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.

Design and in vitro assessment of chitosan nanocapsules for the pulmonary delivery of rifabutin.

Tuberculosis (TB) is a life-threatening disease and a main cause of death worldwide. It mainly affects the lungs, and it is attributed to the infection with Mycobacterium tuberculosis (MTB). Current treatments consist of the oral administration of combinations of antibiotics including rifabutin, in high doses and for long periods of time. These therapeutic regimens are associated with many side effects and high rates of drug resistance. To overcome these problems, this study aims at developing a nanosystem for the improved delivery of antibiotics, with potential application in pulmonary delivery. Chitosan-based nanomaterials are widely used in biomedical applications, due to their biodegradability and biocompatibility, as well as their potential antimicrobial effects and lack of toxicity. In addition, this polymer is particularly attractive for mucosal delivery due to its bioadhesive properties. Therefore, the structure of the proposed nanocarrier consists of a chitosan shell and a lipid core with a combination of different oils and surfactants to allow optimal association of the hydrophobic drug rifabutin. These nanocapsules were characterized in terms of size, polydispersity index, surface charge, morphology, encapsulation efficiency and biological stability. The release kinetics of the drug-loaded nanostructures was evaluated in simulated lung media. Moreover, in vitro studies in different cell models (A549 and Raw 264.7 cells) demonstrated the safety of the nanocapsules as well as their efficient internalization. An antimicrobial susceptibility test was performed to evaluate the efficacy of the rifabutin-loaded nanocapsules against Mycobacterium phlei. This study indicated complete inhibition for antibiotic concentrations within the expected susceptibility range of Mycobacterium (≤ 0.25-16 mg/L).

Nagimycins A and B, Antibacterial Ansamycin-Related Macrolactams from Streptomyces sp. NA07423.

Chemical investigation of Streptomyces sp. NA07423 led to the discovery of two unreported macrolactams, nagimycins A (1) and B (2). Their structures were elucidated by NMR, HRESIMS, X-ray crystallography, and comparison of experimental and theoretical ECD spectra. The nagimycins have a unique butenolide moiety rarely found in ansamycin antibiotics. Genome analysis revealed the putative biosynthetic gene cluster for nagimycins, and a likely biosynthetic pathway was proposed. Notably, compounds 1 and 2 exhibited potent antibacterial activity against two pathogenic Xanthomonas bacteria.

Structural diversity and biological relevance of benzenoid and atypical ansamycins and their congeners.

Covering: 2011 to 2021The structural division of ansamycins, including those of atypical cores and different lengths of the ansa chains, is presented. Recently discovered benzenoid and atypical ansamycin scaffolds are presented in relation to their natural source and biosynthetic routes realized in bacteria as well as their muta and semisynthetic modifications influencing biological properties. To better understand the structure-activity relationships among benzenoid ansamycins structural aspects together with mechanisms of action regarding different targets in cells, are discussed. The most promising directions for structural optimizations of benzenoid ansamycins, characterized by predominant anticancer properties, were discussed in view of their potential medical and pharmaceutical applications. The bibliography of the review covers mainly years from 2011 to 2021.

Heat Shock Protein 90 Inhibitor Effects on Pancreatic Cancer Cell Cultures.

OBJECTIVES: Pancreatic ductal adenocarcinoma is one of the deadliest cancers for which few curative therapies are available to date. Heat shock protein 90 (Hsp90) inhibitors have shown activity against numerous cancers in vitro; therefore, we tested whether they could be used to target pancreatic ductal adenocarcinoma. METHODS: Inhibitors of Hsp90 ATPase activity were applied on low-passage pancreatic cell line cultures (Panc10.05, Panc215, A6L) in a dose-response manner, and the inhibitor in vitro effect on cell growth was evaluated. Seven of novel Hsp90 inhibitors based on resorcinol fragment and 5 commercially available Hsp90 inhibitors (17-AAG, AT-13387, AUY-922, ganetespib, and rifabutin) as well as control compound triptolide were tested yielding IC50 values in 2- and 3-dimensional assays. RESULTS: The novel Hsp90 inhibitors exhibited strong effects on all 3 tested pancreatic cell line cultures (Panc10.05, Panc215, A6L) reaching the IC50 of 300 to 600 nM in 2- and 3-dimensional assays. CONCLUSIONS: Novel Hsp90 inhibitors can be developed as antipancreatic cancer agents. Their chemical structures are simpler, and they are likely to exhibit lower side effects than the much more complex inhibitors used as controls.

