Study of the Structure-Activity Relationship of an Anti-Dormant Mycobacterial Substance 3-(Phenethylamino)Demethyl(oxy)aaptamine to Create a Probe Molecule for Detecting Its Target Protein.

The current tuberculosis treatment regimen is long and complex, and its failure leads to relapse and emergence of drug resistance. One of the major reasons underlying the extended chemotherapeutic regimen is the ability of Mycobacterium tuberculosis to attain a dormant state. Therefore, the identification of new lead compounds with chemical structures different from those of conventional anti-tuberculosis drugs is essential. The compound 3-(phenethylamino)demethyl(oxy)aaptamine (PDOA, 1), isolated from marine sponge of Aaptos sp., is known as an anti-dormant mycobacterial substance, and has been reported to be effective against the drug resistant strains of M. tuberculosis. However, its target protein still remains unclear. This study aims to clarify the structure-activity relationship of 1 using 15 synthetic analogues, in order to prepare a probe molecule for detecting the target protein of 1. We succeeded in creating the compound 15 with a photoaffinity group that retained antimicrobial activity, which proved to be a suitable probe molecule for identifying the target protein of 1.

Anti-mycobacterial activity of heat and pH stable high molecular weight protein(s) secreted by a bacterial laboratory contaminant.

BACKGROUND: Tuberculosis currently stands as the second leading cause of deaths worldwide due to single  infectious agent after Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The current challenges of drug resistance in tuberculosis highlight an urgent need to develop newer anti-mycobacterial compounds. In the present study, we report the serendipitous discovery of a bacterial laboratory contaminant (LC-1) exhibiting a zone of growth inhibition on an agar plate seeded with Mycobacterium tuberculosis. RESULTS: We utilized microbiological, biochemical and biophysical approaches to characterize LC-1 and anti-mycobacterial compound(s) in its secretome. Based on 16S rRNA sequencing and BIOLOG analysis, LC-1 was identified as Staphylococcus hominis, a human bacterial commensal. Anti-mycobacterial activity was initially found in 30 kDa retentate that was obtained by ultrafiltration of culture filtrate (CF). SDS-PAGE analysis of peak fractions obtained by size exclusion chromatography of 30 kDa retentate confirmed the presence of high molecular weight (≥ 30 kDa) proteins. Peak fraction-1 (F-1) exhibited inhibitory activity against M. bovis BCG, but not against M. smegmatis, E. coli and S. aureus. The active fraction F-1 was inactivated by treatment with Proteinase K and α-chymotrypsin. However, it retained its anti-mycobacterial activity over a wide range of heat and pH treatment. The anti-mycobacterial activity of F-1 was found to be maintained even after a long storage (~12 months) at - 20 °C. Mass spectrometry analysis revealed that the identified peptide masses do not match with any previously known bacteriocins. CONCLUSIONS: The present study highlights the anti-mycobacterial activity of high molecular weight protein(s) present in culture filtrate of LC-1, which may be tested further to target M. tuberculosis. The heat and pH stability of these proteins add to their characteristics as therapeutic proteins and may contribute to their long shelf life. LC-1 being a human commensal can be tested in future for its potential as a probiotic to treat tuberculosis.

(R)-(+)-Lasiodiplodin isolated from the endophytic fungus Sordaria tamaensis exhibits potent antimycobacterial and anti-inflammatory activities in vitro and in vivo: a dual approach for the treatment of severe pulmonary tuberculosis.

