UHPLC-QQQ-MS and RP-HPLC Detection of Bioactive Alizarin and Scopoletin Metabolites from Morinda citrifolia Root Extracts and Their Antitubercular, Antibacterial, and Antioxidant Activities.

Morinda citrifolia is a medicinal plant that has been traditionally used in various therapeutic applications. All parts of M. citrifolia including fruits, leaves, stems, roots, and flowers contain various biologically active phytochemicals. This study aimed to evaluate the antitubercular, antibacterial, and antioxidant activities of M. citrifolia root extracts and spectroscopically analyze the bioactive metabolites. M. citrifolia root extracts were prepared via maceration. The minimum inhibitory concentration (MIC) for antitubercular activity, the inhibition zone for antibacterial activity, and the antioxidant activities in terms of half-maximal inhibitory concentration (IC50) values were determined. 1H-NMR, RP-HPLC, and UHPLC-QQQ-MS analyses were performed to evaluate the secondary metabolites. The results showed that the dichloromethane root extract exhibited relatively good inhibition of M. tuberculosis with an MIC value of 50 µg/mL. All extracts were mostly active against five tested bacterial strains. The ethanolic and dichloromethane root extracts showed the highest antioxidant power against DPPH (IC50 = 0.82 mg/mL) and NO (IC50 = 0.64 mg/mL) radicals, respectively. The 1H-NMR-based screening of the secondary metabolites of all M. citrifolia root extracts confirmed the presence of triterpenes, steroids, phenolics, flavonoids, tannins, and anthraquinones as major bioactive components. Alizarin and scopoletin were detected in the extracts via UHPLC-QQQ-MS, and the alizarin (0.552-3.227 g/100 g dry weight) and scopoletin (0.092-0.554 g/100 g dry weight) contents were quantified via RP-HPLC. The antimicrobial and antioxidant activities of M. citrifolia root extracts and the identification of the main bioactive ingredients are the initial studies that can be beneficial for further in vivo studies and biomedical applications of its bioactive compounds.

Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity.

Mutations in DNA gyrase confer resistance to fluoroquinolones, second-line antibiotics for Mycobacterium tuberculosis infections. Identification of new agents that inhibit M. tuberculosis DNA gyrase ATPase activity is one strategy to overcome this. Here, bioisosteric designs using known inhibitors as templates were employed to define novel inhibitors of M. tuberculosis DNA gyrase ATPase activity. This yielded the modified compound R3-13 with improved drug-likeness compared to the template inhibitor that acted as a promising ATPase inhibitor against M. tuberculosis DNA gyrase. Utilization of compound R3-13 as a virtual screening template, supported by subsequent biological assays, identified seven further M. tuberculosis DNA gyrase ATPase inhibitors with IC50 values in the range of 0.42-3.59 µM. The most active compound 1 showed an IC50 value of 0.42 µM, 3-fold better than the comparator ATPase inhibitor novobiocin (1.27 µM). Compound 1 showed noncytotoxicity to Caco-2 cells at concentrations up to 76-fold higher than its IC50 value. Molecular dynamics simulations followed by decomposition energy calculations identified that compound 1 occupies the binding pocket utilized by the adenosine group of the ATP analogue AMPPNP in the M. tuberculosis DNA gyrase GyrB subunit. The most prominent contribution to the binding of compound 1 to M. tuberculosis GyrB subunit is made by residue Asp79, which forms two hydrogen bonds with the OH group of this compound and also participates in the binding of AMPPNP. Compound 1 represents a potential new scaffold for further exploration and optimization as a M. tuberculosis DNA gyrase ATPase inhibitor and candidate anti-tuberculosis agent.

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity.

Mycobacterium tuberculosis protein kinase B (PknB) is essential to mycobacterial growth and has received considerable attention as an attractive target for novel anti-tuberculosis drug development. Here, virtual screening, validated by biological assays, was applied to select candidate inhibitors of M. tuberculosis PknB from the Specs compound library (www.specs.net). Fifteen compounds were identified as hits and selected for in vitro biological assays, of which three indoles (2, AE-848/42799159; 4, AH-262/34335013; 10, AP-124/40904362) inhibited growth of M. tuberculosis H37Rv with minimal inhibitory concentrations of 6.2, 12.5, and 6.2 µg/mL, respectively. Two compounds, 2 and 10, inhibited M. tuberculosis PknB activity in vitro, with IC50 values of 14.4 and 12.1 µM, respectively, suggesting this to be the likely basis of their anti-tubercular activity. In contrast, compound 4 displayed anti-tuberculosis activity against M. tuberculosis H37Rv but showed no inhibition of PknB activity (IC50 > 128 µM). We hypothesize that hydrolysis of its ethyl ester to a carboxylate moiety generates an active species that inhibits other M. tuberculosis enzymes. Molecular dynamics simulations of modeled complexes of compounds 2, 4, and 10 bound to M. tuberculosis PknB indicated that compound 4 has a lower affinity for M. tuberculosis PknB than compounds 2 and 10, as evidenced by higher calculated binding free energies, consistent with experiment. Compounds 2 and 10 therefore represent candidate inhibitors of M. tuberculosis PknB that provide attractive starting templates for optimization as anti-tubercular agents.

