The anti-tubercular callyaerins target the Mycobacterium tuberculosis-specific non-essential membrane protein Rv2113.

Spread of antimicrobial resistances urges a need for new drugs against Mycobacterium tuberculosis (Mtb) with mechanisms differing from current antibiotics. Previously, callyaerins were identified as promising anti-tubercular agents, representing a class of hydrophobic cyclopeptides with an unusual (Z)-2,3-di-aminoacrylamide unit. Here, we investigated the molecular mechanisms underlying their antimycobacterial properties. Structure-activity relationship studies enabled the identification of structural determinants relevant for antibacterial activity. Callyaerins are bacteriostatics selectively active against Mtb, including extensively drug-resistant strains, with minimal cytotoxicity against human cells and promising intracellular activity. By combining mutant screens and various chemical proteomics approaches, we showed that callyaerins target the non-essential, Mtb-specific membrane protein Rv2113, triggering a complex dysregulation of the proteome, characterized by global downregulation of lipid biosynthesis, cell division, DNA repair, and replication. Our study thus identifies Rv2113 as a previously undescribed Mtb-specific drug target and demonstrates that also non-essential proteins may represent efficacious targets for antimycobacterial drugs.

Nature-Inspired (di)Azine-Bridged Bisindole Alkaloids with Potent Antibacterial In Vitro and In Vivo Efficacy against Methicillin-Resistant Staphylococcus aureus.

Natural bisindole alkaloids such as Hyrtinadine A and Alocasin A, which are known to exhibit diverse bioactivities, provide promising chemical scaffolds for drug development. By optimizing the Masuda borylation-Suzuki coupling sequence, a library of various natural product-derived and non-natural (di)azine-bridged bisindoles was created. While unsubstituted bisindoles were devoid of antibacterial activity, 5,5'-chloro derivatives were highly active against methicillin-resistant Staphylococcus aureus (MRSA) and further Gram-positive pathogens at minimal inhibitory concentrations ranging from 0.20 to 0.78 µM. These compounds showed strong bactericidal killing effects but only moderate cytotoxicity against human cell lines. Furthermore, the two front-runner compounds 4j and 4n exhibited potent in vivo efficacy against MRSA in a mouse wound infection model. Although structurally related bisindoles were reported to specifically target pyruvate kinase in MRSA, antibacterial activity of 4j and 4n is independent of pyruvate kinase. Rather, these compounds lead to bacterial membrane permeabilization and cellular efflux of low-molecular-weight molecules.

3-O-Methyl-Alkylgallates Inhibit Fatty Acid Desaturation in Mycobacterium tuberculosis.

In the quest for new antibacterial lead structures, activity screening against Mycobacterium tuberculosis identified antitubercular effects of gallic acid derivatives isolated from the Nigerian mistletoe Loranthus micranthus Structure-activity relationship studies indicated that 3-O-methyl-alkylgallates comprising aliphatic ester chains with four to eight carbon atoms showed the strongest growth inhibition in vitro against M. tuberculosis, with a MIC of 6.25 µM. Furthermore, the most active compounds (3-O-methyl-butyl-, 3-O-methyl-hexylgallate, and 3-O-methyl-octylgallate) were devoid of cytotoxicity against various human cell lines. Furthermore, 3-O-methyl-butylgallate showed favorable absorption, distribution, metabolism, and excretion (ADME) criteria, with a Papp of 6.2 × 10-6 cm/s, and it did not inhibit P-glycoprotein (P-gp), CYP1A2, CYP2B6 or CYP3A4. Whole-genome sequencing of spontaneous resistant mutants indicated that the compounds target the stearoyl-coenzyme A (stearoyl-CoA) delta-9 desaturase DesA3 and thereby inhibit oleic acid synthesis. Supplementation assays demonstrated that oleic acid addition to the culture medium antagonizes the inhibitory properties of gallic acid derivatives and that sodium salts of saturated palmitic and stearic acid did not show compensatory effects. The moderate bactericidal effect of 3-O-methyl-butylgallate in monotreatment was synergistically enhanced in combination treatment with isoniazid, leading to sterilization in liquid culture.