OBJECTIVES: This study aimed to evaluate endophytic fungi isolated from Tocoyena bullata and Humiria balsamifera plant species for their antimycobacterial and anti-inflammatory activities, focusing on severe pulmonary tuberculosis cases which are often associated with exacerbated inflammation. METHODS: Mycobacterium suspensions were incubated with the samples for 5 days. RAW 264.7 macrophages stimulated with LPS were also incubated with them for 24 h to assess the inhibition of inflammatory mediator production and cytotoxicity. C57BL/6 mice were infected with Mtb M299 and treated for 15 days with lasiodiplodin (Lasio). KEY FINDINGS: Endophytic fungus Sordaria tamaensis, obtained from T. bullata, was the most promising. Its ethanolic extract impaired mycobacterial growth with MIC50 (µg/ml): 1.5 ± 0.6 (BCG), 66.8 ± 0.1 (H37Rv) and 80.0 ± 0.1 (M299). (R)-(+)-Lasio showed MIC50 92.2 ± 1.8 µg/ml (M299). In addition, Lasio was able to inhibit NO, IL-1ß and TNF-α production and was not cytotoxic for macrophages. M. tuberculosis-infected C57BL/6 animals treated by Lasio reduced the number of acid-fast bacilli, lung pathology, leucocyte influx and proinflammatory cytokine production in the lungs. The class IIa fructose 1,6-bisphosphate aldolase was the predicted hypothetical target of Lasio. CONCLUSIONS: (R)-(+)-Lasio stood out as a promising anti-TB compound, exhibiting anti-inflammatory and antimycobacterial effects, as well as low cytotoxicity.

Antitubercular metabolites from the marine-derived fungus strain Aspergillus fumigatus MF029.

During the systematic screening of bioactive compounds from our marine natural product library, crude extract of the marine-derived fungus strain Aspergillus fumigatus MF029 exhibited moderate bioactivities against Bacillus subtilis, Staphylococcus aureus, methicillin-resistant S. aureus, and Mycobacterium bovis bacillus Calmette-Guérin (BCG). Further chemical investigation resulted in the identification of two new compounds, chaetominine A (1) and sphingofungin I (2), together with four known compounds, emodin (3), chaetominine (4), sphingofungin D (5) and trypacidin (6). Trypacidin displayed potential antitubercular activity with MIC value of 1.25 µg/mL.

Using Proteolytic Hypomorphs to Detect Small Molecule Mechanism of Action.

With increasing prevalence of antimicrobial resistance, a fundamental goal of antibiotic discovery is to uncover new small molecules that prevent growth of pathogenic bacteria through diverse mechanisms of action. This goal is particularly pertinent for tuberculosis, caused by Mycobacterium tuberculosis. In this chapter, we describe the application of a chemical-genetic method, PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), for sensitively detecting small molecule bioactivity using a pooled panel of hypomorphs (strains depleted in a particular essential gene) of M. tuberculosis. We describe statistical and heuristic approaches to assign small molecule mechanism of action from the resulting chemical-genetic interaction profiles.

Bioactive Dimeric Tetrahydroxanthones with 2,2'- and 4,4'-Axial Linkages from the Entomopathogenic Fungus Aschersonia confluens.

Thirteen tetrahydroxanthone dimers, atrop-ascherxanthone A (1), ascherxanthones C-G (2-6), and confluxanthones A-G (7-13), were isolated from the entomopathogenic fungus Aschersonia confluens BCC53152. The chemical structures were determined based on analysis of NMR spectroscopic and mass spectrometric data. The absolute configurations of compounds 1 and 7 were confirmed by single-crystal X-ray diffraction experiments, while the configurations of other compounds were assigned based upon evidence from NOESY and NOEDIFF experiments, modified Mosher's method, and ECD spectroscopic data together with biogenetic considerations. Compounds 1, 3-5, 7-11, and 13 showed antimalarial activity against Plasmodium falciparum (K1, multidrug-resistant strain) (IC50 0.6-6.1 µM), antitubercular activity against Mycobacterium tuberculosis H37Ra (MIC 6.3-25.0 µg/mL), and cytotoxicity against NCI-H187 (IC50 0.5-3.5 µM) and Vero (IC50 0.9-6.1 µM) cells. All tested compounds except for compound 9 exhibited cytotoxicity against KB cells (IC50 1.3-9.7 µM).

