Genetics-directed drug discovery for combating Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb), the pathogen of tuberculosis (TB), is one of the most infectious bacteria in the world. The traditional strategy to combat TB involves targeting the pathogen directly; however, the rapid evolution of drug resistance lessens the efficiency of this anti-TB method. Therefore, in recent years, some researchers have turned to an alternative anti-TB strategy, which hinders Mtb infection through targeting host genes. In this work, using a theoretical genetic analysis, we identified 170 Mtb infection-associated genes from human genetic variations related to Mtb infection. Then, the agents targeting these genes were identified to have high potential as anti-TB drugs. In particular, the agents that can target multiple Mtb infection-associated genes are more druggable than the single-target counterparts. These potential anti-TB agents were further screened by gene expression data derived from connectivity map. As a result, some agents were revealed to have high interest for experimental evaluation. This study not only has important implications for anti-TB drug discovery, but also provides inspirations for streamlining the pipeline of modern drug discovery.     

Design,synthesis and anti-tuberculosis activity of 1-adamantyl-3-heteroaryl ureas with improved in vitro pharmacokinetic properties

Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (sEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: (1) to increase selectivity for anti-TB activity over human sEH activity, (2) to optimize PK profiles including solubility and (3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead isoxazole, oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub μg/mL minimum inhibitory concentrations.     

非一线抗结核药物耐药机制及耐药性诊断研究进展

结核病(Tuberculosis, TB)至今仍是世界三大传染疾病之一。2014年,TB导致的死亡人数已经超过HIV。二线抗TB药物是临床治疗耐多药TB(Multidrug-resistant TB, MDR-TB)的主要药物,然而某些MDR-TB患者由于未及时诊断、治疗方案不合理、所处区域医疗条件差等原因,逐渐发展成为广泛耐药TB(Extensively drug-resistant TB, XDR-TB),使治疗更加困难,其死亡率甚至与肺癌接近。目前结核分枝杆菌(Mycobacterium tuberculosis)的耐药性机制研究已经转向非一线药物,如二线、三线和一些新研发的抗TB药物,揭示这些非一线药物的耐药机制对于耐药TB的治疗和新型抗TB药物的研发具有重要意义。本文对目前临床上使用的主要非一线药物的耐药机制研究进行了综述,并对目前常用的TB耐药性诊断方法的优缺点进行了归纳比较。     

Structure-activity relationships of tyrosinase inhibitory combinatorial library of 2,5-disubstituted-1,3,4-oxadiazole analogues

Here the tyrosinase inhibition studies of library of 2,5-disubstituted-1,3,4-oxadiazoles have been reported and their structure-activity relationship (SAR) also have been discussed. The library of the oxadiazoles was synthesized under the microwave irradiation and was structures of these were characterized by different spectral techniques. From this study it could be concluded that for a better inhibition of tyrosinase, electronegative substitution is essential as most probably the active site of the enzyme contain some hydrophobic site and position is also very important for the inhibition purposes due to the conformational space. The electronegativity of the compounds is somewhat proportional to the inhibitory activity. The compound 3e (3'-[5-(4'-bromophenyl)-1,3,4-oxadiazol-2-yl]pyridine) exhibited most potent (IC50 = 2.18 microM) inhibition against the enzyme tyrosinase which is more potent than the standard potent inhibitor L-mimosine (IC50 = 3.68 microM). This molecule can be the best candidate as a lead compound for further development of drug for the treatments of several skin disorders.     

Nature-inspired Novel Drug Design Paradigm Using Nanosilver: Efficacy on Multi-Drug-Resistant Clinical Isolates of Tuberculosis

Despite discovery of the pathogen more than 100 years ago, tuberculosis (TB) continues to be a major killer disease worldwide. Currently a third of world population is infected and multiple-drug-resistant (mdr) TB registers maximum mortality by a single pathogen. Nanomedicine provides enormous opportunity for developing novel drugs. We have recently demonstrated surface-modified-lipophilic-nanosilica as drug to combat malaria and 100% lethal virus, BmNPV. Nanosilver possesses inherent antibacterial properties, but toxicity is a major concern. We hypothesized that capping with nature-inspired biomolecules, bovine serum albumin (BSA) and Poly-n-vinyl-pyrrolidone (PVP) used as blood volume extender, might insure biosafety. BSA-nano-Ag was found to be more stable than PVP-nano-Ag at physiological pH. In this first ever study on clinical isolates collected from TB endemic areas, we report, BSA-nano-Ag act as potent anti-TB drug. Further study with (human serum albumin)-nano-Ag and core-shell-nano-Ag could increase the biocompatibility of oral TB drug formulations without compromising on the efficacy of the drug.     