Metabolites from the endophytic fungus Cylindrocarpon sp. isolated from tropical plant Sapium ellipticum.

Three new polyketides, cylindrocarpones A-C (1-3), two new pyridone alkaloids, cylindrocarpyridones A-B (5-6), a new pyrone cylindropyrone (7), together with seven know compounds were isolated from the endophytic fungus, Cylindrocarpon sp., obtained from the tropical plant Sapium ellipticum. The structures of the new compounds were elucidated by extensive analysis of their spectroscopic data (1D and 2D NMR, HRESIMS). The absolute configuration of 19-O-methyl-pyrrocidine B (13) was confirmed by X-ray analysis. All isolated compounds were screened for their cytotoxic and antibacterial activities. Pyrrocidine A (12) exhibited potent cytotoxicity against the human ovarian cancer cell line A2780 with an IC50 value of 1.7 µM. 19-O-Methyl-pyrrocidine B (13) showed moderate antibacterial activity against S. aureus ATCC25923 and ATCC700699 with MIC values of 50 and 25 µM, respectively.

(Some) current concepts in antibacterial drug discovery.

The rise of multidrug resistance in bacteria rendering pathogens unresponsive to many clinical drugs is widely acknowledged and considered a critical global healthcare issue. There is broad consensus that novel antibacterial chemotherapeutic options are extremely urgently needed. However, the development pipeline of new antibacterial drug lead structures is poorly filled and not commensurate with the scale of the problem since the pharmaceutical industry has shown reduced interest in antibiotic development in the past decades due to high economic risks and low profit expectations. Therefore, academic research institutions have a special responsibility in finding novel treatment options for the future. In this mini review, we want to provide a broad overview of the different approaches and concepts that are currently pursued in this research field.

Chlorflavonin Targets Acetohydroxyacid Synthase Catalytic Subunit IlvB1 for Synergistic Killing of Mycobacterium tuberculosis.

The flavonoid natural compound chlorflavonin was isolated from the endophytic fungus Mucor irregularis, which was obtained from the Cameroonian medicinal plant Moringa stenopetala. Chlorflavonin exhibited strong growth inhibitory activity in vitro against Mycobacterium tuberculosis (MIC90 1.56 µM) while exhibiting no cytotoxicity toward the human cell lines MRC-5 and THP-1 up to concentrations of 100 µM. Mapping of resistance-mediating mutations employing whole-genome sequencing, chemical supplementation assays, and molecular docking studies as well as enzymatic characterization revealed that chlorflavonin specifically inhibits the acetohydroxyacid synthase catalytic subunit IlvB1, causing combined auxotrophies to branched-chain amino acids and to pantothenic acid. While exhibiting a bacteriostatic effect in monotreatment, chlorflavonin displayed synergistic effects with the first-line antibiotic isoniazid and particularly with delamanid, leading to a complete sterilization in liquid culture in combination treatment. Using a fluorescent reporter strain, intracellular activity of chlorflavonin against Mycobacterium tuberculosis inside infected macrophages was demonstrated and was superior to streptomycin treatment.

Cyclic Cystine-Bridged Peptides from the Marine Sponge Clathria basilana Induce Apoptosis in Tumor Cells and Depolarize the Bacterial Cytoplasmic Membrane.

