Dual-Mechanism Confers Self-Resistance to the Antituberculosis Antibiotic Capreomycin.

Capreomycin (CMN) is an important second-line antituberculosis antibiotic isolated from Saccharothrix mutabilis subspecies capreolus. The gene cluster for CMN biosynthesis has been identified and sequenced, wherein the cph gene was annotated as a phosphotransferase likely engaging in self-resistance. Previous studies reported that Cph inactivates two CMNs, CMN IA and IIA, by phosphorylation. We, herein, report that (1) Escherichia coli harboring the cph gene becomes resistant to both CMN IIA and IIB, (2) phylogenetic analysis regroups Cph to a new clade in the phosphotransferase protein family, (3) Cph shares a three-dimensional structure akin to the aminoglycoside phosphotransferases with a high binding affinity (KD) to both CMN IIA and IIB at micromolar levels, and (4) Cph utilizes either ATP or GTP as a phosphate group donor transferring its γ-phosphate to the hydroxyl group of CMN IIA. Until now, Cph and Vph (viomycin phosphotransferase) are the only two known enzymes inactivating peptide-based antibiotics through phosphorylation. Our biochemical characterization and structural determination conclude that Cph confers the gene-carrying species resistance to CMN by means of either chemical modification or physical sequestration, a naturally manifested belt and braces strategy. These findings add a new chapter into the self-resistance of bioactive natural products, which is often overlooked while designing new bioactive molecules.

The Long Noncoding RNA Hepatocyte Nuclear Factor 4α Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications.

The maintenance of homeostasis of cytochromes P450 enzymes (P450s) under both physiologic and xenobiotic exposure conditions is ensured by the action of positive and negative regulators. In the current study, the hepatocyte nuclear factor 4α (HNF4A) antisense RNA 1 (HNF4A-AS1), an antisense long noncoding RNA of HNF4A, was found to be a negative regulator of the basal and rifampicin (RIF)-induced expression of nuclear receptors and downstream P450s. In Huh7 cells, knockdown of HNF4A-AS1 resulted in elevated expression of HNF4A, pregnane X receptor (PXR), and P450s (including CYP3A4) under both basal and RIF-induced conditions. Conversely, overexpression of HNF4A-AS1 led to decreased basal expression of constitutive androstane receptor, aryl hydrocarbon receptor, PXR, and all studied P450s. Of note, significantly diminished induction levels of PXR and CYP1A2, 2C8, 2C19, and 3A4 by RIF were also observed in HNF4A-AS1 plasmid-transfected Huh7 cells. Moreover, the negative feedback of HNF4A on HNF4A-AS1-mediated gene expression was validated using a loss-of-function experiment in this study. Strikingly, our data showed that increased enrichment levels of histone 3 lysine 4 trimethylation and HNF4A in the CYP3A4 promoter contribute to the elevated CYP3A4 expression after HNF4A-AS1 knockdown. Overall, the current study reveals that histone modifications contribute to the negative regulation of nuclear receptors and P450s by HNF4A-AS1 in basal and drug-induced levels. SIGNIFICANCE STATEMENT: Utilizing loss-of-function and gain-of-function experiments, the current study systematically investigated the negative regulation of HNF4A-AS1 on the expression of nuclear receptors (including HNF4A, constitutive androstane receptor, aryl hydrocarbon receptor, and pregnane X receptor) and P450s (including CYP1A2, 2E1, 2B6, 2D6, 2C8, 2C9, 2C19, and 3A4) in both basal and rifampicin-induced levels in Huh7 cells. Notably, this study is the first to reveal the contribution of histone modification to the HNF4A-AS1-mediated expression of CYP3A4 in Huh7 cells.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Development and validation of a simple chromatographic method for simultaneous determination of clindamycin phosphate and rifampicin in skin permeation studies.

