Expression of Syo_1.56 SARP Regulator Unveils Potent Elasnin Derivatives with Antibacterial Activity.

Actinomycetes are prolific producers of natural products, particularly antibiotics. However, a significant proportion of its biosynthetic gene clusters (BGCs) remain silent under typical laboratory conditions. This limits the effectiveness of conventional isolation methods for the discovery of novel natural products. Genetic interventions targeting the activation of silent gene clusters are necessary to address this challenge. Streptomyces antibiotic regulatory proteins (SARPs) act as cluster-specific activators and can be used to target silent BGCs for the discovery of new antibiotics. In this study, the expression of a previously uncharacterized SARP protein, Syo_1.56, in Streptomyces sp. RK18-A0406 significantly enhanced the production of known antimycins and led to the discovery of 12 elasnins (1-12), 10 of which were novel. The absolute stereochemistry of elasnin A1 was assigned for the first time to be 6S. Unexpectedly, Syo_1.56 seems to function as a pleiotropic rather than cluster-specific SARP regulator, with the capability of co-regulating two distinct biosynthetic pathways, simultaneously. All isolated elasnins were active against wild-type and methicillin-resistant Staphylococcus aureus with IC50 values of 0.5-20 µg/mL, some of which (elasnins A1, B2, and C1 and proelasnins A1, and C1) demonstrated moderate to strong antimalarial activities against Plasmodium falciparum 3D7. Elasnins A1, B3, and C1 also showed in vitro inhibition of the metallo-ß-lactamase responsible for the development of highly antibiotic-resistant bacterial strains.

Production of Kinanthraquinone D with Antimalarial Activity by Heterologous Gene Expression and Biotransformation in Streptomyces lividans TK23.

Two new compounds, kinanthraquinone C (1) and kinanthraquinone D (2), were isolated along with two known compounds, kinanthraquinone (3) and kinanthraquinone B (4), produced by the heterologous expression of the kiq biosynthetic gene cluster and its pathway-specific regulator, kiqA, in Streptomyces lividans TK23. The chemical structures of compounds 1 and 2 were determined using mass spectrometry and nuclear magnetic resonance analyses. To examine a biosynthetic pathway of compounds 1 and 2, incubation experiments were conducted using S. lividans TK23 to supply the compounds 3 and 4. These experiments indicated that compounds 3 and 4 were converted to compounds 2 and 1, respectively, by the endogenous enzymes of S. lividans TK23. Compounds 2, 3, and 4 had antimalarial activities at half-maximal inhibitory concentration values of 0.91, 1.2, and 15 µM, respectively, without cytotoxicity up to 30 µM.

Identification of microbial metabolites that accelerate the ubiquitin-dependent degradation of c-Myc.

Myc belongs to a family of proto-oncogenes that encode transcription factors. The overexpression of c-Myc causes many types of cancers. Recently, we established a system for screening c-Myc inhibitors and identified antimycin A by screening the RIKEN NPDepo chemical library. The specific mechanism of promoting tumor cell metastasis by high c-Myc expression remains to be explained. In this study, we screened approximately 5,600 microbial extracts using this system and identified a broth prepared from Streptomyces sp. RK19-A0402 strongly inhibits c-Myc transcriptional activity. After purification of the hit broth, we identified compounds closely related to the aglycone of cytovaricin and had a structure similar to that of oligomycin A. Similar to oligomycin A, the hit compounds inhibited mitochondrial complex V. The mitochondria dysfunction caused by the compounds induced the production of reactive oxygen species (ROS), and the ROS activated GSK3α/ß that phosphorylated c-Myc for ubiquitination. This study provides a successful screening strategy for identifying natural products as potential c-Myc inhibitors as potential anticancer agents.

cis-Decalin-containing tetramic acids as inhibitors of insect steroidogenic glutathione S-transferase Noppera-bo.

