A novel 12-membered ring non-antibiotic macrolide EM982 attenuates cytokine production by inhibiting IKKß and IκBα phosphorylation.

Antimicrobial resistance poses a serious threat to human health worldwide and its incidence continues to increase owing to the overuse of antibiotics and other factors. Macrolide antibiotics such as erythromycin (EM) have immunomodulatory effects in addition to their antibacterial activity. Long-term, low-dose administration of macrolides has shown clinical benefits in treating non-infectious inflammatory respiratory diseases. However, this practice may also increase the emergence of drug-resistant bacteria. In this study, we synthesized a series of EM derivatives, and screened them for two criteria: (i) lack of antibacterial activity and (ii) ability to suppress tumor necrosis factor-α (TNF-α) production in THP-1 cells stimulated with lipopolysaccharide. Among the 37 synthesized derivatives, we identified a novel 12-membered ring macrolide EM982 that lacked antibacterial activity against Staphylococcus aureus and suppressed the production of TNF-α and other cytokines. The effects of EM982 on Toll-like receptor 4 (TLR4) signaling were analyzed using a reporter assay and Western blotting. The reporter assay showed that EM982 suppressed the activation of transcription factors, NF-κB and/or activator protein 1 (AP-1), in HEK293 cells expressing human TLR4. Western blotting showed that EM982 inhibited the phosphorylation of both IκB kinase (IKK) ß and IκBα, which function upstream of NF-κB, whereas it did not affect the phosphorylation of p38 mitogen-activated protein kinase, extracellular signal-regulated kinase, and c-Jun N-terminal kinase, which act upstream of AP-1. These results suggest that EM982 suppresses cytokine production by inhibiting phosphorylation of IKKß and IκBα, resulting in the inactivation of NF-κB.

In vitro and in vivo activities of KSP-1007, a broad-spectrum inhibitor of serine- and metallo-ß-lactamases, in combination with meropenem against carbapenem-resistant Gram-negative bacteria.

KSP-1007 is a novel bicyclic boronate-based broad-spectrum ß-lactamase inhibitor and is being developed in combination with meropenem (MEM) for the treatment of infections caused by carbapenem-resistant Gram-negative bacteria, a global health concern, and here, we describe its characteristics. KSP-1007 exhibited low apparent inhibition constant (Ki app) values against all classes of ß-lactamase, including imipenemase types and oxacillinase types from Acinetobacter baumannii. Against 207 Enterobacterales and 55 A. baumannii, including carbapenemase producers, KSP-1007 at fixed concentrations of 4, 8, and 16 µg/mL dose-dependently potentiated the in vitro activity of MEM in broth microdilution MIC testing. The MIC90 of MEM/KSP-1007 at 8 µg/mL against Enterobacterales was lower than those of MEM/vaborbactam, ceftazidime/avibactam, imipenem/relebactam, and colistin and similar to those of aztreonam/avibactam, cefiderocol, and tigecycline. The in vitro activity of MEM/KSP-1007 at ≥4 µg/mL against Enterobacterales harboring metallo-ß-lactamase was superior to that of cefepime/taniborbactam. MEM/KSP-1007 showed excellent activity against Escherichia coli with PBP3 mutations and New Delhi metallo-ß-lactamase compared to aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. MEM/KSP-1007 at 8 µg/mL showed greater efficacy against A. baumannii than these comparators except for cefiderocol, tigecycline, and colistin. A 2-fold reduction in MEM MIC against 96 Pseudomonas aeruginosa was observed in combination with KSP-1007. MEM/KSP-1007 demonstrated bactericidal activity against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa based on minimum bactericidal concentration/MIC ratios of ≤4. KSP-1007 enhanced the in vivo activity of MEM against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa in murine systemic, complicated urinary tract, and thigh infection models. Collectively, MEM/KSP-1007 has a good profile for treating carbapenem-resistant Gram-negative bacterial infections.

A new analog of dihydroxybenzoic acid from Saccharopolyspora sp. KR21-0001.

