Efficacy of rifapentine and other rifamycins against Coxiella burnetii in vitro.

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii. Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii. Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL-1. Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized, and rifapentine, not previously reported against C. burnetii. These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL-1. In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL-1. Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL-1 and should be further explored as alternative Q fever treatments given their efficacy in vitro. IMPORTANCE: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii. Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro. Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii.

PKC-α is involved in the signaling of phagocytosis induced by two snake venom secretory PLA2S in macrophages.

Phagocytosis, an essential process for host defense, requires the coordination of a variety of signaling reactions. MT-II, an enzymatically inactive Lys49 phospholipase A2 (PLA2) homolog, and MT-III, a catalytically-active Asp49 PLA2, are known to activate phagocytosis in macrophages. In this study, the signaling pathways mediating phagocytosis, focusing on protein kinases, were investigated. Macrophages from male Swiss mice peritoneum were obtained 96 h after intraperitoneal thioglycolate injection. Phagocytosis was evaluated using non-opsonized zymosan particles in the presence or absence of specific inhibitors, as well as PKC and PKC-α localization by confocal microscopy. Moreover, protein kinase C (PKC) activity was assessed by γP32 ATP in macrophages stimulated by both PLA2s. Data showed that both sPLA2s increased phagocytosis. Cytochalasin D, staurosporine/H7, wortmannin, and herbimycin, inhibitors of actin polymerization, PKC, phosphoinositide 3-kinase (PI3K), and protein tyrosine kinase (PTK), respectively, significantly reduced phagocytosis induced by both PLA2s. PKC activity was increased in macrophages stimulated by both PLA2s. Actin polymerization and talin were evidenced by immunofluorescence and talin was recruited 5 min after both PLA2s stimulation. PKC and PKC-α localization within the cell were increased after 60 min of MT-II and MT-III stimulation. These data suggest that the effect of both PLA2s depends on actin cytoskeleton rearrangements and the activation of PKC, PI3K, and PTK signaling events required for phagocytosis.

Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.

Genome-Driven Discovery of Hygrocins in Streptomyces rapamycinicus.

Ansamycins, represented by the antituberculosis drug rifamycin, are an important family of natural products. To obtain new ansamycins, Streptomyces rapamycinicus IMET 43975 harboring an ansamycin biosynthetic gene cluster was fermented in a 50 L scale, and subsequent purification work led to the isolation of five known and four new analogues, where hygrocin W (2) belongs to benzoquinonoid ansamycins, and the other three hygrocins, hygrocins X-Z (6-8), are new seco-hygrocins. The structures of ansamycins (1-8) were determined by the analysis of spectroscopic (1D/2D NMR and ECD) and MS spectrometric data. The Baeyer-Villiger enzyme which catalyzed the ester formation in the ansa-ring was confirmed through in vivo CRISPR base editing. The discovery of these compounds further enriches the structural diversity of ansamycins.

Identification and Heterologous Expression of the Kendomycin B Biosynthetic Gene Cluster from Verrucosispora sp. SCSIO 07399.

Verrucosispora sp. SCSIO 07399, a rare marine-derived actinomycete, produces a set of ansamycin-like polyketides kendomycin B-D (1-3) which possess potent antibacterial activities and moderate tumor cytotoxicity. Structurally, kendomycin B-D contain a unique aliphatic macrocyclic ansa scaffold in which the highly substituted pyran ring is connected to the quinone moiety. In this work, a type I/type III polyketide synthase (PKS) hybrid biosynthetic gene cluster coding for assembly of kendomycin B (kmy), and covering 33 open reading frames, was identified from Verrucosispora sp. SCSIO 07399. The kmy cluster was found to be essential for kendomycin B biosynthesis as verified by gene disruption and heterologous expression. Correspondingly, a biosynthetic pathway was proposed based on bioinformatics, cluster alignments, and previous research. Additionally, the role of type III PKS for generating the precursor unit 3,5-dihydroxybenzoic acid (3,5-DHBA) was demonstrated by chemical complementation, and type I PKS executed the polyketide chain elongation. The kmy cluster was found to contain a positive regulatory gene kmy4 whose regulatory effect was identified using real-time quantitative PCR (RT-qPCR). These advances shed important new insights into kendomycin B biosynthesis and help to set the foundation for further research aimed at understanding and exploiting the carbacylic ansa scaffold.