Marine Pharmacology in 2016-2017: Marine Compounds with Antibacterial, Antidiabetic, Antifungal, Anti-Inflammatory, Antiprotozoal, Antituberculosis and Antiviral Activities; Affecting the Immune and Nervous Systems, and Other Miscellaneous Mechanisms of Action.

The review of the 2016-2017 marine pharmacology literature was prepared in a manner similar as the 10 prior reviews of this series. Preclinical marine pharmacology research during 2016-2017 assessed 313 marine compounds with novel pharmacology reported by a growing number of investigators from 54 countries. The peer-reviewed literature reported antibacterial, antifungal, antiprotozoal, antituberculosis, and antiviral activities for 123 marine natural products, 111 marine compounds with antidiabetic and anti-inflammatory activities as well as affecting the immune and nervous system, while in contrast 79 marine compounds displayed miscellaneous mechanisms of action which upon further investigation may contribute to several pharmacological classes. Therefore, in 2016-2017, the preclinical marine natural product pharmacology pipeline generated both novel pharmacology as well as potentially new lead compounds for the growing clinical marine pharmaceutical pipeline, and thus sustained with its contributions the global research for novel and effective therapeutic strategies for multiple disease categories.

Blutaparon portulacoides ethanolic extract reduced IL-1ß and inflammatory parameters induced by the Mycobacterium complex and carrageenan in mice.

Information on the health benefits of ethanolic extracts obtained from Blutaparon portulacoides stem (EEBP) hasn´t been consistently described in the literature until the present moment. This study investigated the antimycobacterial, anti-inflammatory and toxicological effects of EEBP in models of inflammation/infection, as well as its chemical composition. Chemical analysis of EEBP by electrospray ionization-mass spectrometry/HPLC-MS/MS identified 3,5,3'-Trihydroxy-4'-methoxy-6,7-methylenedioxy-flavone, gomphrenol, ferulic, vanillic, and caffeic acids. The minimum inhibitory concentration of EEBP and isoniazid in the presence of Mycobacterium tuberculosis was 123.4 and 0.030 µg/ml, respectively. EEBP oral administration (p.o.) (300-1000 mg/kg) or dexamethasone subcutaneous injection (s.c.) (1 mg/kg) significantly inhibited leukocytes and proteins resulting from carrageenan-induced pleurisy in Swiss mice. In the BCG-induced pleurisy model, the oral treatments performed once a day for 7 days, with EEBP (30 and 100 mg/kg) and isoniazid (25 mg/kg), inhibited the increase in plasmatic IL-1ß levels and in pleural exudate from C57BL-6 mice, and reduced M. tuberculosis growth in organs (colony forming units assays). EEBP (30-300 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) significantly prevented carrageenan-induced oedema and mechanical hyperalgesia in Swiss mice. The treatments (once a day for 22 days) with EEBP (30 mg/kg, p.o.) and dexamethasone (1 mg/s.c.) substantially inhibited oedema and mechanical- and cold-hyperalgesia at 11, 16 and 22 days after the administration of Freund's Complete Adjuvant in C57bL6 mice. No evidence of physio-pathologic was observed in Wistar rats acutely treated with EEBP (2000 mg/kg, p.o.). This study confirms the anti-inflammatory and antibiotic properties of EEBP, opening possibilities for the development of safe new drugs with dual anti-inflammatory/antimycobacterial activities which could be favorable from a pharmacoeconomic perspective.

Soybean lectin induces autophagy through P2RX7 dependent activation of NF-κB-ROS pathway to kill intracellular mycobacteria.

