as 26-membered cyclic lactams with polyene features. Bombyxamycin A showed significant antibacterial and antiproliferative effects. The bombyxamycin biosynthetic gene cluster was identified by genetic analysis. Gene deletion experiments confirmed that the cytochrome P450 BomK is responsible for the generation of 2, which unprecedentedly bears tetrahydrofuran in its macrocyclic ring.
Subject(s)
Bombyx/chemistry , Cytochrome P-450 Enzyme System/genetics , Intestines/physiology , Lactams, Macrocyclic/isolation & purification , Animals , Cytochrome P-450 Enzyme System/chemistry , Lactams, Macrocyclic/chemistry , Lactams, Macrocyclic/metabolism , Molecular Structure , Multigene FamilyABSTRACT
Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC0-8h in CYP3A4-humanized mice was â¼1.8-fold lower than in wild-type and Cyp3a-/- mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC0-8h levels seen in wild-type and Cyp3a-/- mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8â¯h in wild-type, Cyp3a-/-, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.