Identification of Potent DNA Gyrase Inhibitors Active against Mycobacterium tuberculosis.

Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (www.specs.net). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 µg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 µM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Mycobacterium haemophilum scleritis: two case reports and review of literature.

BACKGROUND: Mycobacterium haemophilum is a rare and emerging nontuberculous mycobacteria (NTM). It normally causes localized or disseminated systemic diseases, particularly skin infections and arthritis in severely immunocompromised patients. There have been 5 cases of M. haemophilum ocular infections reported in the literature. Only 1 case presented with scleritis with keratitis. Here, we reported 2 cases of M. haemophilum scleritis. One of them was immunocompetent host and had keratitis with radial keratoneuritis as a presenting sign. CASE PRESENTATION: Case 1: A 52-year-old Thai female with rheumatoid arthritis presented with scleritis. Conjunctival scraping was carried out and the culture result was positive for M. haemophilum. Despite receiving systemic and topical antibiotics, her clinical symptoms and signs worsened. Surgical debridement was performed. After surgery, the lesion was significantly improved and finally turned to conjunctival scarring. Case 2: A 32-year old healthy Thai male without underlying disease presented with nodular scleritis and keratouveitis with multiple radial keratoneuritis. Surgical debridement of the scleral nodule was performed. Initial microbiological investigations were negative. Herpes ocular infections was suspected. Topical antibiotics, oral acyclovir, low-dose topical steroids and systemic steroids were started. The scleral inflammation subsided but later the keratitis relapsed, requiring corneal biopsy. Histopathology of the specimen revealed acid-fast bacteria and M. haemophilum was identified by polymerase chain reaction (PCR) and sequencing. The diagnosis of Mycobacterial keratitis was made. Although using the combination of systemic and topical antibiotics, his clinical status progressively deteriorated. Multiple therapeutic penetrating keratoplasties were required to eradicate the infection. No recurrence was found during the 1-year follow-up in both cases. CONCLUSIONS: M. haemophilum can cause scleritis and keratitis, even in immunocompenent host. Radial keraoneuritis is first described in M. haemophilum keratitis. NTM keratitis should be considered in the differential diagnosis of patients with radial keratoneuritis. Increased awareness and early diagnosis using appropriate culture conditions and molecular techniques are important for the proper treatment of this infection. Prompt surgical intervention appears to be vital for successful management of M. haemophilum scleritis and keratitis.

Discovery of New and Potent InhA Inhibitors as Antituberculosis Agents: Structure-Based Virtual Screening Validated by Biological Assays and X-ray Crystallography.

The enoyl-acyl carrier protein reductase InhA of Mycobacterium tuberculosis is an attractive, validated target for antituberculosis drug development. Moreover, direct inhibitors of InhA remain effective against InhA variants with mutations associated with isoniazid resistance, offering the potential for activity against MDR isolates. Here, structure-based virtual screening supported by biological assays was applied to identify novel InhA inhibitors as potential antituberculosis agents. High-speed Glide SP docking was initially performed against two conformations of InhA differing in the orientation of the active site Tyr158. The resulting hits were filtered for drug-likeness based on Lipinski's rule and avoidance of PAINS-like properties and finally subjected to Glide XP docking to improve accuracy. Sixteen compounds were identified and selected for in vitro biological assays, of which two (compounds 1 and 7) showed MIC of 12.5 and 25 µg/mL against M. tuberculosis H37Rv, respectively. Inhibition assays against purified recombinant InhA determined IC50 values for these compounds of 0.38 and 0.22 µM, respectively. A crystal structure of the most potent compound, compound 7, bound to InhA revealed the inhibitor to occupy a hydrophobic pocket implicated in binding the aliphatic portions of InhA substrates but distant from the NADH cofactor, i.e., in a site distinct from those occupied by the great majority of known InhA inhibitors. This compound provides an attractive starting template for ligand optimization aimed at discovery of new and effective compounds against M. tuberculosis that act by targeting InhA.

In Vitro Activities of Enantiopure and Racemic 1'-Acetoxychavicol Acetate against Clinical Isolates of Mycobacterium tuberculosis.