New Kendomycin Derivative Isolated from Streptomyces sp. Cl 58-27.

In the course of screening new streptomycete strains, the strain Streptomyces sp. Cl 58-27 caught our attention due to its interesting secondary metabolite production profile. Here, we report the isolation and characterization of an ansamycin natural product that belongs structurally to the already known kendomycins. The structure of the new kendomycin E was elucidated using NMR spectroscopy, and the corresponding biosynthetic gene cluster was identified by sequencing the genome of Streptomyces sp. Cl 58-27 and conducting a detailed analysis of secondary metabolism gene clusters using bioinformatic tools.

Spiropiperidyl rifabutins: expanded in vitro testing against ESKAPE pathogens and select bacterial biofilms.

The expanded microbiological evaluation of a series of rifastures, novel spiropiperidyl rifamycin derivatives, against clinically relevant ESKAPE bacteria has identified several analogs with promising in vitro bioactivities against antibiotic-resistant strains of Enterococcus faecium and Staphylococcus aureus. Thirteen of the rifastures displayed minimum inhibitory concentrations (MICs) below 1 µg/ml against the methicillin- and vancomycin-resistant forms of S. aureus and E. faecium (MRSA, VRSA, VRE). Aryl-substituted rifastures 1, 11, and 12 offered the greatest bioactivity, with MICs reaching ≤0.063 µg ml-1 for these human pathogens. Further analysis indicates that diphenyl rifasture 1 had greater antibiofilm activity against S. aureus and lower cytotoxicity in mammalian HEK cells than rifabutin.

Ansamycin derivatives from the marine-derived Streptomyces sp. SCSGAA 0027 and their cytotoxic and antiviral activities.

Fourteen ansamycin derivatives including seven new herbimycins G-L (1-6) and divergolide O (7), and seven known analogues were isolated from a culture broth of the marine-derived Streptomyces sp. SCSGAA 0027. Their complete structures were determined by detailed analysis of spectroscopic data and quantum chemical calculations. Compounds 1-5 and 7 featured an additional eight-membered O-heterocycle that has rarely been reported for ansamycins, and the Z,Z- and E,E-configurations for Δ2,Δ4 were reported for the first time in geldanamycin analogues. Compound 1 exhibited weak inhibition activity towards Hsp90α with an IC50 value of 96 µM, 2-5 showed mild cytotoxicity against four human cancer cell lines with IC50 values ranging from 13 µM to 86 µM, and 7 had moderate anti-HSV-1 activity with an IC50 value of 19 µM and very weak cytotoxicity towards Vero cell. The possible biosynthetic pathways for 1-5 were proposed. And their structure-bioactivity relationship was also discussed.

Kendomycin Cytotoxicity against Bacterial, Fungal, and Mammalian Cells Is Due to Cation Chelation.

Kendomycin is a small-molecule natural product that has gained significant attention due to reported cytotoxicity against pathogenic bacteria and fungi as well as a number of cancer cell lines. Despite significant biomedical interest and attempts to reveal its mechanism of action, the cellular target of kendomycin remains disputed. Herein it is shown that kendomycin induces cellular responses indicative of cation stress comparable to the effects of established iron chelators. Furthermore, addition of excess iron and copper attenuated kendomycin cytotoxicity in bacteria, yeast, and mammalian cells. Finally, NMR analysis demonstrated a direct interaction with cations, corroborating a close link between the observed kendomycin polypharmacology across different species and modulation of iron and/or copper levels.