Rifampicin (RIF) and clindamycin phosphate (CDM) are the main drugs currently used in combination to treat severe infectious diseases in hair follicles. This work describes a simple, rapid and sensitive method for simultaneous analysis of RIF and CDM in the different skin layers using high performance liquid chromatography (HPLC). The efficient chromatographic separation of CDM and RIF was succeeded using a C18 column (150 mm x 4.6 mm, 5 µm) with gradient elution using a mobile phase composed of 0.01 M phosphoric acid and methanol at a flow rate of 1 mL min-1. Determinations were performed using UV-vis detector at 200 nm and 238 nm for CDM and RIF, respectively. The method was precise, accurate and linear (r2 > 0.999) with regression curve in the concentration range from 0.5 to 20.0 µg mL-1 and recovery rates from the skin layers higher than 85%. The retention times for CDM and RIF were approximately 7.4 and 12.2 min, respectively. The presence of skin components did not interfere with the analysis. The validated method was therefore appropriate for quantification of both CDM and RIF and thus may be feasible to be used in skin permeation studies.

Molecular insight into multiple RpoB clinical mutants of Mycobacterium tuberculosis: An attempt to probe structural variations in rifampicin binding site underlying drug resistance.

Rifampicin (RMP) resistant strains are still persisting as a threat to the global TB control program. Therefore, understanding the RMP resistant mechanism is need of the hour. The current study has investigated the role of each RMP binding site (RBS) residues, deploying computational alanine scanning mutagenesis (CASM) to unravel the critical and non-critical binding site residues. In addition, conformational shifts in RBS cavity of different RNAP ß-subunit (RpoB) systems have also been analyzed. Our initial findings showed that in addition to reported mutational sites, Q510, Q513, R529, P564 and P566 were also critical binding site residues, which upon mutation destabilize the RMP binding. Study also indicated that R3, R4 and R5 regions of RpoB were very significant for the functioning of RMP. The concerted interactions of these regions with RMP hold it into the centre position of RBS and restrict the entrance of elongating RNA transcript. Whereas, after mutation, due to repositioning of RMP and changes in its interactions, the overall cavity becomes significantly hollow which may confer space for phosphodiester bond formation during transcription elongation. Our findings provide valuable details to forestall RMP resistance and may help in the development of new leads against the RMP resistant strains.

Poly(ethylene carbonate)-containing polylactic acid microparticles with rifampicin improve drug delivery to macrophages.

OBJECTIVE: Pulmonary delivery of antibiotics will decrease the required dose for efficient treatment of lung infections and reduce systemic side effects of the drug. The objective was to evaluate the applicability of poly(ethylene carbonate) (PEC) for the preparation of inhalable, antibiotic-containing particles. METHODS: Rifampicin (RF)-loaded microparticles were prepared by electrospraying a carrier matrix of polylactic acid (PLA) with 0%, 5% and 10% PEC. KEY FINDINGS: Prepared particles had an aerodynamic diameter between 4 and 5 µm. Within 60 min, PEC-containing particles released 35-45% of RF, whereas PLA particles released only 15% of RF. Irrespective of particle composition, uptake of RF by macrophages was improved to 40-60% when formulated in microparticles compared to 0.4% for RF in solution, and intracellular localisation of particles was confirmed using confocal microscopy. Effect on macrophage and alveolar cell viability was similar for all particles whereas the minimal inhibitory concentrations against Pseudomonas aeruginosa and Escherichia coli for RF-containing PEC particles were twofold lower than for PLA particles, explained by the faster release of RF from PEC-containing particles. CONCLUSIONS: The inclusion of PEC in PLA microparticles increased the release of RF and the inhibitory effect against two bacteria species while displaying physical particle properties similar to PLA particles.

Development of Novel Octanoyl Chitosan Nanoparticles for Improved Rifampicin Pulmonary Delivery: Optimization by Factorial Design.