Genetic polymorphisms of NAT2, CYP2E1 and GST enzymes and the occurrence of antituberculosis drug-induced hepatitis in Brazilian TB patients

Isoniazid (INH), one of the most important drugs used in antituberculosis (anti-TB) treatment, is also the major drug involved in hepatotoxicity. Differences in INH-induced toxicity have been attributed to genetic variability at several loci, such as NAT2, CYP2E1, GSTM1 and GSTT1, that code for drug-metabolising enzymes. Our goal was to examine the polymorphisms in these enzymes as susceptibility factors to anti-TB drug-induced hepatitis in Brazilian individuals. In a case-control design, 167 unrelated active tuberculosis patients from the University Hospital of the Federal University of Rio de Janeiro, Brazil, were enrolled in this study. Patients with a history of anti-TB drug-induced acute hepatitis (cases with an increase to 3 times the upper limit of normal serum transaminases and symptoms of hepatitis) and patients with no evidence of anti-TB hepatic side effects (controls) were genotyped for NAT2, CYP2E1, GSTM1 and GSTT1 polymorphisms. Slow acetylators had a higher incidence of hepatitis than intermediate/rapid acetylators [22% (18/82) vs. 9.8% (6/61), odds ratio (OR), 2.86, 95% confidence interval (CI), 1.06-7.68, p = 0.04). Logistic regression showed that slow acetylation status was the only independent risk factor (OR 3.59, 95% CI, 2.53-4.64, p = 0.02) for the occurrence of anti-TB drug-induced hepatitis during anti-TB treatment with INH-containing schemes in Brazilian individuals.     

Treating tuberculosis with high doses of anti-TB drugs: mechanisms and outcomes

Tuberculosis (TB) is considered as one of the most serious threats to public health in many parts of the world. The threat is even more severe in the developing countries where there is a lack of advanced medical amenities and contemporary anti-TB drugs. In such situations, dosage optimization of existing medication regimens seems to be the only viable option. Therapeutic drug monitoring study results suggest that high-dose treatment regimens can compensate the low serum concentration of anti-TB drugs and shorten the therapy duration. The article presents a critical review on the possible changes that occur in the host and the pathogen upon the administration of standard and high-dose regimens. Some of the most common factors that are responsible for low anti-TB drug concentrations in the serum are differences in hosts’ body weight, metabolic processing of the drug, malabsorption and/or drug–drug interaction. Furthermore, failure to reach the cavitary pulmonary and extrapulmonary tissues also contributes to the therapeutic inefficiency of the drugs. In such conditions, administration of higher doses can help in compensating the pathogenic outcomes of enhancement of the pathogen’s physical barriers, efflux pumps and genetic mutations. The present article also presents a summary of the recorded treatment outcomes of clinical trials that were conducted to test the efficacy of administration of high dose of anti-tuberculosis drugs. This review will help physicians across the globe to understand the underlying pathophysiological changes (including side effects) that dictate the clinical outcomes in patients administered with standard and/or high dose anti-TB drugs.     

Preparation,Characterization, and In Vitro Cytotoxicity Evaluation of a Novel Anti-Tuberculosis Reconstruction Implant

Background

Reconstruction materials currently used in clinical for osteoarticular tuberculosis (TB) are unsatisfactory due to a variety of reasons. Rifampicin (RFP) is a well-known and highly effective first-line anti-tuberculosis (anti-TB) drug. Poly-DL-lactide (PDLLA) and nano-hydroxyapatite (nHA) are two promising materials that have been used both for orthopedic reconstruction and as carriers for drug release. In this study we report the development of a novel anti-TB implant for osteoarticular TB reconstruction using a combination of RFP, PDLLA and nHA.

Methods

RFP, PDLLA and nHA were used as starting materials to produce a novel anti-TB activity implant by the solvent evaporation method. After manufacture, the implant was characterized and its biodegradation and drug release profile were tested. The in vitro cytotoxicity of the implant was also evaluated in pre-osteoblast MC3T3-E1 cells using multiple methodologies.