Decalin-containing tetramic acid is a bioactive scaffold primarily produced by filamentous fungi. The structural diversity of this group of compounds is generated by characteristic enzymes of fungal biosynthetic pathways, including polyketide synthase/nonribosomal peptide synthetase hybrid enzymes and decalin synthase, which are responsible for the construction of a linear polyenoyl tetramic acid structure and stereoselective decalin formation via the intramolecular Diels-Alder reaction, respectively. Compounds that differed only in the decalin configuration were collected from genetically engineered mutants derived from decalin-containing tetramic acid-producing fungi and used for a structure-activity relationship study. Our evaluation of biological activities, such as cytotoxicity against several cancer cell lines and antibacterial, antifungal, antimalarial, and mitochondrial inhibitory activities, demonstrated that the activity for each assay varies depending on the decalin configurations. In addition to these known biological activities, we revealed that the compounds showed inhibitory activity against the insect steroidogenic glutathione S-transferase Noppera-bo. Engineering the decalin configurations would be useful not only to find derivatives with better biological activities but also to discover overlooked biological activities.

Wakodecaline C, new tetrahydrofuran-fused decalin metabolite isolated from fungus Pyrenochaetopsis sp. RK10-F058.

A new decalin-containing secondary metabolite, wakodecaline C, was isolated from a fungus Pyrenochaetopsis sp. RK10-F058 by screening structurally interesting metabolites based on LC/MS profiling. The structure including the absolute configuration was determined by a combination of spectroscopic methods including NMR and mass spectrometry, chemical reaction, and calculation of ECD spectra. Wakodecaline C has unique structural features containing a tetrahydrofuran-fused decalin skeleton and tetramic acid moiety, which are connected through a double bond. The compound showed moderate cytotoxicity against HL-60 cells and antimalarial activity against the Plasmodium falciparum 3D7 strain.

Naturally Occurring 8ß,13ß-kaur-15-en-17-al and Anti-Malarial Activity from Podocarpus polystachyus Leaves.

Despite much interest and studies toward the genus Podocarpus, the anti-malarial evaluation of Podocarpus polystachyus's phytoconstituents remains lacking. Herein, the phytoconstituents of P. polystachyus leaves and their anti-malarial effect against Plasmodium falciparum were investigated for the first time. One new natural product, 8ß,13ß-kaur-15-en-17-al (1), along with three known compounds, 8ß,13ß-kaur-15-en-17-ol (2) and 13ß-kaur-16-ene (3), and α-tocopherol hydroquinone (4) were isolated via HR-ESI-MS and NMR analyses. Compounds 1 and 2 inhibited P. falciparum growth at 12 and 52 µM of IC50, respectively. Their anti-malarial activity was associated with the in silico P. falciparum lactate dehydrogenase (PfLDH) inhibition. Molecular docking of ligands 1 and 2 with the putative target PfLDH revealed ~-2 kcal/mol of binding energies more negative than the control. Molecular dynamic simulations (100 ns) showed equal or smaller deviation values (RMSD, RMSF, Rg) and stronger interactions of PfLDH-1 and PfLDH-2 complexes via at least one consistent H-bond than the control. Additionally, a slightly increased PfLDH H-bond profile in their interactions improved the PfLDH dynamic and structural stabilities. Overall, this study supports the relevance of 1 and 2 as plasmodial growth inhibitors with their putative anti-PfLDH activity, which could be a potential scaffold for developing anti-malarial drugs.

Phenylboronic Ester-Activated Aryl Iodide-Selective Buchwald-Hartwig-Type Amination toward Bioactivity Assay.