A novel hydrophobic chitosan derivative, octanoyl chitosan (OC) with improved organic solubility was synthesized, characterized, and employed for the preparation of rifampicin (Rif) encapsulated nanoparticle formulations for pulmonary delivery. OC was characterized to confirm acyl group substitution and cytotoxicity in A549 epithelial lung cells. OC nanoparticles were produced by the double emulsion solvent evaporation technique without cross-linking and characterized for particle size distribution, morphology, crystallinity, thermal stability, aerosol delivery, and drug release rate. OC was successfully synthesized with substitution degree of 44.05 ± 1.75%, and solubility in a range of organic solvents. Preliminary cytotoxicity studies of OC showed no effect on cell viability over a period of 24 h on A549 cell lines. OC nanoparticles were optimized using a 32 full factorial design. An optimized batch of OC nanoparticles, smooth and spherical in morphology, had mean hydrodynamic diameter of 253 ± 19.06 nm (PDI 0.323 ± 0.059) and entrapment efficiency of 64.86 ± 7.73% for rifampicin. Pulmonary deposition studies in a two-stage impinger following aerosolization of nanoparticles from a jet nebulizer gave a fine particle fraction of 43.27 ± 4.24%. In vitro release studies indicated sustained release (73.14 ± 3.17%) of rifampicin from OC nanoparticles over 72 h, with particles demonstrating physical stability over 2 months. In summary, the results confirmed the suitability of the developed systems for pulmonary delivery of drugs with excellent aerosolization properties and sustained-release characteristics.

Fidaxomicin jams Mycobacterium tuberculosis RNA polymerase motions needed for initiation via RbpA contacts.

Fidaxomicin (Fdx) is an antimicrobial RNA polymerase (RNAP) inhibitor highly effective against Mycobacterium tuberculosis RNAP in vitro, but clinical use of Fdx is limited to treating Clostridium difficile intestinal infections due to poor absorption. To identify the structural determinants of Fdx binding to RNAP, we determined the 3.4 Å cryo-electron microscopy structure of a complete M. tuberculosis RNAP holoenzyme in complex with Fdx. We find that the actinobacteria general transcription factor RbpA contacts fidaxomycin, explaining its strong effect on M. tuberculosis. Additional structures define conformational states of M. tuberculosis RNAP between the free apo-holoenzyme and the promoter-engaged open complex ready for transcription. The results establish that Fdx acts like a doorstop to jam the enzyme in an open state, preventing the motions necessary to secure promoter DNA in the active site. Our results provide a structural platform to guide development of anti-tuberculosis antimicrobials based on the Fdx binding pocket.

Inhibition of D-Ala:D-Ala ligase through a phosphorylated form of the antibiotic D-cycloserine.

D-cycloserine is an antibiotic which targets sequential bacterial cell wall peptidoglycan biosynthesis enzymes: alanine racemase and D-alanine:D-alanine ligase. By a combination of structural, chemical and mechanistic studies here we show that the inhibition of D-alanine:D-alanine ligase by the antibiotic D-cycloserine proceeds via a distinct phosphorylated form of the drug. This mechanistic insight reveals a bimodal mechanism of action for a single antibiotic on different enzyme targets and has significance for the design of future inhibitor molecules based on this chemical structure.

Molecular screening of xerophilic Aspergillus strains producing mycophenolic acid.

Mycophenolic acid (MPA) is the fungal secondary metabolite displaying several biological properties. Up to now, screening of fungal strains producing MPA has mainly been the result of the search of this molecule in their culture medium by chemical methods. Here we developed a molecular approach by targeting the expression level of the MpaC gene encoding the polyketide synthase, one of the key enzymes involved in the MPA synthesis. Thirty xerophilic Aspergillus strains were identified using the RNA polymerase II subunit and the ß-tubulin genes. Seven Aspergillus species were evidenced. The expression level of the MpaC gene was quantified and compared to the MPA production rate. Only Aspergillus pseudoglaucus and all the eight strains of this species produced MPA. While the MpaC gene was not expressed or weakly expressed in the MPA non-producing strains, all the A. pseudoglaucus strains presented a high level of expression of this gene. The highest expression level of the MpaC gene among the MPA non-producing strains was significantly lower than the lowest expression level of this gene in the MPA producing strains. To our knowledge, this is the first study that demonstrates the effectiveness of molecular approach for the screening of MPA-producing species.