Results

A RFP/PDLLA/nHA composite was successfully synthesized using the solvent evaporation method. The composite has a loose and porous structure with evenly distributed pores. The production process was steady and no chemical reaction occurred as proved by Fourier Transform Infrared Spectroscopy (FTIR) and X-Ray Diffraction (XRD). Meanwhile, the composite blocks degraded and released drug for at least 12 weeks. Evaluation of in vitro cytotoxicity in MC3T3-E1 cells verified that the synthesized composite blocks did not affect cell growth and proliferation.

Conclusion

It is feasible to manufacture a novel bioactive anti-TB RFP/PDLLA/nHA composite by the solvent evaporation method. The composite blocks showed appropriate properties such as degradation, drug release and biosafety to MC3T3-E1 cells. In conclusion, the novel composite blocks may have great potential for clinical applications in repairing bone defects caused by osteoarticular TB.     

Epidemiology of anti-tuberculosis drug resistance patterns and trends in tuberculosis referral hospital in Addis Ababa, Ethiopia

ABSTRACT: BACKGROUND: Drug-resistant TB has emerged as a major challenge facing TB prevention and control efforts. In Ethiopia, the extent/trend of drug resistance TB is not well known. The aim of this study was to determine the pattern and trend of resistance to first line anti-TB drugs among culture positive retreatment cases at St.Peter's TB Specialized Hospital. FINDINGS: A hospital based retrospective study was used to assess the pattern of anti-TB drug resistance among previously treated TB patients referred to St.Peter's TB Specialized Hospital from January 2004-December 2008 Gregorian calendar(GC) for better diagnosis and treatment. Among 376 culture positive for M. tuberculosis one hundred and two (27.1%) were susceptible to all of the four first line anti-TB drugs -Isoniazid (INH), Rifampicin (RIF), Ethambutol (ETB) & Streptomycin (STM). While 274 (72.9%) were resistant to at least one drug. Resistance to STM (67.3%) was found to be the most common and the prevalence of MDR-TB was 174 (46.3%). Trend in resistance rate among re-treatment cases from 2004 to 2008 showed a significant increase for any drug as well as for INH, RIF, and MDR resistance (P <0.05 for trend). CONCLUSIONS: There has been an increasing trend in drug resistance in recent years, particularly in retreatment cases. Therefore, establishing advanced diagnostic facilities for early detection of MDR-TB and expanding second line treatment center to treat MDR-TB patients and to prevent its transmission is recommended.     

In silico Structure Modeling and Comparative Analysis of Characterization Properties of Protein Polymers Useful for Protein-Based Nano Particulate Drug Delivery Systems (NPDDS): A Bioinformatics Approach

With an increasing interest in nanoparticulate delivery systems, there is a greater need to identify biomaterials that are biocompatible and safe for human applications. Protein polymers from animal and plant sources are promising materials for designing nanocarriers. Composition of the protein plays an important role for specific drug delivery applications such as drug release, targeting, and stimuli responsive drug release. An important issue in protein polymers is characteristics such as size, charge, and hydrophobicity may play a significant role in phagocytic uptake and initiating a subsequent immune response. This remains to be investigated systematically by analyzing factors that influence nanoparticle characteristics of protein and reduce phagocytic uptake and does not initiate immune response too. Although protein polymers are biodegradable, it is essential to ensure that there must not be premature enzymatic breakdown of the protein nanoparticles in the systemic circulation. Surface modification of the protein nanoparticles can be used to address this issue to propose the necessary modification in the surface of the protein would be great contribution in the nano particulate drug delivery systems (NPPDS). Of the various proteins, gelatin and albumin have been widely studied for drug delivery applications. Plant proteins are yet to be investigated widely for drug delivery applications so there is need to find out the plant proteins capable to act as nanoparticles. The commercial success of albumin-based nanoparticles has created an interest in other proteins. An increased understanding of the physicochemical properties coupled with the developments in rDNA technology will open up new opportunities for protein-based nanoparticulate systems. In the present studies several proteins currently useful for drug delivery system were structurally modeled and has been analyzed to propose the essential characteristics of protein for protein-based NPDDS.     

Optimization of anti-Trypanosoma cruzi oxadiazoles leads to identification of compounds with efficacy in infected mice

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure–activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a–h and 6a–h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC₅₀ of 9.5 ± 2.8 and 3.5 ± 1.8 μM for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC₅₀ of 11.3 ± 2.8 μM. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50 mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection.     