Prediction of potential inhibitors for RNA-dependent RNA polymerase of SARS-CoV-2 using comprehensive drug repurposing and molecular docking approach.

The pandemic prevalence of COVID-19 has become a very serious global health issue. Scientists all over the world have been seriously attempting in the discovery of a drug to combat SARS-CoV-2. It has been found that RNA-dependent RNA polymerase (RdRp) plays a crucial role in SARS-CoV-2 replication, and thus could be a potential drug target. Here, comprehensive computational approaches including drug repurposing and molecular docking were employed to predict an effective drug candidate targeting RdRp of SARS-CoV-2. This study revealed that Rifabutin, Rifapentine, Fidaxomicin, 7-methyl-guanosine-5'-triphosphate-5'-guanosine and Ivermectin have a potential inhibitory interaction with RdRp of SARS-CoV-2 and could be effective drugs for COVID-19. In addition, virtual screening of the compounds from ZINC database also allowed the prediction of two compounds (ZINC09128258 and ZINC09883305) with pharmacophore features that interact effectively with RdRp of SARS-CoV-2, indicating their potentiality as effective inhibitors of the enzyme. Furthermore, ADME analysis along with analysis of toxicity was also undertaken to check the pharmacokinetics and drug-likeness properties of the two compounds. Comparative structural analysis of protein-inhibitor complexes revealed that the amino acids Y32, K47, Y122, Y129, H133, N138, D140, T141, S709 and N781 are crucial for drug surface hotspot in the RdRp of SARS-CoV-2.

On-PKS Baeyer-Villiger-Type O-Atom Insertion Catalyzed by Luciferase-Like Monooxygenase OvmO during Olimycin Biosynthesis.

A silent ansamycin biosynthetic gene cluster (ovm) was activated in Streptomyces olivaceus SCSIO T05 following mutagenesis and media optimization. A new shunt product, olimycin C (1a) was produced by the ovmO-inactivated mutant strain, along with a minor product, olimycin D (1b). The production of these linear olimycin counterparts suggest that luciferase-like monooxygenase (LLM) OvmO catalyzes an on-PKS Baeyer-Villiger-type oxidation during assembly of the olimycin A (2) linear polyketide backbone.

Rifabutin Is Active against Mycobacterium abscessus in Mice.

There is no reliable cure for Mycobacterium abscessus lung disease. Rifampin is not used clinically due to poor in vitro potency. In contrast, we have shown that rifabutin, another approved rifamycin used to treat tuberculosis, is potent in vitro against M. abscessus Here, we report that rifabutin is as active as clarithromycin against M. abscessus K21 in NOD.CB17-Prkdcscid/NCrCrl mice. This suggests that rifabutin should be considered a repurposing candidate for patients with M. abscessus disease.

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.

Inhalable chitosan microparticles for simultaneous delivery of isoniazid and rifabutin in lung tuberculosis treatment.

The direct delivery of antibiotics to the lung has been considered an effective approach to treat pulmonary tuberculosis, which represents approximately 80% of total cases. In this sense, this work aimed at producing inhalable chitosan microparticles simultaneously associating isoniazid and rifabutin, for an application in pulmonary tuberculosis therapy. Spray-dried chitosan microparticles were obtained with adequate flow properties for deep lung delivery (aerodynamic diameter of 4 µm) and high drug association efficiencies (93% for isoniazid and 99% for rifabutin). The highest concentration of microparticles that was tested (1 mg/mL) decreased the viability of macrophage-differentiated THP-1 cells to around 60% after 24 h exposure, although no deleterious effect was observed in human alveolar epithelial (A549) cells. The release of LDH was, however, increased in both cells. Chitosan microparticles further evidenced capacity to activate macrophage-like cells, inducing cytokine secretion well above basal levels. Moreover, the propensity of macrophages to internalize microparticles was demonstrated, with uptake levels over 90%. Chitosan microparticles also inhibited bacterial growth by 96%, demonstrating that the microencapsulation preserved drug antibacterial activity in vitro. Overall, the obtained data suggest the potential of chitosan microparticles for inhalable lung tuberculosis therapy.

Nano-Rifabutin entrapment within glucan microparticles enhances protection against intracellular Mycobacterium tuberculosis.