Clementine juice has the potential for drug interactions - In vitro comparison with grapefruit and mandarin juice.

Adverse drug interactions due to grapefruit juice are well known prompting warnings even in drug labels. Similar issues have not been reported for clementines and available data is scarce, despite of genetic descent. We observed substantially increased tacrolimus trough concentrations in a renal transplant patient consuming high clementine amounts and, thus, scrutinised the effects of clementine juice on drug metabolism and drug transporters in vitro and compared it to the effects of mandarin and grapefruit juice. All citrus juices profoundly induced several drug transporters and drug metabolising enzymes, whereas the effects of grapefruit juice were most pronounced (e.g. 156-fold and 34-fold induction of cytochrome P450 (CYP) 3A4 mRNA by grapefruit juice and clementine juice, respectively). However, the juices also inhibited e.g. CYP3A4, raising the question which effect prevails in vivo. Using an enzymatic activity assay, we demonstrated that at least in vitro CYP3A4 inhibition prevails for both grapefruit and clementine juice, whereas for CYP1A2 induction appears to predominate. Thus, inhibition of CYP3A4 is presumably the underlying reason for the observed increase in the concentrations of the CYP3A4 substrate tacrolimus in the patient. Taken together, our data indicate that clementine juice as well as grapefruit juice and to a lesser extent also mandarin juice can induce several important drug metabolising enzymes and drug transporters, but also inhibit some of these proteins. Our data indicate that clementine juice similar to grapefruit juice bears the potential for profound interactions with drugs potentially leading to adverse drug effects e.g. through over-exposure to CYP3A4 substrates.

Design of a nanostructured lipid carrier intended to improve the treatment of tuberculosis.

This work aimed to design, develop, and characterize a lipid nanocarrier system for the selective delivery of rifabutin (RFB) to alveolar macrophages. Lipid nanoparticles, specifically nanostructured lipid carriers (NLC), were synthetized by the high-shear homogenization and ultrasonication techniques. These nanoparticles were designed to exhibit both passive and active targeting strategies to be efficiently internalized by the alveolar macrophages, traffic to the acidified phagosomes and phagolysosomes, and release bactericidal concentrations of the antituberculosis drug intracellularly. NLC that could entrap RFB were prepared, characterized, and further functionalized with mannose. Particles' diameter, zeta potential, morphology, drug% entrapping efficiency, and drug release kinetics were evaluated. The mannose coating process was confirmed by Fourier transform infrared. Further, the cytotoxicity of the formulations was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay in A549, Calu-3, and Raw 264.7 cells. The diameter of NLC formulations was found to be in the range of 175-213 nm, and drug entrapping efficiency was found to be above 80%. In addition, high storage stability for the formulations was expected since they maintained the initial characteristics for 6 months. Moreover, the drug release was pH-sensitive, with a faster drug release at acidic pH than at neutral pH. These results pose a strong argument that the developed nanocarrier can be explored as a promising carrier for safer and more efficient management of tuberculosis by exploiting the pulmonary route of administration.

Rifamycins, Alone and in Combination.

Rifamycins inhibit RNA polymerase of most bacterial genera. Rifampicin remains part of combination therapy for treating tuberculosis (TB), and for treating Gram-positive prosthetic joint and valve infections, in which biofilms are prominent. Rifabutin has use for AIDS patients in treating mycobacterial infections TB and Mycobacterium avium complex (MAC), having fewer drug-drug interactions that interfere with AIDS medications. Rifabutin is occasionally used in combination to eradicate Helicobacter pylori (peptic ulcer disease). Rifapentine has yet to fulfill its potential in reducing time of treatment for TB. Rifaximin is a monotherapeutic agent to treat gastrointestinal (GI) disorders, such as hepatic encephalopathy, irritable bowel syndrome, and travelers' diarrhea. Rifaximin is confined to the GI tract because it is not systemically absorbed on oral dosing, achieving high local concentrations, and showing anti-inflammatory properties in addition to its antibacterial activity. Resistance issues are unavoidable with all the rifamycins when the bioburden is high, because of mutations that modify RNA polymerase.