Cytotoxic Kendomycins Containing the Carbacylic Ansa Scaffold from the Marine-Derived Verrucosispora sp. SCSIO 07399.

Three new kendomycin analogues, kendomycins B-D (1-3), were discovered from the marine-derived actinomycete Verrucosispora sp. SCSIO 07399. The structures of 1-3 were elucidated using diverse spectroscopic data analyses, X-ray crystallography, and semisynthetic derivatization. In vitro antimicrobial assays revealed that 1-3 all display good antibacterial activities against six Gram-positive bacteria with MIC values ranging from 0.5 to 8.0 µg/mL. Additionally, 1-3 were found to be moderately cytotoxic against MGC803, A549, HeLa, HepG2, MCF-7, and RKO human tumor cell lines; IC50 values ranged from 2.2 to 44 µM.

Targeted Discovery of Pentaketide Ansamycin Aminoansamycins A-G.

Ansamycins are a class of macrolactams with diverse bioactivities, characterized by the unique 3-amino-5-hydroxybenzoic acid moiety. In this study, the ansamycin gene cluster aas in Streptomyces sp. S35 was activated by the constitutive coexpression of two pathway-specific regulator genes aas1 and aas10, and seven novel pentaketide ansamycin aminoansamycins A-G (1-7) were identified. Compound 4 with better antiproliferative activity indicated that the anthranilate analogues are probably promising building blocks for the production of unnatural ansamycins with improved activity.

Screening and identification of inhibitors of endoplasmic reticulum stress-induced activation of the IRE1α-XBP1 branch.

Endoplasmic reticulum (ER) stress and the subsequent adaptive cellular response, termed the unfolded protein response (UPR), have been implicated in several diseases, including cancer. In this review, I present a brief introduction to ER stress and the UPR and then summarize the importance of the IRE1α-XBP1 branch as a target for anticancer drug discovery. In addition, I introduce our approach to the identification of inhibitors against the IRE1α-XBP1 branch from microbial cultures. As a result of our screening, toyocamycin has been identified and toyocamycin showed anticancer activity against multiple myeloma.

Acute stimulatory effect of tumor necrosis factor on the basolateral 50 pS K channels in the thick ascending limb of the rat kidney.

The aim of the present study was to investigate the acute effect and mechanism of tumor necrosis factor (TNF) on basolateral 50 pS K channels in the thick ascending limb (TAL) of the rat kidney. The TAL tubules were isolated from the rat kidney, and the activity of the 50 pS K channels was recorded using the patch­clamp technique. The results indicated that the application of TNF (10 nM) significantly activated the 50 pS K channels and the TNF effect was concentration­dependent. Inhibition of protein kinase A, phospholipase A2 and protein tyrosine kinase using pathway inhibitors (H89, AACOCF3 and Herbimycin A, respectively) did not abolish the stimulatory effect of TNF, indicating that none of these pathways mediated the TNF effect. By contrast, the phenylarsine oxide inhibitor against protein tyrosine phosphatase (PTP) decreased the activity of the 50 pS K channels and blocked the stimulatory effect of TNF on these channels. Furthermore, western blot analysis demonstrated that the application of TNF (10 nM) in the TAL increased the phosphorylation of PTP, an indication of PTP activity stimulation. Thus, it was concluded that the acute application of TNF may stimulate the basolateral 50 pS K channel in the TAL and the stimulatory effect of TNF may be mediated by the PTP­dependent pathway.

Pharmacological induction of heat shock proteins ameliorates toxicity of mutant PKCγ in spinocerebellar ataxia type 14.