In this study, a phenylboronic ester-activated aryl iodide-selective Buchwald-Hartwig-type amination was developed. When the reaction of aryl iodides and aryl/aliphatic amines using Ni(acac)2 is carried out in the presence of phenylboronic ester, the Buchwald-Hartwig-type amination proceeds smoothly to afford the corresponding amines in high yields. This reaction does not proceed in the absence of phenylboronic ester. A wide variety of aryl iodides can be applied in the presence of aryl chlorides and bromides, which remain intact during the reaction. The mechanistic studies of this reaction suggest that the phenylboronic ester acts as an activator for the amines to form the ″ate complex″. Chemical kinetics studies show that the reaction of aryl iodides, base, and Ni(acac)2 follows first-order kinetics, while that of amines and phenylboronic ester follows zero-order kinetics. The bioactivity screening of the corresponding products showed that some amination products exhibit antifungal activity.

New antimalarials identified by a cell-based phenotypic approach: Structure-activity relationships of 2,3,4,9-tetrahydro-1H-ß-carboline derivatives possessing a 2-((coumarin-5-yl)oxy)alkanoyl moiety.

The identification, structure-activity relationships (SARs), and biological effects of new antimalarials consisting of a 2,3,4,9-tetrahydro-1H-ß-carboline core, a coumarin ring, and an oxyalkanoyl linker are described. A cell-based phenotypic approach was employed in this search for novel antimalarial drugs with unique modes of action. Our screening campaign of the RIKEN compound library succeeded in the identification of the known tetrahydro-ß-carboline derivative (4e) as a hit compound showing significant in vitro activity. SAR studies on this chemical series led to the discovery of compound 4h having a (R)-methyl group on the oxyacetyl linker with potent inhibition of parasite growth (IC50 = 2.0 nM). Compound 4h was also found to exhibit significant in vivo antimalarial effects in mouse models. Furthermore, molecular modeling studies on 4e, 4h, and its diastereomer (4j) suggested that the (R)-methyl group of 4h forces the preferential adoption of a specific conformer which is considered to be an active conformer.

Isolation of new lucilactaene derivatives from P450 monooxygenase and aldehyde dehydrogenase knockout Fusarium sp. RK97-94 strains and their biological activities.

Fusarium sp. RK97-94 is a producer of potent antimalarial compounds such as lucilactaene and its derivatives. The biosynthetic gene cluster of lucilactaene was identified but only a knockout mutant of methyltransferase (luc1) was reported in previous papers. Herein, we report on isolation and identification of prelucilactaene G (1), and prelucilactaene H (2) from the aldehyde dehydrogenase knockout strain (∆luc3) culture broth, as well as prelucilactaene A (3), prelucilactaene B (4), and two isomeric mixtures of prelucilactaene E (5) and prelucilactaene F (6), from the P450 monooxygenase knockout strain (∆luc2) culture broth. Our data, unlike the previous ones, suggest the involvement of the aldehyde dehydrogenase (Luc3) in lucilactaene biosynthesis, and support the involvement of the P450 monooxygenase (Luc2) in C-20 hydroxylation rather than C-13-C-14 epoxidation or C-15 hydroxylation. Isolated compounds displayed moderate to strong antimalarial activities, and the structure-activity relationship of lucilactaene derivatives was examined.

ß-Methyltryptamine Provoking the Crucial Role of Strictosidine Synthase Tyr151-OH for Its Stereoselective Pictet-Spengler Reactions to Tryptoline-type Alkaloids.

Strictosidine synthase (STR), the gate enzyme for monoterpenoid indole alkaloid biosynthesis, catalyzes the Pictet-Spengler reaction (PSR) of various tryptamine derivatives with secologanin assisted by "indole sandwich" stabilization. Continuous exploration with ß-methyltryptamine (IPA) stereoselectively delivered the C6-methylstrictosidines and C6-methylvincosides by enzymatic and nonenzymatic PSR, respectively. Unexpectedly, the first "nonindole sandwich" binding mode was witnessed by the X-ray structures of STR1-ligand complexes. Site-directed mutagenesis revealed the critical cryptic role of the hydroxyl group of Tyr151 in IPA biotransformation. Further computational calculations demonstrated the adjustable IPA position in STR1 upon the binding of secologanin, and Tyr151-OH facilitates the productive PSR binding mode via an advantageous hydrogen-bond network. Further chemo-enzymatic manipulation of C6-methylvincosides successfully resulted in the discovered antimalarial framework (IC50 = 0.92 µM).