Development and Evaluation of Chitosan Microparticles Based Dry Powder Inhalation Formulations of Rifampicin and Rifabutin.

BACKGROUND: The lung is the primary entry site and target for Mycobacterium tuberculosis; more than 80% of the cases reported worldwide are of pulmonary tuberculosis. Hence, direct delivery of anti-tubercular drugs to the lung would be beneficial in reducing both, the dose required, as well as the duration of therapy for pulmonary tuberculosis. In the present study, microsphere-based dry powder inhalation systems of the anti-tubercular drugs, rifampicin and rifabutin, were developed and evaluated, with a view to achieve localized and targeted delivery of these drugs to the lung. METHODS: The drug-loaded chitosan microparticles were prepared by an ionic gelation method, followed by spray-drying to obtain respirable particles. The microparticles were evaluated for particle size and drug release. The drug-loaded microparticles were then adsorbed onto an inhalable lactose carrier and characterized for in vitro lung deposition on an Andersen Cascade Impactor (ACI) followed by in vitro uptake study in U937 human macrophage cell lines. In vivo toxicity of the developed formulations was evaluated using Sprague Dawley rats. RESULTS: Both rifampicin and rifabutin-loaded microparticles had MMAD close to 5 µm and FPF values of 21.46% and 29.97%, respectively. In vitro release study in simulated lung fluid pH 7.4 showed sustained release for 12 hours for rifampicin microparticles and up to 96 hours for rifabutin microparticles, the release being dependent on both swelling of the polymer and solubility of the drugs in the dissolution medium. In vitro uptake studies in U937 human macrophage cell line suggested that microparticles were internalized within the macrophages. In vivo acute toxicity study of the microparticles in Sprague Dawley rats revealed no significant evidence for local adverse effects. CONCLUSION: Thus, spray-dried microparticles of the anti-tubercular drugs, rifampicin and rifabutin, could prove to be an improved, targeted, and efficient system for treatment of tuberculosis.

Guía de la Sociedad Española de Infectología Pediátrica sobre tuberculosis en la embarazada y el recién nacido (II): profilaxis y tratamiento / Spanish Society for Pediatric Infectious Diseases guidelines on tuberculosis in pregnant women and neonates (II): Prophylaxis and treatment

En la embarazada expuesta a tuberculosis (TB) no se recomienda profilaxis primaria con isoniazida salvo en gestantes inmunodeprimidas, con enfermedades crónicas o factores de riesgo obstétrico y antecedente de contacto íntimo mantenido con un enfermo bacilífero. En la infección tuberculosa latente (ITBL) se iniciará profilaxis con isoniazida si existe contacto reciente con TB o factores de riesgo de progresión a TB activa. En caso contrario, se retrasará hasta al menos 3 semanas después del parto. El tratamiento de la enfermedad tuberculosa es el mismo que fuera de la gestación. Los recién nacidos de madres con historia gestacional de TB diseminada o extrapulmonar, con TB activa en el parto o con contacto TB posnatal conocido, asintomáticos y con pruebas diagnósticas negativas, deben recibir profilaxis primaria con isoniazida durante al menos 12 semanas. Transcurrido ese tiempo se repiten los test diagnósticos, y si son negativos, se interrumpe la profilaxis. En la ITBL, se administrará isoniazida durante 9 meses. En la enfermedad tuberculosa, el tratamiento es el mismo que en el niño mayor pero mantenido al menos 9 meses. Se recomienda aislamiento respiratorio en la TB congénita y en la TB posnatal con baciloscopia positiva en jugo gástrico o aspirado bronquial. La separación madre-hijo solo está indicada en madres que han recibido tratamiento durante menos de 2 semanas, presentan baciloscopia positiva o tienen TB resistente. La lactancia materna no está contraindicada y en las situaciones de separación la madre puede extraerse la leche para que sea administrada en biberón al recién nacido