Amyloid and amyloid-like protein aggregations are hallmarks of multiple, varied neurodegenerative disorders, including Alzheimer's and Parkinson's diseases. We previously reported that spinocerebellar ataxia type 14 (SCA14), a dominant-inherited neurodegenerative disease that affects cerebellar Purkinje cells, is characterized by the intracellular formation of neurotoxic amyloid-like aggregates of genetic variants of protein kinase Cγ (PKCγ). A number of protein chaperones, including heat shock protein 70 (Hsp70), promote the degradation and/or refolding of misfolded proteins and thereby prevent their aggregation. Here, we report that, in various SCA14-associated, aggregating PKCγ variants, endogenous Hsp70 is incorporated into aggregates and that expression of these PKCγ mutants up-regulates Hsp70 expression. We observed that PKCγ binds Hsp70 and that this interaction is enhanced in the SCA14-associated variants, mediated by the kinase domain that is involved in amyloid-like fibril formation as well as the C2 domain of PKCγ. Pharmacological up-regulation of Hsp70 by the Hsp90 inhibitors celastrol and herbimycin A attenuated the aggregation of mutant PKCγ in primary cultured Purkinje cells. Up-regulation of Hsp70 diminished net PKCγ aggregation by preventing aggregate formation, resulting in decreased levels of apoptotic cell death among primary cultured Purkinje cells expressing the PKCγ variant. Of note, herbimycin A also ameliorated abnormal dendritic development. Extending our in vitro observations, administration of celastrol to mice up-regulated cerebellar Hsp70. Our findings identify heat shock proteins as important endogenous regulators of pathophysiological PKCγ aggregation and point to Hsp90 inhibition as a potential therapeutic strategy in the treatment of SCA14.

Macrocyclic Compounds from Ansamycin Antibiotic Class as Inhibitors of PD1-PDL1 Protein-Protein Interaction.

The ability of tumors to escape from immune destruction is attributed to the protein-protein interaction between programmed cell death protein 1 (PD1) and programmed cell death ligand 1 (PDL1) proteins expressed by immune T cells and cancer cells, respectively. Therefore, pharmacological inhibition of the PD1-PDL1 interaction presents an important therapeutic target against a variety of tumors expressing PDL1 on their cell surface. Recently, five antibodies have been approved and several are in clinical trials against the PD1-PDL1 protein-protein interaction target. In contrast, there are very few reports of small-molecule inhibitors of PD1-PDL1 interaction, and most of them have relatively modest or weak inhibition activities, emphasizing the difficulty in designing small-molecule inhibitors against this challenging target. Therefore, we focused our attention on macrocycles that are known to exhibit target activity comparable to large macromolecules despite having molecular weights closer to small, drug-like molecules. In this context, our present study led to the identification of several macrocyclic compounds from the ansamycin antibiotics class to be inhibitors of PD1-PDL1 interaction. Importantly, one of these macrocyclic antibiotics, Rifabutin, showed an IC50 value of ca. 25 µM. This is remarkable considering it has a relatively low molecular weight and still is capable of inhibiting PD1-PDL1 protein-protein interaction whose binding interface spans over ca. 1970 Å2. Thus, these macrocycles may serve as guiding points for discovery and optimization of more potent, selective small-molecule inhibitors of PD1-PDL1 interaction, one of the most promising therapeutic targets against cancer.

Activity of Rifabutin and Hemi-synthetic Derivatives Against Mycobacterium abscessus.