BACKGROUND: Host-directed therapy is considered a novel anti-tuberculosis strategy in tackling the tuberculosis burden through autophagy induction by various inducers to curtail the growth of intracellular Mycobacterium tuberculosis. METHODS: In this study, we investigated the anti-tubercular role of soybean lectin, a lectin isolated from Glycine max (Soybean). Effect of SBL on intracellular mycobacterial viability through autophagy and the mechanism involved in differentiated THP-1 cells was studied using different experimental approaches. RESULTS: We initially performed a time kinetic experiment with the non-cytotoxic dose of SBL (20 µg/ml) and observed autophagy induction after 24 h of treatment. Abrogation of autophagy in the presence of 3-MA and an increase in LC3 puncta formation upon Baf-A1 addition elucidated the specific effect on autophagy and autophagic flux. SBL treatment also led to autophagy induction in mycobacteria infected macrophages that restricted the intracellular mycobacterial growth, thus emphasizing the host defensive role of SBL induced autophagy. Mechanistic studies revealed an increase in P2RX7 expression, NF-κB activation and reactive oxygen species generation upon SBL treatment. Inhibition of P2RX7 expression suppressed NF-κB dependent ROS level in SBL treated cells. Moreover, SBL induced autophagy was abrogated in the presence of either different inhibitors or P2RX7 siRNA, leading to the reduced killing of intracellular mycobacteria. CONCLUSION: Taken together, these results conclude that SBL induced autophagy exerts an anti-mycobacterial effect in P2RX7-NF-κB dependent manner through the generation of ROS. GENERAL SIGNIFICANCE: This study has provided a novel anti-mycobacterial role of SBL, which may play an important role in devising new therapeutic interventions.

Identification of fusarielin M as a novel inhibitor of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB).

The secreted Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an essential virulence factor required for the intracellular survival of Mtb within host macrophages. MptpB has become a promising target for the development of novel anti-tuberculosis (TB) drugs. In this study, two new fusarielins, fusarielins M (1) and N (2), and a biogenetically related known compound, fusarielin G (3) were isolated from the marine-derived fungus Fusarium graminearum SYSU-MS5127. Their inhibitory effects on MptpB were evaluated. Among these compounds, fusarielin M substantially inhibited MptpB with a half-maximal inhibitory concentration (IC50) of 1.05 ± 0.08 µM, and an inhibition constant (Ki) of 1.03 ± 0.39 µM. Surface plasmon resonance analysis was used to characterize the interaction between fusarielin M and MptpB in vitro. Fusarielin M also exhibited cellular activity in blocking MptpB-mediated Erk1/2 and p38 inactivation in macrophages. Importantly, fusarielin M (20 µM) substantially reduced intracellular mycobacterial growth within macrophages, causing a 62% reduction in the bacterial burden. The binding mode of fusarielin M was further explored via molecular docking which suggested that fusarielin M binds to the active site of MptpB, forming a hydrogen bond with the side chain of Asp165; this is unique in the P-loop of MptpB compared to conventional human PTPs. The contact between fusarielin M and Asp165 in the catalytic loop provides a potential basis for inhibitor selectivity. Therefore, fusarielin M shows great potential as an anti-TB drug candidate.

Nonpeptidal compounds from the insect Polyphaga plancyi and their biological evaluation.

Five new nitrogen-containing compounds (1-3, 5, and 6), two compounds which was firstly isolated from natural origin (7 and 10), along with six known ones, were isolated from the ethanol extract of the whole bodies of Polyphaga plancyi. The structures of the new compounds including their absolute configurations at stereogenic centers were assigned on the basis of spectroscopic analyses and computational methods. Racemic 10 was separated by chiral HPLC. Biological activities of these isolates against extracellular matrix components in rat renal proximal tubular cells, EV71, COX-2, ROCK2, JAK3, and tuberculosis were evaluated. Importantly, 8 was found to be a selective Smad3 phosphorylation inhibitor.

Novel Antimicrobials from Uncultured Bacteria Acting against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world's population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets.IMPORTANCE Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery.

Three promising antimycobacterial medicinal plants reviewed as potential sources of drug hit candidates against multidrug-resistant tuberculosis.