In pregnant women who have been exposed to tuberculosis (TB), primary isoniazid prophylaxis is only recommended in cases of immunosuppression, chronic medical conditions or obstetric risk factors, and close and sustained contact with a patient with infectious TB. Isoniazid prophylaxis for latent tuberculosis infection (LTBI) is recommended in women who have close contact with an infectious TB patient or have risk factors for progression to active disease. Otherwise, it should be delayed until at least three weeks after delivery. Treatment of TB disease during pregnancy is the same as for the general adult population. Infants born to mothers with disseminated or extrapulmonary TB in pregnancy, with active TB at delivery, or with postnatal exposure to TB, should undergo a complete diagnostic evaluation. Primary isoniazid prophylaxis for at least 12 weeks is recommended for those with negative diagnostic tests and no evidence of disease. Repeated negative diagnostic tests are mandatory before interrupting prophylaxis. Isoniazid for 9 months is recommended in LTBI. Treatment of neonatal TB disease is similar to that of older children, but should be maintained for at least 9 months. Respiratory isolation is recommended in congenital TB, and in postnatal TB with positive gastric or bronchial aspirate acid-fast smears. Separation of mother and infant is only necessary when the mother has received treatment for less than 2 weeks, is sputum smear-positive, or has drug-resistant TB. Breastfeeding is not contraindicated, and in case of mother-infant separation expressed breast milk feeding is recommended

Primera serie de pacientes con TB-XDR y pre-XDR tratados con esquemas que incluyeron meropenem-clavulanato en Argentina / First Series of Patients With XDR and Pre-XDR TB Treated With Regimens That Included Meropenen-clavulanate in Argentina

La tuberculosis extensamente resistente (TB-XDR) y pre-XDR comprometen seriamente el pronóstico de la enfermedad, y su tratamiento requiere inevitablemente el uso de fármacos del grupo V (Organización Mundial de la Salud [OMS]). Se analizó retrospectivamente la evolución de todos los pacientes con TB pre-XDR y XDR asistidos en un servicio especializado durante 2012 y 2013, medicados con regímenes que incluyeron por lo menos 6 meses de meropenem-clavulanato (MPC), capreomicina, moxifloxacina, linezolid, clofazimina, isoniacida en alta dosis, PAS y en un caso bedaquilina. Fueron tratados 10 pacientes, 9 de ellos con un extenso patrón de resistencia a un mínimo de 6 fármacos y uno por reacciones adversas e interacciones medicamentosas que generaron una situación análoga. Ocho de los 10 pacientes tratados, hicieron la conversión bacteriológica del esputo (2 cultivos mensuales consecutivos negativos) en un lapso de 2 a 7 meses, en tanto que 2 fallecieron. No se observaron reacciones adversas atribuibles a la administración prolongada del MPC

XDR (extensively drug-resistant) and pre-XDR tuberculosis (TB) seriously compromise prognosis and treatment possibilities, and inevitably require the use of group V drugs (World Health Organization). The progress of all patients with XDR and pre-XDR TB seen in a specialized unit during 2012 and 2013 and treated with regimens that included at least 6 months of meropenem-clavulanate (MPC), capreomycin, moxifloxacin, linezolid, clofazimine, high-dose isoniazid, PAS, and bedaquiline in 1 case, were retrospectively analyzed. Ten patients were treated, 9 with an extensive pattern of resistance to at least 6 drugs, and 1 because of adverse reactions and drug interactions leading to a similar situation. Eight of the 10 patients treated achieved bacteriological sputum conversion (2 consecutive negative monthly cultures) over a period of 2–7 months, while 2 died. No adverse reactions attributable to prolonged administration of MPC were observed

Attenuation of anti-tuberculosis therapy induced hepatotoxicity by Spirulina fusiformis, a candidate food supplement.