BACKGROUND: Mycobacterium abscessus causes a wide range of clinical diseases that are difficult to treat. This microorganism is resistant not only to the classical antituberculosis agents but also to most of the antimicrobials that are currently available, resulting in limited therapeutic options and treatment failure. This scenario stresses the need to search for new drugs with activity against M. abscessus. OBJECTIVE: To evaluate in vitro the antimycobacterial activity and cytotoxicity of rifabutin (RFB 1) and ten derivatives (2-11) against M. abscessus ATCC 19977. METHOD: The minimum inhibitory concentration (MIC) of the molecules was determined by the microdilution broth method according to the guideline described in CLSI. The toxicity evaluation was carried in 96-well microplates, using the cell line J774A.1 (ATCC TIB-67). RESULT: From the eleven molecules tested, RFB 1 and RFB 4 were the compounds showing higher activities against M. abscessus, with MICs of 0.9 and 1.0 µM, respectively. The R1 and R2 moieties seem to have deciding influence over the final activity. Furthermore, N-oxide derivatives 9, 10, and 11 were also active against M. abscessus, with MICs of 7.2 µM, 1.8 µM and 3.8 µM, respectively. An explanatory hypothesis for the better activities of compounds RFB 1, RFB 4, RFB 10 and RFB 11 considers the likely hydrogen bonding between ligands and receptor, balancing the global flexibility and interaction energies. RFB 1 and its most effective derivatives were found to be not toxic. CONCLUSION: Besides RFB 1, its derivatives 4, 10 and 11 show potential for clinical development in the M. abscessus treatment.

Tyrosine kinase inhibition is an important factor for gene expression of CRTH2 in human eosinophils and lymphocytes: A novel mechanism for explaining eosinophils recruitment by the neuro-immune axis in allergic rhinitis.

We recently shown a novel neuro-immune competition between vasoactive intestinal peptide (VIP) and PGD2 for CRTH2 receptor, and that genistein augmented VIP and PGD2-induced eosinophil chemotaxis. However, there are neither studies on the CRTH2 gene expression in allergic rhinitis (AR) nor in the effect of tyrosine kinase inhibitors in CRTH2 gene regulation. Our Objectives were to study the gene expression modulation of CRTH2 receptor in AR patients and the effect of tyrosine kinase inhibitors (TKIs) on CRTH2 gene modulation. Nasal provocation tests, ELISA, qRT-PCR, western blot, flow cytometry and chemotaxis assays in modified micro-Boyden chambers, were all used, to achieve our objectives. Herein we show that AR patients increased the amounts of VIP and PGD2 in their nasal secretions in the early phase reaction, however CRTH2 gene expression from leukocytes recovered in their nasal secretions was upregulated only during the late phase reaction. The TKIs; Genistein, Erbstatin and Herbimycin A, induced the gene expression of CRTH2 and increased the protein content of CRTH2 in both human lymphocytes and eosinophils. This was functional as PGD2/VIP-induced eosinophil chemotaxis was augmented by the TKIs and inhibited by pervanadate, the tyrosine phosphatase inhibitor. These results open channels for therapeutic modalities targeting CRTH2 molecules in AR.

C15-methoxyphenylated 18-deoxy-herbimycin A analogues, their in vitro anticancer activity and heat shock protein 90 binding affinity.

Benzoquinone ansamycins are important leads for the discovery of novel inhibitors of heat shock protein 90 (Hsp90), a promising target of cancer chemotherapeutics. Intrinsic hepatotoxicity caused by the benzoquinone moiety appeared to be a serious limitation to the development of these compounds. To solve this problem by rational structure optimization, a short series of C18-deoxy analogues of herbimycin A were designed based on putative interactions between the compound and the protein. Chemical synthesis of the target molecules were attempted by following the established synthetic route to the natural product, but resulted in the isolation of four serendipitous C15 phenylated final products. In vitro antiproliferative activity and Hsp90 binding affinity of the compounds were determined, suggesting the C18-oxygen of herbimycin A is removable and bulky lipophilic groups can be accommodated at C15 without loss of activity.

Ansalactams B-D Illustrate Further Biosynthetic Plasticity within the Ansamycin Pathway.

Further chemical investigation of a marine-derived bacterium of the genus Streptomyces has led to the isolation of ansalactams B-D (1-3) along with the previously reported metabolite ansalactam A (4). Ansalactams B-D are significantly modified ansamycins, representing three new carbon skeletons and further illustrating the biosynthetic plasticity of the ansalactam class. Unlike ansalactam A, ansalactams B and D are penta- and hexacyclic metabolites, while ansalactam C illustrates an open polyene chain with a terminal carboxylic acid.