Design,synthesis, biological evaluation,structure-activity relationship,and toxicity of clinafloxacin-azole conjugates as novel antitubercular agents

Based on the advantages of azole molecules and fluoroquinolone drugs, we designed and synthesized 34 clinafloxacin-azole conjugates using fragment-based drug design and drug combination principles. The in vitro activities of the synthesized conjugates against Mycobacterium tuberculosis (H37Rv), Hela cell as well as Gram-positive and Gram-negative bacteria were assayed. The bioassay results revealed that most of the target molecules had anti-tuberculosis (anti-TB) activity, of which 14 compounds had very strong anti-TB activity [minimum inhibitory concentration (MIC)?<?2?μM]. In addition, the compounds with strong activity towards H37Rv had weak activity towards Gram-negative and Gram-positive bacteria, showing obvious selectivity towards H37Rv. Predicted toxicity data indicated that 27 molecules were less toxic or equivalent to that of the original drug (clinafloxacin). Especially, it is demonstrated that compound TM2l exhibited the strongest anti-TB activity (MIC?=?0.29?μM), low antibacterial activity, negligible toxicity, and good drug-likeness values, which can be considered as an ideal lead molecule for future optimization.     

Exploring anti-TB leads from natural products library originated from marine microbes and medicinal plants

Multidrug-resistant tuberculosis (MDR-TB) and TB–HIV co-infection have become a great threat to global health. However, the last truly novel drug that was approved for the treatment of TB was discovered 40?years ago. The search for new effective drugs against TB has never been more intensive. Natural products derived from microbes and medicinal plants have been an important source of TB therapeutics. Recent advances have been made to accelerate the discovery rate of novel TB drugs including diversifying strategies for environmental strains, high-throughput screening (HTS) assays, and chemical diversity. This review will discuss the challenges of finding novel natural products with anti-TB activity from marine microbes and plant medicines, including biodiversity- and taxonomy-guided microbial natural products library construction, target- and cell-based HTS, and bioassay-directed isolation of anti-TB substances from traditional medicines.     

New perspectives on natural products in TB drug research

The challenge of discovering new, urgently needed anti-TB drugs from natural sources requires a truly interdisciplinary research. Cutting-edge mycobacteriology and innovative natural products chemistry tools have to be developed and employed in tandem, in order to meet these demands. The present review provides cross-linkage to the most recent literature on anti-TB active natural products and summarizes the recent developments in both fields and their potential to impact the early steps of the TB drug discovery process.     

纳米材料在结核病诊疗中的应用

结核病是由结核分枝杆菌引起的慢性感染性疾病,经过呼吸道感染后侵犯机体器官,严重威胁全球公共卫生。传统结核诊疗手段存在诊断效率低、易误诊漏诊、易产生耐药、治疗效果和患者依从性差等瓶颈问题,亟需开发快速、准确的结核即时诊断（POC）方法和安全、高效的结核治疗方案,切实解决结核防治难题。本文总结了纳米材料在结核病诊疗领域的研究进展及应用前景,旨在为开发新一代安全、快速、有效的结核病诊疗方法提供参考。     

Treatment of Mycobacterium tuberculosis-Infected Macrophages with Poly(Lactic-Co-Glycolic Acid) Microparticles Drives NFκB and Autophagy Dependent Bacillary Killing

The emergence of multiple-drug-resistant tuberculosis (MDR-TB) has pushed our available repertoire of anti-TB therapies to the limit of effectiveness. This has increased the urgency to develop novel treatment modalities, and inhalable microparticle (MP) formulations are a promising option to target the site of infection. We have engineered poly(lactic-co-glycolic acid) (PLGA) MPs which can carry a payload of anti-TB agents, and are successfully taken up by human alveolar macrophages. Even without a drug cargo, MPs can be potent immunogens; yet little is known about how they influence macrophage function in the setting of Mycobacterium tuberculosis (Mtb) infection. To address this issue we infected THP-1 macrophages with Mtb H37Ra or H37Rv and treated with MPs. In controlled experiments we saw a reproducible reduction in bacillary viability when THP-1 macrophages were treated with drug-free MPs. NFκB activity was increased in MP-treated macrophages, although cytokine secretion was unaltered. Confocal microscopy of immortalized murine bone marrow-derived macrophages expressing GFP-tagged LC3 demonstrated induction of autophagy. Inhibition of caspases did not influence the MP-induced restriction of bacillary growth, however, blockade of NFκB or autophagy with pharmacological inhibitors reversed this MP effect on macrophage function. These data support harnessing inhaled PLGA MP-drug delivery systems as an immunotherapeutic in addition to serving as a vehicle for targeted drug delivery. Such “added value” could be exploited in the generation of inhaled vaccines as well as inhaled MDR-TB therapeutics when used as an adjunct to existing treatments.     