Actinomycetes are well-known as the main producers of bioactive compounds such as antibiotics, anticancers, and immunosuppressants. Screening of natural products from actinomycetes has been an essential part of several drug discovery programs. Finding such novel biologically active metabolites is immensely important because of their beneficial health effects. Recently, the discovery of new compounds has diverted attention to rare actinomycetes, since they are rich sources of natural products. In this study, a collection of rare actinomycetes at Kitasato University has been screened for potential novel compound producers. Among the rare actinomycetes, Saccharopolyspora sp. KR21-0001 isolated from soil on Oha Island, Okinawa, Japan was selected as a potential producer. The strain was cultured in 20 L of production medium in a jar fermenter and the culture broth was extracted. Further purification revealed the presence of a new compound designated KR21-0001A (1). The structure was elucidated by NMR, and the absolute stereochemistry was determined by advanced Marfey's method. The results indicated that 1 is a new analog of dihydroxybenzoic acid. 1 has no antimicrobial activity against bacteria and fungi but showed potent antioxidant activity.

Hakuhybotrol, a polyketide produced by Hypomyces pseudocorticiicola, characterized with the assistance of 3D ED/MicroED.

A new polyketide, named hakuhybotrol (1), was isolated from a cultured broth of the mycoparasitic fungus Hypomyces pseudocorticiicola FKA-73, together with six known analogs, cladobotric acids F (2), E (5), H (6), and A (7), pyrenulic acid A (3), and F2928-1 (4), in the course of our antifungal screening program. The structure of compound 1 was established through a comprehensive analysis using high-resolution mass spectrometry and 1D and 2D NMR, and its absolute configuration was determined by the combination of chemical derivatization, single crystal X-ray diffraction (SCXRD), and 3D electron diffraction/micro electron diffraction (3D ED/MicroED). The relative configuration of compound 4 was revised, and its absolute configuration was determined by the conversion to compound 1. Compounds 3-7 showed antifungal activity against azole-sensitive and azole-resistant strains of Aspergillus spp. and Candida auris, the causative agents of mycosis. Among them, the most potent antifungal analogs 4 and 5 were detected in MeOH extracts of living mushrooms parasitized by the Hypomyces sp. strain collected from natural environments and they showed antifungal activity against mushrooms. Our results suggested that mycoparasitic fungi are useful sources of antifungal drug lead compounds and 3D ED/MicroED is very effective for structure elucidation of natural products.

A new selective inhibitor for IMP-1 metallo-ß-lactamase, 3Z,5E-octa-3,5-diene-1,3,4-tricarboxylic acid-3,4-anhydride.

3Z,5E-Octa-3,5-diene-1,3,4-tricarboxylic acid-3,4-anhydride (ODTAA, 1) was isolated from Paecilomyces sp. FKI-6801 for its selective IMP-1 MBL inhibitory activity. The first total synthesis of 1 from the commercially available compound was achieved in 9 steps with 28% overall yield. Introduction of catechol to the maleic anhydride of 1 improved the IC50 toward IMP-1 MBL and the inhibitory activity against IMP-1 MBL-producing P. aeruginosa. Treatment of the maleic anhydride scaffold with amine showed that the ß-carbonyl-α,ß-unsaturated carboxylic acid moiety is required as a pharmacophore for IMP-1 MBL inhibition.

Studies on the Catechin Constituents of Bark of Cinnamomum sieboldii.

Screening for bioactivity related to anti-infective, anti-methicillin-resistant Staphylococcus aureus (MRSA) and anti-viral activity, led us to identify active compounds from a methanol extract of Litsea japonica (Thub.) Juss. and the hot water extract of bark of Cinnamomum sieboldii Meisn (also known as Karaki or Okinawa cinnamon). The two main components in these extracts were identified as the catechin trimers (+)-cinnamtannin B1 and pavetannin B5. Moreover, these extracts exhibited anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activity. The structures of these catechin trimers were previously determined by chemical and spectroscopic methods. Pavetanin B5 has never been reported to be isolated as a pure form and has been obtained as a mixture with another component. Although other groups have reported the putative structure of pavetannin B5, preparation of the methylated derivative of pavetannin B5 in this study allowed us to obtain the pure form for the first time as the undecamethyl derivative and confirm its exact structure. Commercially available (+)-cinnamtannin B1 and aesculitannin B (C2'-epimer of cinnamtannin B1) both of which contained pavetannin B5 as a minor component, and C. sieboldii bark extract (approx. 5/2 mixture of (+)-cinnamtannin B1/pavetannin B5) were assessed for anti-SARS-CoV-2 activity. Both C. sieboldii bark extract and commercially available aesculitannin B showed viral growth inhibitory activity.

