The ctpF Gene Encoding a Calcium P-Type ATPase of the Plasma Membrane Contributes to Full Virulence of Mycobacterium tuberculosis

Identification of alternative attenuation targets of Mycobacterium tuberculosis (Mtb) is pivotal for designing new candidates for live attenuated anti-tuberculosis (TB) vaccines. In this context, the CtpF P-type ATPase of Mtb is an interesting target; specifically, this plasma membrane enzyme is involved in calcium transporting and response to oxidative stress. We found that a mutant of MtbH37Rv lacking ctpF expression (MtbΔctpF) displayed impaired proliferation in mouse alveolar macrophages (MH-S) during in vitro infection. Further, the levels of tumor necrosis factor and interferon-gamma in MH-S cells infected with MtbΔctpF were similar to those of cells infected with the parental strain, suggesting preservation of the immunogenic capacity. In addition, BALB/c mice infected with Mtb∆ctpF showed median survival times of 84 days, while mice infected with MtbH37Rv survived 59 days, suggesting reduced virulence of the mutant strain. Interestingly, the expression levels of ctpF in a mouse model of latent TB were significantly higher than in a mouse model of progressive TB, indicating that ctpF is involved in Mtb persistence in the dormancy state. Finally, the possibility of complementary mechanisms that counteract deficiencies in Ca²⁺ transport mediated by P-type ATPases is suggested. Altogether, our results demonstrate that CtpF could be a potential target for Mtb attenuation.     

Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.     

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal β-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11β, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.     

Preliminary Evaluation of Artemisinin–Cholesterol Conjugates as Potential Drugs for the Treatment of Intractable Forms of Malaria and Tuberculosis

To evaluate the feasibility of developing drugs that may be active against both malaria and tuberculosis (TB) by using in part putative cholesterol transporters in the causative pathogens and through enhancement of passive diffusion in granulomatous TB, artemisinin–cholesterol conjugates were synthesized by connecting the component molecules through various linkers. The compounds were screened in vitro against Plasmodium falciparum (Pf) and Mycobacterium tuberculosis (Mtb). Antimalarial activities (IC₅₀) against Pf drug‐sensitive NF54, and drug‐resistant K1 and W2 strains ranged from 0.03–2.6, 0.03–1.9, and 0.02–1.7 μm . Although the compounds are less active than the precursor artemisinin derivatives, the cholesterol moiety renders the compounds relatively insoluble in the culture medium, and variation in solubilities among the different compounds may reflect in the range of efficacies observed. Activities against Mtb H37Rv were assessed using a standardized colony‐forming unit (CFU) assay after 24 h pretreatment of cultures with each of the compounds. Percentage inhibition ranged from 3–38 % and 18–52 % at 10 and 80 μm , respectively. Thus, in contrast to the comparator drug artemether, the conjugates display enhanced activities. The immediate aims include the preparation of conjugates with enhanced aqueous solubilities, assays against malaria and TB in vivo, and for TB, assays using an infected macrophage model and assessment of granuloma influx.     

Hydrazone-Tethered 5-(Pyridin-4-yl)-4H-1,2,4-triazole-3-thiol Hybrids: Synthesis,Characterisation, in silico ADME Studies,and in vitro Antimycobacterial Evaluation and Cytotoxicity

Compounds containing arylpyrrole-, 1,2,4-triazole- and hydrazone structural frameworks have been widely studied and demonstrated to exhibit a wide range of pharmacological properties. Herein, an exploratory series of new 1,2,4-triazole derivatives designed by amalgamation of arylpyrrole and 1,2,4-triazole structural units via a hydrazone linkage is reported. The synthesised compounds were tested in vitro for their potential activity against Mycobacterium tuberculosis (MTB) H₃₇Rv strain. The most promising compound 13 – the derivative without the benzene ring appended to the pyrrole unit displayed acceptable activity (MIC₉₀=3.99 μM) against MTB H₃₇Rv, while other compounds from the series exhibited modest to weak antimycobacterial activity with MIC₉₀ values in the range between 7.0 and >125 μM. Furthermore, in silico results, predicated using the SwissADME web tool, show that the prepared compounds display desirable ADME profile with parameters within acceptable range.     