Therapy using Isoniazid (INH) and Rifampicin (RIF) leads to induction of hepatotoxicity in some individuals undergoing anti-tuberculosis treatment. In this study, we assessed the effect of Spirulina fusiformis on INH and RIF induced hepatotoxicity in rats compared with hepatoprotective drug Silymarin. Induction of hepatotoxicity was measured by changes in the liver marker enzymes (aspartate transaminase, alanine transaminase, and alkaline phosphatase). The antioxidant status was also analyzed in liver tissue homogenate and plasma by measurement of superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, and lipid peroxidation levels. We also aimed to study the binding and interactions of the transcription factors Pregnane X Receptor (PXR) and Farnesoid X Receptor (FXR) with INH, RIF, and representative active compounds of Spirulina fusiformis by in silico methods. The administration of INH and RIF resulted in significant (p < 0.05) decrease in the antioxidant levels and total protein levels. There was also a significant (p < 0.05) increase in the levels of liver marker enzymes. Spirulina fusiformis was seen to protect the parameters from significant changes upon challenge with INH and RIF in a dose-dependent manner. This was corroborated by histological examination of the liver. The results of the in silico analyses further support the wet lab results.

Redesign of substrate specificity and identification of the aminoglycoside binding residues of Eis from Mycobacterium tuberculosis.

The upsurge in drug-resistant tuberculosis (TB) is an emerging global problem. The increased expression of the enhanced intracellular survival (Eis) protein is responsible for the clinical resistance to aminoglycoside (AG) antibiotics of Mycobacterium tuberculosis . Eis from M. tuberculosis (Eis_Mtb) and M. smegmatis (Eis_Msm) function as acetyltransferases capable of acetylating multiple amines of many AGs; however, these Eis homologues differ in AG substrate preference and in the number of acetylated amine groups per AG. The AG binding cavity of Eis_Mtb is divided into two narrow channels, whereas Eis_Msm contains one large cavity. Five bulky residues lining one of the AG binding channels of Eis_Mtb, His119, Ile268, Trp289, Gln291, and Glu401, have significantly smaller counterparts in Eis_Msm, Thr119, Gly266, Ala287, Ala289, and Gly401, respectively. To identify the residue(s) responsible for AG binding in Eis_Mtb and for the functional differences from Eis_Msm, we have generated single, double, triple, quadruple, and quintuple mutants of these residues in Eis_Mtb by mutating them into their Eis_Msm counterparts, and we tested their acetylation activity with three structurally diverse AGs: kanamycin A (KAN), paromomyin (PAR), and apramycin (APR). We show that penultimate C-terminal residue Glu401 plays a critical role in the overall activity of Eis_Mtb. We also demonstrate that the identities of residues Ile268, Trp289, and Gln291 (in Eis_Mtb nomenclature) dictate the differences between the acetylation efficiencies of Eis_Mtb and Eis_Msm for KAN and PAR. Finally, we show that the mutation of Trp289 in Eis_Mtb into Ala plays a role in APR acetylation.

Reactive rifampicin derivative able to damage transcription complex.

Rifampicin (Rif) is powerful broad spectrum antibiotic that targets bacterial RNA polymerase (RNAP) by blocking the transcript exit channel. The performance of the drug can be further enhanced by tagging with active chemical groups that produce collateral damage. We explored this principle by tethering Rif to Fe(2+)-EDTA chelate. Modified drug retained high binding affinity to RNAP and caused localized cleavage of the enzyme and promoter DNA. Analysis of the degradation products revealed the cleavage of RNAP ß subunit at the sites involved in the drug binding, while DNA was selectively seized in the vicinity of the transcription start site. The synthesized Rif derivative exemplifies "aggressive" types of drugs that can be especially useful for TB treatment by attacking the nongrowing dormant form of the mycobacterium, which is hardly susceptible to "passive" drugs.