In the process of evaluating the effect of several plant extracts against Mycobacterium tuberculosis using the Microplate Alamar Blue Assay (MABA), an extract of Thai herb Alpinia galanga rhizome and its major component, 1'-acetoxychavicol acetate (ACA), exhibited marked anti-tuberculosis activity. The minimal inhibition concentrations (MICs) of the S-enantiomer of ACA (S-ACA) against M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294 strains were 0.2 µg/mL and 0.7 µg/mL, respectively. More than 95% of 100 drug-sensitive and 50 drug-resistant mycobacterial clinical isolates were inhibited by extracted S-ACA at 1.0 µg/mL. All of the remaining isolates were inhibited at 2.0 µg/mL. In contrast to the S-enantiomer, synthetic racemic 1'-R,S-ACA (rac-ACA) showed MICs of 0.5 µg/mL and 2.7 µg/mL for M. tuberculosis H37Ra ATCC 25177 and H37Rv ATCC 27294, respectively, suggesting that the anti-tuberculosis effect might be primarily due to the S-form. These observations were in line with the MICs of rac-ACA against 98% of 93 drug-resistant clinical isolates, which showed the effective inhibitory dose at 2.0 µg/mL. After exposure to 2.7 µg/mL of rac-ACA for at least 3 h, the tubercle bacilli were completely killed. These demonstrated that ACA had potent anti-TB activity.

Antimycobacterial activity of natural products and synthetic agents: pyrrolodiquinolines and vermelhotin as anti-tubercular leads against clinical multidrug resistant isolates of Mycobacterium tuberculosis.

Various classes of natural products and synthetic compounds were tested against reference strains and clinical multidrug resistant isolates of Mycobacterium tuberculosis. Vermelhotin (19), a natural tetramic acid from fungi, was the most active toward clinical MDR TB isolates (MIC 1.5-12.5 µg/mL). Synthetic compounds (i.e. benzoxazocines, coumarins, chromenes, and pyrrolodiquinoline derivatives) were prepared by green chemistry approaches. Under microwave irradiation, a one-pot synthesis of pyrrolodiquinoline 85 was achieved by homocoupling of 1-methylquinolinium iodide; the structure of 85 was confirmed by single-crystal X-ray analysis. Compound 85 and its derivative 86 exhibited potent anti-tubercular activity (MIC 0.3-6.2 µg/mL) against clinical MDR TB isolates, and they displayed weak cytotoxicity toward normal cell line. The scaffold of 85 and 86 is potential for antimycobacterial activity.

Cutaneous Mycobacterium haemophilum infections in immunocompromised patients in a tertiary hospital in Bangkok, Thailand: under-reported/under-recognized infection.

INTRODUCTION: Mycobacterium haemophilum is one of the non-tuberculous mycobacteria (NTM) that can cause cutaneous infection. As acid-fast staining cannot distinguish NTM from Mycobacterium tuberculosis, and as skin culture for M. haemophilum is not performed routinely, the diagnosis of M. haemophilum infection in Thailand is rarely made. CASE PRESENTATION: Between 2006 and 2009, five patients with M. haemophilum infection were diagnosed in Ramathibodi Hospital, a tertiary care centre in Bangkok, Thailand. The patients were aged 3, 29, 47, 75 and 76 years, and four were immunocompromised. Three patients received immunosuppressive medication. Most patients presented with subacute cutaneous infection. A suboptimal response to conventional antibiotics raised suspicions of M. haemophilum cutaneous infections, which can occur in immunocompromised patients. Diagnoses of these cases were made by skin culture for mycobacteria at an incubating temperature of around 30 °C with iron supplementation, DNA sequencing, or PCR/restriction enzyme analysis. Rifampicin, ofloxacin and clarithromycin were active against all isolates, whereas ethambutol and streptomycin were inactive. CONCLUSION: Skin culture should be performed under special conditions or molecular technique should be used to identify M. haemophilum in susceptible patients.

Potent activity against multidrug-resistant Mycobacterium tuberculosis of α-mangostin analogs.

A new series of mangostin analogs of natural α-mangostin from mangosteen was prepared and their antimycobacterial activity was evaluated in vitro against Mycobacterium tuberculosis H(37)Ra. The results showed that the monoalkyl tetrahydro α-mangostin analogs displayed increased antimycobacterial activity as compared with the lead natural xanthone, α-mangostin. Among the tested compounds, 6-methoxytetrahydro α-mangostin (16) exhibited the most potent antimycobacterial activity with minimum inhibitory concentration (MIC) of 0.78 µg/mL. The activity of the monoalkylated and monoacylated tetrahydro α-mangostins decreases as the length of carbon chain increases. The methyl ether analog was also active against the multidrug-resistant (MDR) strains with pronounced MICs of 0.78-1.56 µg/mL.