Recently, yeast-derived glucan particles (GP) have emerged as novel drug delivery agents that provide for receptor-mediated uptake by phagocytic cells expressing ß-glucan receptors. In our previous study, we prepared GP loaded with high payload (40.5 + 1.9%) of rifabutin (RB) nano-particles [(RB-NPs)-GP]. We investigated the anti-mycobacterial efficacy and cellular activation responses within Mycobacterium tuberculosis (M. tuberculosis) infected J774 macrophage cells following exposure to the (RB-NPs)-GP formulation. The exposure was seen to augment a robust innate immune response including the induction of reactive oxygen and nitrogen species, autophagy and apoptosis within M. tuberculosis infected macrophage. Further, the efficacy testing of these particles in murine macrophage exhibited that the (RB-NPs)-GP formulation enhanced the efficacy of RB drug by ∼2.5 fold. The study suggests that the set of innate responses conducive to killing intracellular bacteria evoked by (RB-NPs)-GP play a pivotal role in impeding the intracellular M. tuberculosis survival, resulting in enhanced efficacy of the formulation. Our results establish that the (RB-NPs)-GP formulation not only activate M. tuberculosis infected, immune-suppressed macrophage, but also adds significantly to the efficacy of loaded drug, and thus forms a promising approach that should be explored further as an alternative or adjunct form of TB therapy. Highlights Nano-Rifabutin loaded Glucan microparticles [(RB-NPs)-GP] administered to M. tuberculosis infected macrophage. (RB-NPs)-GP induces appropriate innate immune responses in host macrophage. Mycobactericidal Effect of Rifabutin was markedly enhanced by its nano-entrapment in GP. Intracellular drug delivery supplements the innate response in M. tuberculosis infected macrophage.

Delimiting the knowledge space and the design space of nanostructured lipid carriers through Artificial Intelligence tools.

Nanostructured lipid carriers (NLC) are biocompatible and biodegradable nanoscale systems with extensive application for controlled drug release. However, the development of optimal nanosystems along with a reproducible manufacturing process is still challenging. In this study, a two-step experimental design was performed and databases were successfully modelled using Artificial Intelligence techniques as an innovative method to get optimal, reproducible and stable NLC. The initial approach, including a wide range of values for the different variables, was followed by a second set of experiments with variable values in a narrower range, more suited to the characteristics of the system. NLC loaded with rifabutin, a hydrophobic drug model, were produced by hot homogenization and fully characterized in terms of particle size, size distribution, zeta potential, encapsulation efficiency and drug loading. The use of Artificial Intelligence tools has allowed to elucidate the key parameters that modulate each formulation property. Stable nanoparticles with low sizes and polydispersions, negative zeta potentials and high drug loadings were obtained when the proportion of lipid components, drug, surfactants and stirring speed were optimized by FormRules® and INForm®. The successful application of Artificial Intelligence tools on NLC formulation optimization constitutes a pioneer approach in the field of lipid nanoparticles.

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.

Pentaketide Ansamycin Microansamycins A-I from Micromonospora sp. Reveal Diverse Post-PKS Modifications.

Overexpression of the pathway-specific positive regulator gene mas13 activated the cryptic gene cluster mas, resulting in the isolation of nine novel pentaketide ansamycins, namely, microansamycins A-I (1-9). These results not only revealed a biosynthetic gene cluster of pentaketide ansamycins for the first time but also presented an unprecedented scenario of diverse post-PKS modifications in ansamycin biosynthesis.

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.

Synthesis of 99mTc-Rifabutin: A Potential Tuberculosis Radiodiagnostic Agent.

BACKGROUND: Rifabutin (RFN) is bactericidal antibiotic with a very broad spectrum of activity against gram positive & gram negative organisms including Pseudomonas aeruginosa and specifically Mycobacterium tuberculosis. RFN inhibits DNA dependent RNA polymerase activity in susceptible cells. In the instant work, the therapeutic characteristics of RFN were intended for diagnostic rationale by labeling it with 99mTc (Technetium-99m). OBJECTIVE: The 99mTc labeled RFN (99mTc-RFN) was investigated for labeling capacity, steadiness in saline & serum, in vitro Mycobacterium tuberculosis (MBT) uptake & distribution in MBT stained animal model rats. METHOD: It was found that 99mTc-RFN prepared by mixing 2 mg of RFN, 2.5 mCi sodium pertechnetate, 150 µg stannous chloride at pH 5.4 gave highest yield after 30 minutes and was intact above 90 % after 240 min at room temperature in saline. RESULT: The 99mTc-RFN showed a stable profile in serum at 37 °C and impurities appeared up 16 h was 15.20 %. The maximum in vitro MBT uptake observed in live strain was 71.75 ± 0.75 %. The premier uptake observed in the MBT infected site (target site) was 14.15 ± 0.00 %, in animal model rat. CONCLUSION: Higher labeling capacity, steadiness in saline & serum, higher MBT uptake, maximum uptake in the MBT infected sites and precise imaging posed 99mTc- RFN as an alternate radio-drug for tuberculosis scintigraphy.