Establishment of a Monoclonal Antibody against Human NTCP That Blocks Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infects 240 million people worldwide. Current therapy profoundly suppresses HBV replication but requires long-term maintenance therapy. Therefore, there is still a medical need for an efficient HBV cure. HBV enters host cells by binding via the preS1 domain of the viral L protein to the Na+/taurocholate cotransporting polypeptide (NTCP). Thus, NTCP should be a key target for the development of anti-HBV therapeutics. Indeed, myrcludex B, a synthetic form of the myristoylated preS1 peptide, effectively reduces HBV/hepatitis D virus (HDV) infection and has been approved as Hepcludex in Europe for the treatment of patients with chronic HDV infection. We established a monoclonal antibody (MAb), N6HB426-20, that recognizes the extracellular domain of human NTCP and blocks HBV entry in vitro into human liver cells but has much less of an inhibitory effect on bile acid uptake. In vivo, administration of the N6HB426-20 MAb prevented HBV viremia for an extended period of time after HBV inoculation in a mouse model system without strongly inhibiting bile acid absorption. Among the extracellular loops (ECLs) of NTCP, regions of amino acids (aa) 84 to 87 in ECL1 and aa 157 to 165 near ECL2 of transmembrane domain 5 are critically important for HBV/HDV infection. Epitope mapping and the three-dimensional (3D) model of the NTCP structure suggested that the N6HB426-20 MAb may recognize aa 276/277 at the tip of ECL4 and interfere with binding of HBV to the region from aa 84 to 87. In summary, we identified an in vivo neutralizing NTCP-targeting antibody capable of preventing HBV infection. Further improvements in efficacy of this drug will pave the way for its clinical applications. IMPORTANCE A number of entry inhibitors are being developed to enhance the treatment of HBV patients with oral nucleoside/nucleotide analogues (NA). To amplify the effectiveness of NA therapy, several efforts have been made to develop therapeutic MAbs with neutralizing activity against HBs antigens. However, the neutralizing effect of these MAbs may be muted by a large excess of HBsAg-positive noninfectious particles in the blood of infected patients. The advantage of NTCP-targeted HBV entry inhibitors is that they remain effective regardless of viral genotype, viral mutations, and the presence of subviral particles. Although N6HB426-20 requires a higher dose than myrcludex to obtain equivalent suppression of HBV in a model mouse system, it maintained the inhibitory effect for a long time postadministration in proportion to the half-life of an IgG MAb. We believe that further improvements will make this antibody a promising treatment option for patients with chronic hepatitis B.

Dihydrolucilactaene, a Potent Antimalarial Compound from Fusarium sp. RK97-94.

A recently discovered secondary metabolism regulator, NPD938, was used to alter the secondary metabolite profile in Fusarium sp. RK97-94. Three lucilactaene analogues were detected via UPLC-ESI-MS analysis in NPD938-treated culture. The three metabolites were successfully purified and identified as dihydroNG391 (1), dihydrolucilactaene (2), and 13α-hydroxylucilactaene (3) via extensive spectroscopic analyses. DihydroNG391 (1) exhibited weak in vitro antimalarial activity (IC50 = 62 µM). In contrast, dihydrolucilactaene (2) and 13α-hydroxylucilactaene (3) showed very potent antimalarial activity (IC50 = 0.0015 and 0.68 µM, respectively). These findings provide insight into the structure-activity relationship of lucilactaene and its analogues as antimalarial lead compounds.

2-Methylthio-N7-methyl-cis-zeatin, a new antimalarial natural product isolated from a Streptomyces culture.