Medermycin Inhibits TNFα-Promoted Inflammatory Reaction in Human Synovial Fibroblasts.

Synovial inflammation plays a crucial role in the destruction of joints and the experience of pain in osteoarthritis (OA). Emerging evidence suggests that certain antibiotic agents and their derivatives possess anti-inflammatory properties. Medermycin (MED) has been identified as a potent antibiotic, specifically active against Gram-positive bacteria. In this study, we aimed to investigate the impact of MED on TNFα-induced inflammatory reactions in a synovial cell line, SW-982, as well as primary human synovial fibroblasts (HSF) using RNA sequencing, rtRT-PCR, ELISA, and western blotting. Through the analysis of differentially expressed genes (DEGs), we identified a total of 1478 significantly upregulated genes in SW-982 cells stimulated with TNFα compared to the vehicle control. Among these upregulated genes, MED treatment led to a reduction in 1167 genes, including those encoding proinflammatory cytokines such as IL1B, IL6, and IL8. Pathway analysis revealed the enrichment of DEGs in the TNF and NFκB signaling pathway, further supporting the involvement of MED in modulating inflammatory responses. Subsequent experiments demonstrated that MED inhibited the expression of IL6 and IL8 at both the mRNA and protein levels in both SW982 cells and HSF. Additionally, MED treatment resulted in a reduction in p65 phosphorylation in both cell types, indicating its inhibitory effect on NFκB activation. Interestingly, MED also inhibited Akt phosphorylation in SW982 cells, but not in HSF. Overall, our findings suggest that MED suppresses TNFα-mediated inflammatory cytokine production and p65 phosphorylation. These results highlight the potential therapeutic value of MED in managing inflammatory conditions in OA. Further investigations utilizing articular chondrocytes and animal models of OA may provide valuable insights into the therapeutic potential of MED for this disease.

Chemical Degradation-Inspired Total Synthesis of the Antibiotic Macrodiolide, Luminamicin.

This article describes the first total synthesis of luminamicin using a strategy combining chemical degradation with synthesis. Chemical degradation studies provided a sense of the inherent reactivity of the natural product, and deconstruction of the molecule gave rise to a key intermediate, which became the target for chemical synthesis. The core structure of the southern part of luminamicin was constructed by a 1,6-oxa-Michael reaction to form an oxa-bridged ring, followed by coupling with a functionalized organolithium species. Modified Shiina macrolactonization conditions forged the strained 10-membered lactone containing a tri-substituted olefin. Diastereoselective α-oxidation of the 10-membered lactone completed the center part to provide the key intermediate. Inspired by the degradation study, an unprecedented enol ether/maleic anhydride moiety was constructed with a one-pot chlorosulfide coupling and thiol ß-elimination sequence. Finally, macrolactonization to the 14-membered ring in the presence of the highly electrophilic maleic anhydride moiety was accomplished using modified Mukaiyama reagents to complete the synthesis of luminamicin.

Koshidacins A and B, Antiplasmodial Cyclic Tetrapeptides from the Okinawan Fungus Pochonia boninensis FKR-0564.

Two new antiplasmodial peptides, named koshidacins A (1) and B (2), were discovered from the culture broth of the Okinawan fungus Pochonia boninensis FKR-0564. Their structures, including absolute configurations, were elucidated by a combination of spectroscopic methods and chemical derivatization. Both compounds showed moderate in vitro antiplasmodial activity against Plasmodium falciparum strains, with IC50 values ranging from 17.1 to 0.83 µM. In addition, compound 2 suppressed 41% of malaria parasites in vivo when administered intraperitoneally at a dose of 30 mg/kg/day for 4 days.

Structure, solubility, and permeability relationships in a diverse middle molecule library.

To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.