Regimens of current drugs for tuberculosis are lengthy and are associated with many adverse effects. Currently, the emergence of different resistant strains has been observed. This urges a need for the discovery and development of novel drugs. The main sources of drug lead candidates are based on natural products. Zanthoxylum leprieurii, Lantana camara, and Cryptolepis Sanguinolenta are among the plants that have antimycobacterial activity. Recent technological methods, such as metabolomics, can rapidly detect and identify active compounds from medicinal plants. In this review, we aim to provide an overview and discussion of the antimycobacterial activity, phytochemical analysis and toxicity profile of these plants and their products as well as the potential of metabolomic fingerprinting of medicinal plants with a given activity on microbes, in the search for the potential drug hit molecules. The information for this review was extracted from databases such as Excerpta Medica Database, Google Scholar, Springer, and PubMed Central. Primary studies, using a combination of the keywords antimycobacterial medicinal plant, multidrug-resistant tuberculosis, phytochemistry, toxicity, Zanthoxylum leprieurii, Lantana camara, Cryptolepis sanguinolenta, and plant metabolomics/metabolic fingerprinting of plant extracts, have been considered. The above-mentioned plant species showed antimycobacterial activity against drug-resistant strains of M. tuberculosis. They may provide potential candidates for novel drugs against multidrug-resistant tuberculosis. However, extensive work is still needed. To our knowledge, there is no or limited literature that reports the metabolic fingerprints of these plants. The analysis of the metabolite fingerprints of medicinal plants with similar antimicrobial activity could be important to determine whether the activity results from common metabolites within different plant species. This review shows that these plants are potential candidates to provide drug hits against multidrug-resistant tuberculosis strains. Future studies of compound optimization, in vivo safety and efficacy, as well as of the specific mechanisms of action are however required.

Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Emergence of drug-resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is a major barrier to tuberculosis (TB) eradication, as it leads to longer treatment regimens and in many cases treatment failure. Thus, there is an urgent need to explore new TB drugs and combinations, in order to shorten TB treatment and improve outcomes. Here, we evaluated the potential of two Asian and African traditional medicinal plants, Artemisia annua, a natural source of artemisinin (AN), and Artemisia afra, as sources of novel antitubercular agents. AIM OF THE STUDY: Our goal was to measure the activity of A. annua and A. afra extracts against Mtb as potential natural and inexpensive therapies for TB treatment, or as sources of compounds that could be further developed into effective treatments. MATERIALS AND METHODS: The minimum inhibitory concentrations (MICs) of A. annua and A. afra dichloromethane extracts were determined, and concentrations above the MICs were used to evaluate their ability to kill Mtb and Mycobacterium abscessus in vitro. RESULTS: Previous studies showed that A. annua and A. afra inhibit Mtb growth. Here, we show for the first time that Artemisia extracts have a strong bactericidal activity against Mtb. The killing effect of A. annua was much stronger than equivalent concentrations of pure AN, suggesting that A. annua extracts kill Mtb through a combination of AN and additional compounds. A. afra, which produces very little AN, displayed bactericidal activity against Mtb that was substantial but weaker than that of A. annua. In addition, we measured the activity of Artemisia extracts against Mycobacterium abscessus. Interestingly, we observed that while A. annua is not bactericidal, it inhibits growth of M. abscessus, highlighting the potential of this plant in combinatory therapies to treat M. abscessus infections. CONCLUSION: Our results indicate that Artemisia extracts have an enormous potential for treatment of TB and M. abscessus infections, and that these plants contain bactericidal compounds in addition to AN. Combination of extracts with existing antibiotics may not only improve treatment outcomes but also reduce the emergence of resistance to other drugs.

Bioactive Metabolites from the Deep-Sea-Derived Fungus Diaporthe longicolla FS429.