2-Methylthio-N7-methyl-cis-zeatin (1) was isolated from the culture broth of Streptomyces sp. 80H647 along with 2 known purine derivatives, 5'-methylthioinosine (2) and AT-265 (dealanylascamycin, 3). The structure elucidation of compound 1 was accomplished by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) analyses. It inhibited the growth of Plasmodium falciparum 3D7 with a GI50 of 2.4 µm and had no effect on the growth of Arabidopsis at 2 µm. This is the first report of an N7-methylated zeatin-type natural product from Streptomyces and as an antimalarial compound.

Privileged Biorenewable Secologanin-Based Diversity-Oriented Synthesis for Pseudo-Natural Alkaloids: Uncovering Novel Neuroprotective and Antimalarial Frameworks.

Bioprivileged molecules hold great promise for supplementing petrochemicals in sustainable organic synthesis of a diverse bioactive products library. Secologanin, a biorenewable monoterpenoid glucoside with unique structural elements, is the key precursor for thousands of natural monoterpenoid alkaloids. Inspired by its inherent highly congested functional groups, a secologanin-based diversity-oriented synthesis (DOS) strategy for novel pseudo-natural alkaloids was developed. All the reactive units of secologanin were involved in these operation simplicity protocols under mild reaction conditions, including the one-step enantioselective transformation of exocyclic C8, C8/C11, and C8/C9/C10 as well as the chemoenzymatic manipulation of endocyclic C2/C6 via the attack by various nucleophiles. A combinatory scenario of the aforementioned reactions further provided diverse polycyclic products with multiple chiral centers. Preliminary activity screening of these newly constructed molecules led to the discovery of antimalarial and highly potent neuroprotective skeletons. The application of green biorenewable secologanin in diversity-oriented pseudo-natural monoterpenoid alkaloid synthesis might encourage the pursuit of valuable bioactive frameworks.

6,9-Dihydroxytetrangulol, a novel angucyclinone antibiotic accumulated in kiqO gene disruptant in the biosynthesis of kinanthraquinone.

A novel angucyclinone, 6,9-dihydroxytetrangulol, was isolated from Streptomyces lividans TK23 transformed with a kinanthraquinone biosynthetic gene cluster in which the kiqO gene was disrupted. The chemical structure was elucidated by spectroscopic analyses. It showed significant antibacterial activities with an IC50 value of 1.9 µM against Staphylococcus aureus and moderate anticancer activities against HL-60 cells.

Zealpeptaibolin, an 11-mer cytotoxic peptaibol group with 3 Aib-Pro motifs isolated from Trichoderma sp. RK10-F026.

Six new 11-mer peptaibols designed as zealpeptaibolins, A - F were isolated from the soil fungus, Trichoderma sp. RK10-F026, based on the principal component analysis of the MS data from five different culture compositions. Previously, 20-mer peptaibols from the same fungal strain were identified; 11-mer peptaibols in contrast were discovered from a different culture condition, signifying peptaibol production was culture condition-dependent. These peptaibols contained three Aib-Pro motifs in the sequence. The structures were established by NMR and HR-MS experiments including detailed MS/MS fragmentations. The absolute configurations were determined by Marfey's analysis. Zealpeptaibolin F exhibited the strongest cytotoxicity toward K562 leukemia cells with an IC50 value of 0.91 µM.

Glyoxalase I disruption and external carbonyl stress impair mitochondrial function in human induced pluripotent stem cells and derived neurons.