Investigation of the sponge Clathria basilana collected in Indonesia afforded five new peptides, including microcionamides C (1) and D (2), gombamides B (4), C (5), and D (6), and an unusual amide, (E)-2-amino-3-methyl-N-styrylbutanamide (7), along with 11 known compounds, among them microcionamide A (3). The structures of the new compounds were elucidated by one- and two-dimensional NMR spectroscopy as well as by high-resolution mass spectrometry. The absolute configurations of the constituent amino acid residues in 1-7 were determined by Marfey's analysis. Microcionamides A, C, and D (1-3) showed in vitro cytotoxicity against lymphoma (Ramos) and leukemia cell lines (HL-60, Nomo-1, Jurkat J16), as well as against a human ovarian carcinoma cell line (A2780) with IC50 values ranging from 0.45 to 28 µM. Mechanistic studies showed that compounds 1-3 rapidly induce apoptotic cell death in Jurkat J16 and Ramos cells and that 1 and 2 potently block autophagy upon starvation conditions, thereby impairing pro-survival signaling of cancer cells. In addition, microcionamides C and A (1 and 3) inhibited bacterial growth of Staphylococcus aureus and Enterococcus faecium with minimal inhibitory concentrations between 6.2 and 12 µM. Mechanistic studies indicate dissipation of the bacterial membrane potential.

A novel mycobacterial In Vitro infection assay identifies differences of induced macrophage apoptosis between CD4+ and CD8+ T cells.

Macrophages are natural host cells for pathogenic mycobacteria, like Mycobacterium tuberculosis (M.tb). Immune surveillance by T cells and interaction with M.tb infected macrophages is crucial for protection against M.tb reactivation and development of active tuberculosis. Several factors play a role in the control of M.tb infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional in vitro assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional in vitro assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a Mycobacterium bovis BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated in vitro and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. M.tb protein specific CD4+ and CD8+ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4+ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional in vitro assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.

Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of Amorpha fruticosa.

Fourteen new natural products, namely, 2-[(Z)-styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7-trihydroxyflavanone (4), 8-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (5), 6-geranyl-5,7,3'-trihydroxy-4'-methoxyisoflavone (6), 8-geranyl-7,3'-dihydroxy-4'-methoxyisoflavone (7), 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-demethylamorphigenin (9), (6aR,12aR,5'R)-amorphigenin (10), amorphispironones B and C (11 and 12), resokaempferol 3-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside-7-O-α-l-rhamnopyranoside (13), and daidzein 7-O-ß-d-glucopyranosyl-(1→2)-ß-d-glucopyranoside (14), together with 40 known compounds, were isolated from the fruits of Amorpha fruticosa. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic analysis as well as from the mass spectrometry data. ECD calculations were performed to determine the absolute configurations of 11 and 15. Compounds 1, 4-6, and 16-23 showed potent to moderate antibacterial activities against several Gram-positive bacteria with MIC values ranging from 3.1 to 100 µM. In addition, compounds 11 and 24-33 were significantly cytotoxic against the L5178Y mouse lymphoma cell line and exhibited IC50 values from 0.2 to 10.2 µM.

Metabolites from the Fungal Endophyte Aspergillus austroafricanus in Axenic Culture and in Fungal-Bacterial Mixed Cultures.

The endophytic fungus Aspergillus austroafricanus isolated from leaves of the aquatic plant Eichhornia crassipes was fermented axenically on solid rice medium as well as in mixed cultures with Bacillus subtilis or with Streptomyces lividans. Chromatographic analysis of EtOAc extract of axenic cultures afforded two new metabolites, namely, the xanthone dimer austradixanthone (1) and the sesquiterpene (+)-austrosene (2), along with five known compounds (3-7). Austradixanthone (1) represents the first highly oxygenated heterodimeric xanthone derivative. When A. austroafricanus was grown in mixed cultures with B. subtilis or with S. lividans, several diphenyl ethers (8-11) including the new austramide (8) were induced up to 29-fold. The structures of new compounds were unambiguously elucidated using 1D- and 2D-NMR spectroscopy, HRESIMS, and chemical derivatization. Compound 7 exhibited weak cytotoxicity against the murine lymphoma L5178Y cell line (EC50 is 12.6 µM). In addition, compounds 9 and 10, which were enhanced in mixed fungal/bacterial cultures, proved to be active against Staphylococcus aureus (ATCC 700699) with minimal inhibitory concentrations (MICs) of 25 µM each (6.6 µg/mL), whereas compound 11 revealed moderate antibacterial activity against B. subtilis 168 trpC2 with an MIC value of 34.8 µM (8 µg/mL).