The chemical investigation of a methanol extract of the deep-sea-derived fungus Diaporthe longicolla FS429 led to the isolation of two novel diterpenoids longidiacids A and B (1 and 2), two new polyketides (3 and 4), two new cytochalasin analogues longichalasins A and B (6 and 8) and three known analogues 5, 7, 9. Their structures were elucidated through comprehensive spectroscopic analysis, while the absolute configurations were established by the comparison of the experimental and quantum chemical calculated ECD spectra. The structure of compound 7 was confirmed through X-ray diffraction for the first time. In the bioassays compound 8 exhibited antiproliferative effects against SF-268, with an IC50 value of 16.44 µM. Moreover, compounds 1 and 8 were detected to inhibit 35.4% and 53.5% of enzyme activity of Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) at a concentration of 50 µM.

Flavanones from the Twigs and Barks of Artocarpus lakoocha Having Antiplasmodial and Anti-TB Activities.

Chromatographic separation of the acetone extracts from the twigs and barks of Artocarpus lakoocha led to the isolation of the one new flavanone, lakoochanone (1), together with eleven known compounds (2-12). Lakoochanone (1) and moracin C (4) exhibited weak antiplasmodial activity against Plasmodium falciparum Dd2 with IC50 values of 36.7 and 33.9 µM, respectively. Moreover, moracin C (4) and sanggenofuran B (5) showed cytotoxic activity against A2780 cell line with the respective IC50 values of 15.0 and 57.1 µM. In addition, cyclocommunin (7) displayed strong antimycobacterial activity against Mycobacterium tuberculosis H37Ra with the minimum inhibitory concentration (MIC) value of 12.3 µM.

Antitubercular compounds isolated and characterized in Tithonia diversifolia and Couroupita guianensis.

Background: Tuberculosis (TB) has become a public health challenge in the current scenario with a single causative agent, Mycobacterium tuberculosis (MTB) causing the highest morbidity and mortality affecting almost 1.7 million of the population. Furthermore, there has been no novel drug discovery for the past five decades, and the emergence of latent, multiple drug-resistant, and extensively drug-resistant species has given rise to an alarming necessity for a novel compound/s for treating this highly untamable microbe. In developing countries, plant-based drugs have shown promising results in combating TB or its symptoms; naturally occurring secondary metabolites can act as lead-drug molecules or can be co-administered with conventional drugs. Therefore, the present study was focused to identify and characterize potential antimycobacterial compounds found in the screened ethnobotanical plants, Tithonia diversifolia (TD) and Couroupita guianensis (CG). These plants are used for treating respiratory disorders and allergies in the traditional medicinal systems. Methods: These plant leaf extracts were detected and purified using chromatographic techniques for potent antitubercular phytochemicals, and the purified eluents were tested on Mycobacterium smegmatis (MSM) as a surrogate for MTB; further, the fractions inhibiting growth of MSM were characterized through gas chromatography-mass spectrometry. A toxicity test of the purified samples was also assessed by an in vitro 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction and hemolytic assays. Results: The analyzed plant extracts showed the presence of a C-15 sesquiterpene, zingiberene in TD, and a phthalate ester, bis (2-ethylhexyl) phthalate, in CG leaf extracts. The toxicity assessment proved the purified fractions to be moderately toxic at higher concentrations (≥100 µg/mL). Conclusion: Therefore, the identified compounds can be promising antitubercular agents; however, further in vivo investigations will add substantial value to the compounds being pharmacologically useful.

5-Alkylamino-N-phenylpyrazine-2-carboxamides: Design, Preparation, and Antimycobacterial Evaluation.