Antimycobacterial activity of bisbenzylisoquinoline alkaloids from Tiliacora triandra against multidrug-resistant isolates of Mycobacterium tuberculosis.

Bisbenzylisoquinoline alkaloids, tiliacorinine (1), 2'-nortiliacorinine (2), and tiliacorine (3), isolated from the edible plant, Tiliacora triandra, as well as a synthetic derivative, 13'-bromo-tiliacorinine (4), were tested against 59 clinical isolates of multidrug-resistant Mycobacterium tuberculosis (MDR-MTB). The alkaloids 1-4 showed MIC values ranging from 0.7 to 6.2 µg/ml, but they exhibited the MIC value at 3.1 µg/ml against most MDR-MTB isolates. The present work suggests that bisbenzylisoquinoline alkaloids are potential new chemical scaffolds for antimycobacterial activity.

Isoxazole analogs of curcuminoids with highly potent multidrug-resistant antimycobacterial activity.

Curcumin (1), demethoxycurcumin (2) and bisdemethoxycurcumin (3), the curcuminoid constituents of the medicinal plant Curcuma longa L., have been structurally modified to 55 analogs and antimycobacterial activity against Mycobacterium tuberculosis has been evaluated. Among the highly active curcuminoids, the isoxazole analogs are the most active group, with mono-O-methylcurcumin isoxazole (53) being the most active compound (MIC 0.09 microg/mL). It was 1131-fold more active than curcumin (1), the parent compound, and was approximately 18 and 2-fold more active than the standard drugs kanamycin and isoniazid, respectively. Compound 53 also exhibited high activity against the multidrug-resistant M. tuberculosis clinical isolates, with the MICs of 0.195-3.125 microg/mL. The structural requirements for a curcuminoid analog to exhibit antimycobacterial activity are the presence of an isoxazole ring and two unsaturated bonds on the heptyl chain. The presence of a suitable para-alkoxyl group on the aromatic ring which is attached in close proximity to the nitrogen function of the isoxazole ring and a free para-hydroxyl group on another aromatic ring enhances the biological activity.

In vitro activities of cloxyquin (5-chloroquinolin-8-ol) against Mycobacterium tuberculosis.

The in vitro activities of cloxyquin (5-chloroquinolin-8-ol) against 9 standard strains and 150 clinical isolates of Mycobacterium tuberculosis were studied. The MICs ranged from 0.062 to 0.25 microg/ml. The MIC(50) and MIC(90) were 0.125 and 0.25 microg/ml, respectively. These indicate that cloxyquin exhibited good antituberculosis activity, even for multidrug-resistant isolates.

In vitro and in vivo activities of macrolide derivatives against Mycobacterium tuberculosis.

Existing macrolides have never shown definitive clinical efficacy in tuberculosis. Recent reports suggest that ribosome methylation is involved in macrolide resistance in Mycobacterium tuberculosis, a mechanism that newer macrolides have been designed to overcome in gram-positive bacteria. Therefore, selected macrolides and ketolides (descladinose) with substitutions at positions 9, 11,12, and 6 were assessed for activity against M. tuberculosis, and those with MICs of < or = 4 microM were evaluated for cytotoxicity to Vero cells and J774A.1 macrophages. Several compounds with 9-oxime substitutions or aryl substitutions at position 6 or on 11,12 carbamates or carbazates demonstrated submicromolar MICs. For the three macrolide-ketolide pairs, macrolides demonstrated superior activity. Four compounds with low MICs and low cytotoxicity also effected significant reductions in CFU in infected macrophages. Active compounds were assessed for tolerance and the ability to reduce CFU in the lungs of BALB/c mice in an aerosol infection model. A substituted 11,12 carbazate macrolide demonstrated significant dose-dependent inhibition of M. tuberculosis growth in mice, with a 10- to 20-fold reduction of CFU in lung tissue. Structure-activity relationships, some of which are unique to M. tuberculosis, suggest several synthetic directions for further improvement of antituberculosis activity. This class appears promising for yielding a clinically useful agent for tuberculosis.

Concurrent chromomycosis and aspergillosis of the maxillary sinus.

The authors describe a rare case of an infection of both the chromomycosis and aspergillosis of the maxillary sinus in an immunocompetent 72-year-old female who presented with progressive visual loss and dull aching pain of the left eye. Sinuscopy of the left maxillary sinus showed swelling of the mucosa with clay-like materials. Biopsy from the left maxillary sinus showed the typically characteristic morphology of chromomycosis and culture from sinus tissue which yielded Aspergillus. The patient responded to a combination therapy of surgical excision and antifungal agent.