Carbonyl stress, a specific form of oxidative stress, is reported to be involved in the pathophysiology of schizophrenia; however, little is known regarding the underlying mechanism. Here, we found that disruption of GLO1, the gene encoding a major catabolic enzyme scavenging the carbonyl group, increases vulnerability to external carbonyl stress, leading to abnormal phenotypes in human induced pluripotent stem cells (hiPSCs). The viability of GLO1 knockout (KO)-hiPSCs decreased and activity of caspase-3 was increased upon addition of methylglyoxal (MGO), a reactive carbonyl compound. In the GLO1 KO-hiPSC-derived neurons, MGO administration impaired neurite extension and cell migration. Further, accumulation of methylglyoxal-derived hydroimidazolone (MG-H1; a derivative of MGO)-modified proteins was detected in isolated mitochondria. Mitochondrial dysfunction, including diminished membrane potential and dampened respiratory function, was observed in the GLO1 KO-hiPSCs and derived neurons after addition of MGO and hence might be the mechanism underlying the effects of carbonyl stress. The susceptibility to MGO was partially rescued by the administration of pyridoxamine, a carbonyl scavenger. Our observations can be used for designing an intervention strategy for diseases, particularly those induced by enhanced carbonyl stress or oxidative stress.

N-Acetyl-α-hydroxy-ß-oxotryptamine, a racemic natural product isolated from Streptomyces sp. 80H647.

N-acetyl-α-hydroxy-ß-oxotryptamine (1) along with N-acetyl-ß-oxotryptamine (2) and pimprinine (3) were isolated from the culture broth of Streptomyces sp. 80H647. Compound 1 was found to be a racemate via X-ray diffraction analysis and the enantiomers were successfully purified by chiral-phase HPLC. The absolute configuration was assigned by comparison of the calculated and experimental ECD spectra. The α-hydroxy moiety of 1 was vital for cytotoxicity against different cancer cell lines.

A new peptaibol, RK-026A, from the soil fungus Trichoderma sp. RK10-F026 by culture condition-dependent screening.

A new peptaibol, RK-026A (1) was isolated from a fungus, Trichoderma sp. RK10-F026, along with atroviridin B (2), alamethicin II (3), and polysporin B (4) as a cytotoxic compound, which was selected by principal component analysis of the MS data from 5 different culture conditions. The structure of 1 was determined as a new atroviridin B derivative containing Glu at the 18th residue instead of Gln by NMR and HR-MS analyses including the investigation of detailed MS/MS fragmentations. 1 showed cytotoxicity toward K562 leukemia cells at an IC50 value of 4.1 µm.

Discovery of 3-Cinnamamido-N-Substituted Benzamides as Potential Antimalarial Agents.

BACKGROUND: Malaria is one of the most devastating parasitic diseases, yet the discovery of antimalarial agents remains profoundly challenging. Very few new antimalarials have been developed in the past 50 years, while the emergence of drug-resistance continues to appear. OBJECTIVE: This study focuses on the discovery, design, synthesis, and antimalarial evaluation of 3- cinnamamido-N-substituted benzamides. METHODS: In this study, a screening of our compound library was carried out against the multidrugsensitive Plasmodium falciparum 3D7 strain. Derivatives of the hit were designed, synthesized and tested against P. falciparum 3D7 and the in vivo antimalarial activity of the most active compounds was evaluated using the method of Peters' 4-day suppressive test. RESULTS: The retrieved hit compound 1 containing a 3-cinnamamido-N-substituted benzamide skeleton showed moderate antimalarial activity (IC50 = 1.20 µM) for the first time. A series of derivatives were then synthesized through a simple four-step workflow, and half of them exhibited slightly better antimalarial effect than the precursor 1 during the subsequent in vitro assays. Additionally, compounds 11, 23, 30 and 31 displayed potent activity with IC50 values of approximately 0.1 µM, and weak cytotoxicity against mammalian cells. However, in vivo antimalarial activity is not effective, which might be ascribed to the poor solubility of these compounds. CONCLUSION: In this study, the phenotypic screen of our compound library resulted in the first report of a 3-cinnamamide framework with antimalarial activity and 40 derivatives were then designed and synthesized. Subsequent structure-activity studies showed that compounds 11, 23, 30 and 31 exhibited the most potent and selective activity against the P. falciparum 3D7 strain with IC50 values around 0.1 µM. Our work herein sets another example of phenotypic screen-based drug discovery, leading to potentially promising candidates of novel antimalarial agents once given further optimization.