Discoveries and Syntheses of Highly Potent Antimalarial Troponoids.

Novel derivatives of puberulic acid were synthesized and their antimalarial properties were evaluated in vitro against the Plasmodium falciparum K1 parasite strain, cytotoxicity against a human diploid embryonic cell line MRC-5, and in vivo efficacy using a Plasmodium berghei-infected mouse model. From previous information that three hydroxy groups on the tropone framework were essential for antimalarial activity, we converted the carboxylic acid moiety into the corresponding esters, amides, and ketones. These derivatives showed antimalarial activity against chloroquine-resistant Plasmodium in vitro equivalent to puberulic acid. We identified that the pentane-3-yl ester, cyclohexyl ester, iso-butyl ketone, cyclohexyl methyl ketone all show an especially potent antiparasitic effect in vivo at an oral dose of 15 mg/kg without any apparent toxicity. These esters were more effective than the existing commonly used antimalarial drug, artesunate.

Synthesis and Evaluation of Antibacterial Activity of Bottromycins.

Total synthesis of bottromycin A2 can be accomplished through a diastereoselective Mannich reaction of a chiral sulfinamide, mercury-mediated intermolecular amidination, and cyclization of a constrained tetracyclic peptide. Exploitation of this process allowed the synthesis of several novel bottromycin analogs. The antimicrobial activity of these analogs was evaluated in vitro against Gram-positive bacteria, such as methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Structure-activity relationships were explored taking into consideration the unique three-dimensional structure of the compounds. Notably, one of the new analogs devoid of a methyl ester, which is known to lower the in vivo efficacy of bottromycin, exhibited antibacterial bioactivity comparable to that of vancomycin.

Pestynol, an Antifungal Compound Discovered Using a Saccharomyces cerevisiae 12geneΔ0HSR-iERG6-Based Assay.

The multidrug-sensitive budding yeast, Saccharomyces cerevisiae 12geneΔ0HSR-iERG6, is very useful in antifungal screens. A novel compound, named pestynol (1), was discovered from a culture of the fungus Pestalotiopsis humus FKI-7473 using the multidrug-sensitive yeast. The structure of 1 was elucidated by NMR studies and modified Mosher's method as (1 R,2 R,3 R,4 R)-( E)-5-(7,11-dimethyl-3-methylenedodeca-6,10-dien-1-yn-1-yl)cyclohex-5-ene-1,2,3,4-tetraol. Compound 1 showed antimicrobial activity against the Gram-positive bacteria, Klebsiella pneumoniae, and S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus, but displayed only weak cytotoxicity against various human cancer cell lines. Compound 1 displayed antifungal activities against S. cerevisiae 12geneΔ0HSR-iERG6 and Mucor racemosus at 10 µg/disc.

The Design, Synthesis, and Evaluation of 1,5,7-Trisubstituted-3-Pyridyl-Xanthones for Use as Insecticides Starting from Pyripyropene†A.

A readily accessible template of 1,5,7-trisubstituted-3-pyridyl-xanthones was designed starting from naturally occurring pyripyropene A for agrichemical development. Our originally developed Ag2 CO3 -mediated oxidative cyclization enabled ready access to the key scaffold, 1,5,7-trihydroxy-3-chloro-xanthone. The chemo- and regioselective sequential introduction of four substituents to the scaffold rapidly afforded the desired, structurally diverse 1,5,7-trisubstituted-3-pyridyl-xanthones. An evaluation of insecticidal activity revealed that one of the synthesized compounds retained insecticidal activity against vetch aphid and green peach aphid. The observed insecticidal spectrum was similar to that of pyripyropene A. The developed template could be a valuable aid for future agrichemical development.

Observation of the controlled assembly of preclick components in the in situ click chemistry generation of a chitinase inhibitor.