Phenyl Selenide-Based Precursors as Hydrogen Peroxide Inducible DNA Interstrand Cross-Linkers

DNA interstrand crosslinks (ICLs) are highly toxic DNA lesions, and induce cell death by blocking DNA strand separation. Most ICL agents aiming to kill cancer cells, also generate adverse side effects to normal cells. H₂O₂-inducible DNA ICL agents are highly selective for targeting cancer cells, as the concentration of H₂O₂ is higher in cancer cells than normal cells. Previous studies have focused on arylboronate-based precursors, reacting with H₂O₂ to generate reactive quinone methides (QMs) crosslinking DNA. Here we explore phenyl selenide-based precursors 1 – 3 as H₂O₂-inducible DNA ICL agents. The precursors 1 – 3 can be activated by H₂O₂ to generate the good benzylic leaving group and promote production of reactive QMs to crosslink DNA. Moreover, the DNA cross-linking ability is enhanced by the introduction of substituents in the para-position of the phenolic hydroxyl group. From the substituents explored (H, OMe, F), the introduction of electron donating group (OMe) shows a pronounced elevating effect. Further mechanistic studies at the molecular and DNA levels confirm alkylation sites located mainly at dAs, dCs and dGs in DNA. Additionally, cellular experiments reveal that agents 1 – 3 exhibit higher cytotoxicity toward H1299 human lung cancer cells compared to clinically used drugs, by inducing cellular DNA damage, apoptosis and G0/G1 cell cycle arrest. This study provides a strategy to develop H₂O₂-inducible DNA interstrand cross-linkers.     

Identification of a New Diterpene Biosynthetic Gene Cluster that Produces O‐Methylkolavelool in Herpetosiphon aurantiacus

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)‐kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)‐O‐methylkolavelool from (+)‐kolavelool, so this enzyme was an O‐methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)‐O‐methylkolavelool biosynthetic gene cluster.     

Mycobacterium tuberculosis H37Rv Strain Increases the Frequency of CD3+TCR+ Macrophages and Affects Their Phenotype,but Not Their Migration Ability

In mycobacterial infections, the number of cells from two newly discovered subpopulations of CD3⁺ myeloid cells are increased at the infection site; one type expresses the T cell receptor (CD3⁺TCRαβ⁺) and the other does not (CD3⁺TCRαβ⁻). The role of Mycobacterium tuberculosis (Mtb) virulence in generating these subpopulations and the ability of these cells to migrate remains unclear. In this study, monocyte-derived macrophages (MDMs) infected in vitro with either a virulent (H37Rv) or an avirulent (H37Ra) Mtb strain were phenotypically characterized based on three MDM phenotypes (CD3⁻, CD3⁺TCRαβ⁺, and CD3⁺TCRαβ⁻); then, their migration ability upon Mtb infection was evaluated. We found no differences in the frequency of CD3⁺ MDMs at 24 h of infection with either Mtb strain. However, H37Rv infection increased the frequency of CD3⁺TCRαβ⁺ MDMs at a multiplicity of infection of 1 and altered the expression of CD1b, CD1c, and TNF on the surface of cells from both the CD3⁺ MDM subpopulations; it also modified the expression of CCR2, CXCR1, and CCR7, thus affecting CCL2 and IL-8 levels. Moreover, H37Rv infection decreased the migration ability of the CD3⁻ MDMs, but not CD3⁺ MDMs. These results confirm that the CD3⁺ macrophage subpopulations express chemokine receptors that respond to chemoattractants, facilitating cell migration. Together, these data suggest that CD3⁺ MDMs are a functional subpopulation involved in the immune response against Mtb.     