According to the World Health Organization, tuberculosis is still in the top ten causes of death from a single infectious agent, killing more than 1.7 million people worldwide each year. The rising resistance developed by Mycobacterium tuberculosis against currently used antituberculars is an imperative to develop new compounds with potential antimycobacterial activity. As a part of our continuous research on structural derivatives of the first-line antitubercular pyrazinamide, we have designed, prepared, and assessed the in vitro whole cell growth inhibition activity of forty-two novel 5-alkylamino-N-phenylpyrazine-2-carboxamides with various length of the alkylamino chain (propylamino to octylamino) and various simple substituents on the benzene ring. Final compounds were tested against Mycobacterium tuberculosis H37Ra and four other mycobacterial strains (M. aurum, M. smegmatis, M. kansasii, M. avium) in a modified Microplate Alamar Blue Assay. We identified several candidate molecules with micromolar MIC against M. tuberculosis H37Ra and low in vitro cytotoxicity in HepG2 cell line, for example, N-(4-hydroxyphenyl)-5-(pentylamino)pyrazine-2-carboxamide (3c, MIC = 3.91 µg/mL or 13.02 µM, SI > 38) and 5-(heptylamino)-N-(p-tolyl)pyrazine-2-carboxamide (4e, MIC = 0.78 µg/mL or 2.39 µM, SI > 20). In a complementary screening, we evaluated the in vitro activity against bacterial and fungal strains of clinical importance. We observed no antibacterial activity and sporadic antifungal activity against the Candida genus.

Development and validation of a sensitive LC-MS/MS method for the quantitation of IMB-YH-4py5-2H, an antituberculosis candidate, and its application to the pharmacokinetic study.

(E)-N,N-dimethyl-4-oxo-4-(4-(pyridin-4-yl)phenyl)but-2-enamide hydrochloride (IMB-YH-4py5-2H) is a novel Protein Kinase B (PknB) inhibitor with potent activity against Mycobacterium tuberculosis strains. In the present study, a sensitive and specific liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed and validated to determine IMB-YH-4py5-2H in rat plasma. Sample pretreatment was achieved by liquid-liquid extraction with ethyl acetate, and separation was performed on an XTerra MS C18 column (2.1×50 mm, 3.5 µm) with gradient elution (methanol and 0.1% formic acid) at a flow rate of 0.3 mL/min. Detection was performed in multiple reaction monitoring (MRM) mode. Linear calibration curves were obtained over a concentration range of 1-100 ng/mL. The intra-day and inter-day precisions were lower than 8.46%, and the accuracies ranged from -8.71% to 12.36% at all quality control levels. The extraction recoveries were approximately 70%, and the matrix effects were negligible. All quality control samples were stable under different storage conditions. The validated method was successfully applied to a preclinical pharmacokinetic study in Sprague-Dawley rats. IMB-YH-4py5-2H demonstrated improved pharmacokinetic properties (higher exposure level) compared with its leading compound. IMB-YH-4py5-2H was also distributed throughout the lung pronouncedly, especially inside alveolar macrophages, indicating its effectiveness against lower respiratory infections.

Lignans and Neolignans Anti-tuberculosis Identified by QSAR and Molecular Modeling.

BACKGROUND: Tuberculosis is a disease with high incidence and high mortality rate, especially in Brazil. Although there are several medications available for treatment, in cases of resistance, there is a need to use more than one medication. OBJECTIVE: Therefore, cases of toxicity increase and reports of resistance have been worrying the population. In addition, some medications have a short period of effectiveness. To achieve the goal, ligand-based and structure-based approaches were used. METHODS: Thus, in an attempt to discover potent inhibitors against Mycobacterium tuberculosis enzymes, we sought to identify natural products with high therapeutic potential for the treatment of Tuberculosis through QSAR, Molecular Modeling and ADMET studies. RESULTS: The results showed that the models generated from two sets of molecules with known activity against M. tuberculosis enzymes InhA and PS were able to select 11 and 8 compounds, respectively, between Lignans and Neolignans with 50 to 60% activity probability. In addition, molecular docking contributed to confirm the mechanism of action of compounds and increase the accuracy of methodologies. All molecules showed higher binding energy values for the drug Isoniazid. We conclude that compounds 33, 34, 110, 114 and 133 are promising for InhA target and compounds 07, 08, 19, 21, 42, 48, 75 and 141 for target PS. In addition, most molecules did not show any toxicity according to the evaluated parameters. CONCLUSION: Therefore, Lignans and Neolignans may be an alternative for the treatment of Tuberculosis.