Simultaneous LC-MS-MS Determination of Lopinavir and Rifabutin in Human Plasma.

Tuberculosis (TB) with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome represents the most common infectious diseases worldwide. Anti-TB drugs are used concurrently with antiretroviral drug for treatment of TB-HIV co-morbidities. Due to lower risk of interaction with protease inhibitors, rifabutin is preferred over rifampicin in treatment of HIV and TB co-morbidity. A simple and specific liquid chromatography tandem mass spectrometry method was developed for quantification of rifabutin (RBT) and lopinavir (LPV) simultaneously in human plasma. Following extraction using 60% n-hexane in ethyl acetate, the processed samples were chromatographed on a Discovery HS C18 column (5 µm, 50 × 4.6 mm, id) using mobile phase [85% acetonitrile in ammonium acetate buffer (10 mM, pH 4.5)] at a flow rate of 0.7 mL/min. Mass spectrometric detection was performed in positive electrospray ionization mode using multiple reaction monitoring (RBT, m/z 847.7 → 815.4; LPV, m/z 629.6 → 447.4). Raloxifene and phenacetin were used as internal standards for RBT and LPV, respectively. Linearity was established in the range of 1-1,000 ng/mL and 0.5-10 µg/mL (R2 ≥ 0.99) for RBT and LPV, respectively. The recovery of LPV and RBT were always >90 and >50%, respectively. The precisions and accuracies were well within the acceptable limits of variation.

Simultaneous determination of rifabutin and human serum albumin in pharmaceutical formulations by capillary electrophoresis.

Capillary zone electrophoresis (CZE) was used for determination of rifabutin (RFB), an anti-tuberculosis antibiotic drug, in various pharmaceutical formulations. Apart from that, simultaneous determination of RFB and human serum albumin (HSA) was performed. Electrophoretic behaviour of RFB was examined at various pH levels. CE conditions: a quartz capillary tube (internal diameter 75mm, effective length 50cm, total length 60cm), the capillary temperature was 25°Ð¡, the voltage applied to the capillary tube was +20kV, the UV detection wavelength was 214nm, hydrodynamic injection of the sample was performed at 30mbar for 5s, tetraborate buffer solution (0.01М, рН9.2). The obtained results are characterized by high efficiency (number of theoretical plates up to 260,000) and sufficient sensitivity (LOQ starting from 0.02µg/ml for RFB). The obtained data are in good accord with both HPLC results (for RFB) and spectrophotometry (for HSA).

Lipid Nanocarrier-Mediated Drug Delivery System to Enhance the Oral Bioavailability of Rifabutin.

Rifabutin (RFB) is prescribed for the treatment of tuberculosis infections as well as Mycobacterium avium complex (MAC) infection in immunocompromised individuals and HIV patients. With a view to develop a sustained release oral solid lipid nanoformulation (SLN), RFB was encapsulated in glyceryl monostearate (GMS) nanoparticles. The rifabutin solid lipid nanoparticles (RFB-SLNs), prepared by the solvent diffusion evaporation method, had a size of 345 ± 17.96 nm and PDI of 0.321 ± 0.09. The stability of RFB-SLNs was investigated in simulated gastric fluid (SGF) pH 2.0, simulated intestinal fluid (SIF) pH 6.8 and physiological buffer (PBS) pH 7.4. The gastric medium did not affect the SLNs and were found to be stable, while a sustained release was observed in SIF up to 48 h and in PBS up to 7 days. The pharmacokinetic profile of a single oral administration of RFB-SLNs in mice showed maintenance of therapeutic drug concentrations in plasma for 4 days and in the tissues (lungs, liver and spleen) for 7 days. Oral administration of free RFB showed clearance from plasma within 24 h. The relative bioavailability of RFB from SLNs was five fold higher as compared to administration with free RFB. The intent of using lipid nanocarriers is primarily to enhance the oral bioavailability of rifabutin and eventually decrease the dose and dosing frequency for successful management of MAC infection.