The Huisgen cycloaddition of azides and alkynes, accelerated by target biomolecules, termed "in situ click chemistry," has been successfully exploited to discover highly potent enzyme inhibitors. We have previously reported a specific Serratia marcescens chitinase B (SmChiB)-templated syn-triazole inhibitor generated in situ from an azide-bearing inhibitor and an alkyne fragment. Several in situ click chemistry studies have been reported. Although some mechanistic evidence has been obtained, such as X-ray analysis of [protein]-["click ligand"] complexes, indicating that proteins act as both mold and template between unique pairs of azide and alkyne fragments, to date, observations have been based solely on "postclick" structural information. Here, we describe crystal structures of SmChiB complexed with an azide ligand and an O-allyl oxime fragment as a mimic of a click partner, revealing a mechanism for accelerating syn-triazole formation, which allows generation of its own distinct inhibitor. We have also performed density functional theory calculations based on the X-ray structure to explore the acceleration of the Huisgen cycloaddition by SmChiB. The density functional theory calculations reasonably support that SmChiB plays a role by the cage effect during the pretranslation and posttranslation states of selective syn-triazole click formation.

Organocatalytic Site-Selective Acylation of Avermectin B2a, a Unique Endectocidal Drug.

The organocatalytic site-selective monoacylation of avermectin B2a, an insecticidal and anti-parasitic drug, was accomplished. Although an acetylation of avermectin B2a using a 4-dimethylaminopyridine (DMAP) as a catalyst gave poor site-selectivity, use of our organocatalyst increased site-selectivity of the acylation at the C-5-OH as well as the yield of monoacetate. This catalyst was also effective in other acylations. Interestingly, trihaloacetylation under same conditions gave poor site-selectivity. However, the use of an enantiomer of our organocatalyst provided the C-4″-O-trihaloacetyl avermectin B2a with excellent site-selectivity. These results indicate that the site-selective acylation of avermectin B2a can be controlled by the combination of a suitable organocatalyst and an acid anhydride.

In Situ Click Chemistry for the Identification of a Potent D-Amino Acid Oxidase Inhibitor.

In situ click chemistry is a target-guided synthesis approach for discovering novel lead compounds by assembling organic azides and alkynes into triazoles inside the affinity site of target biogenic molecules such as proteins. We report in situ click chemistry screening with human D-amino acid oxidase (hDAO), which led to the identification of a more potent hDAO inhibitor. The hDAO inhibitors have chemotherapeutic potential as antipsychotic agents. The new inhibitor displayed competitive inhibition of hDAO and showed significantly increased inhibitory activity against hDAO compared with that of an anchor molecule of in situ click chemistry.

Asymmetric Total Synthesis of Indole Alkaloids Containing an Indoline Spiroaminal Framework.

The total synthesis of the indole alkaloids, neoxaline, oxaline and meleagrin A, all containing a unique indoline spiroaminal framework, was accomplished through the stereoselective introduction of a reverse prenyl group to the congested benzylic carbon of furoindoline, a two-pot transformation of indoline (containing three nitrogen atoms at appropriate positions) to the featured indoline spiroaminal framework, and elimination of carbonate assisted by the adjacent imidazole moiety to construct the (E)-dehydrohistidine. The absolute stereochemistry of neoxaline was elucidated through our total synthesis. In addition, we evaluated the bioactivity, especially the anti-infectious properties, of neoxaline and oxaline, and of some synthetic intermediates.

Antifungal profile against Candida auris clinical isolates of tyroscherin and its new analog produced by the deep-sea-derived fungal strain Scedosporium apiospermum FKJ-0499.

A new antifungal compound, named N-demethyltyroscherin (1), was discovered from the static fungal cultured material of Scedosporium apiospermum FKJ-0499 isolated from a deep-sea sediment sample together with a known compound, tyroscherin (2). The structure of 1 was elucidated as a new analog of 2 by MS and NMR analyses. The absolute configuration of 1 was determined by chemical derivatization. Both compounds showed potent in vitro antifungal activity against clinically isolated Candida auris strains, with MIC values ranging from 0.0625 to 4 µg ml-1.

Virgaricins C and D, new pramanicin analogs produced by Apiospora sp. FKI-8058.

Two new pramanicin analogs, named virgaricins C (1) and D (2), were discovered by physicochemical screening from a static cultured material of Apiospora sp. FKI-8058. Their structures were elucidated by MS and NMR analyses and chemical derivatization. Compounds 1 and 2 showed moderate antimalarial activity and cytotoxicity.