Mechanism of tissue-selective drug action in the cardiovascular system

Analysis of the human genome project tells us that there may be as few as 3000 genes that are likely to be good drug targets. Although the number of targets is still very large, these data have been interpreted by some to mean that the pharmaceutical industry may someday run out of novel drug targets. Despite the doom and gloom of such analysis, there is considerable reason for optimism. Drugs may exhibit selectivity of action beyond that predicted by target expression alone. Drugs that act at a single molecular target may have very different pharmacology and, as a result, different therapeutic uses. Three well-characterized model systems are highlighted to illustrate this point. The first model system is exemplified by nifedipine and verapamil, both of which act on L-type calcium channels. Both drugs are used to treat hypertension, but only verapamil can be used to produce atrioventricular block in patients with atrial fibrillation. The second model system describes the therapeutic exploitation of unusual conditions that occur in the ischemic myocardium to produce drugs that are more effective for suppressing ischemia-induced arrhythmias. The third model system discusses the mechanisms through which phosphodiesterase-5 (PDE5) inhibitors act selectively to facilitate penile erection while having little effect in the non-penile vasculature that also expresses PDE5.     

结核病疫苗研究进展

随着对抗结核免疫机制的深入研究,新型结核疫苗的研发也更加理性和成熟。近期研究表明,CD4 T细胞多功能至关重要,人类CD8和γδT细胞也有抗结核免疫保护作用,是新型疫苗设计有潜力的T细胞靶点。系统的"组学"技术大规模筛选有可能发现更多强免疫原性的抗原。不同表达时期的多抗原组成的多价疫苗对不同感染时期的结核都有预防作用。针对潜伏感染或已经感染个体配合化学药物使用的新型治疗性疫苗,有望促进清除残留的结核分枝杆菌。     

Anti-tuberculosis lead molecules from natural products targeting <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> ClpC1

Tuberculosis (TB) is a serious and potentially fatal disease caused by Mycobacterium tuberculosis (M. tb). The occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb is a significant public health concern because most of the anti-TB drugs that have been in use for over 40 years are no longer effective for the treatment of these infections. Recently, new anti-TB lead compounds such as cyclomarin A, lassomycin, and ecumicin, which are cyclic peptides from actinomycetes, have shown potent anti-TB activity against MDR and XDR M. tb as well as drug-susceptible M. tb in vitro. The target molecule of these antibiotics is ClpC1, a protein that is essential for the growth of M. tb. In this review, we introduce the three anti-TB lead compounds as potential anti-TB therapeutic agents targeting ClpC1 and compare them with the existing anti-TB drugs approved by the US Food and Drug Administration.     

Detoxifying Antitumoral Drugs via Nanoconjugation: The Case of Gold Nanoparticles and Cisplatin

Nanoparticles (NPs) have emerged as a potential tool to improve cancer treatment. Among the proposed uses in imaging and therapy, their use as a drug delivery scaffold has been extensively highlighted. However, there are still some controversial points which need a deeper understanding before clinical application can occur. Here the use of gold nanoparticles (AuNPs) to detoxify the antitumoral agent cisplatin, linked to a nanoparticle via a pH-sensitive coordination bond for endosomal release, is presented. The NP conjugate design has important effects on pharmacokinetics, conjugate evolution and biodistribution and results in an absence of observed toxicity. Besides, AuNPs present unique opportunities as drug delivery scaffolds due to their size and surface tunability. Here we show that cisplatin-induced toxicity is clearly reduced without affecting the therapeutic benefits in mice models. The NPs not only act as carriers, but also protect the drug from deactivation by plasma proteins until conjugates are internalized in cells and cisplatin is released. Additionally, the possibility to track the drug (Pt) and vehicle (Au) separately as a function of organ and time enables a better understanding of how nanocarriers are processed by the organism.