Characterization of the Rv3377c Gene Product,a Type‐B Diterpene Cyclase,from the Mycobacterium tuberculosis H37 Genome

The Rv3377c gene from the Mycobacterium tuberculosis H37 genome is specifically limited to those Mycobacterium species that cause tuberculosis. We have demonstrated that the gene product of Rv3377c is a diterpene cyclase that catalyzes the formation of tuberculosinol from geranylgeranyl diphosphate (GGPP). However, the characteristics of this enzyme had not previously been studied in detail with homogeneously purified enzyme. The purified enzyme catalyzed the synthesis of tuberculosinyl diphosphate from GGPP, but it did not bring about the synthesis of tuberculosinol. Optimal conditions for the highest activity were found to be as follows: pH 7.5, 30 °C, Mg^II (0.1 mM ), and Triton X‐100 (0.1 %). Under these conditions, the kinetic values of K_M and k_cat were determined to be 11.7±1.9 μM for GGPP and 12.7±0.7 min^?1, respectively, whereas the specific activity was 186 nmol min^?1 mg^?1. The enzyme activity was inhibited at substrate concentrations higher than 50 μM . The catalytic activity was strongly inhibited by 15‐aza‐dihydrogeranylgeraniol and 5‐isopropyl‐N,N,N,2‐tetramethyl‐4‐(piperidine‐1‐carbonyloxy)benzenaminium chloride (Amo‐1618). The DXDTT_293–297 motif, corresponding to the DXDDTA motif conserved among terpene cyclases, was mutated in order to investigate its function. The middle D295 was found to be the most crucial entity for the catalysis. D293 and two threonine residues function synergistically to enhance the acidity of D295, possibly through hydrogen‐bonding networks. The Rv3377c enzyme could also react with (14R/S)‐14,15‐oxidoGGPP to generate 3α‐ and 3β‐hydroxytuberculosinyl diphosphate. Conformational analyses were carried out with deuterium‐labeled GGPP and oxidoGGPP. We found that GGPP and (14R)‐oxidoGGPP adopted a chair/chair conformation, but (14S)‐oxidoGGPP adopted a boat/chair conformation. Interestingly, the conformations of oxidoGGPP for the A‐ring formation are the opposite of those of oxidosqualene when it is used as a substrate by squalene cyclases for the biosynthesis of hopene and tetrahymanol. (3R)‐Oxidosqualene is folded in a boat conformation, whereas (3S)‐2,3‐oxidosqualene folds into a chair conformation, for the formation of the A‐rings of the hopene and tetrahymanol skeletons, respectively.     

In Vitro Activity of Copper(II) Complexes,Loaded or Unloaded into a Nanostructured Lipid System,against Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious disease caused mainly by the bacillus Mycobacterium tuberculosis (Mtb), presenting 9.5 million new cases and 1.5 million deaths in 2014. The aim of this study was to evaluate a nanostructured lipid system (NLS) composed of 10% phase oil (cholesterol), 10% surfactant (soy phosphatidylcholine, sodium oleate), and Eumulgin^® HRE 40 ([castor oil polyoxyl-40-hydrogenated] in a proportion of 3:6:8), and an 80% aqueous phase (phosphate buffer pH = 7.4) as a tactic to enhance the in vitro anti-Mtb activity of the copper(II) complexes [CuCl₂(INH)₂]·H₂O (1), [Cu(NCS)₂(INH)₂]·5H₂O (2) and [Cu(NCO)₂(INH)₂]·4H₂O (3). The Cu(II) complex-loaded NLS displayed sizes ranging from 169.5 ± 0.7095 to 211.1 ± 0.8963 nm, polydispersity index (PDI) varying from 0.135 ± 0.0130 to 0.236 ± 0.00100, and zeta potential ranging from −0.00690 ± 0.0896 to −8.43 ± 1.63 mV. Rheological analysis showed that the formulations behave as non-Newtonian fluids of the pseudoplastic and viscoelastic type. Antimycobacterial activities of the free complexes and NLS-loaded complexes against Mtb H₃₇Rv ATCC 27294 were evaluated by the REMA methodology, and the selectivity index (SI) was calculated using the cytotoxicity index (IC₅₀) against Vero (ATCC^® CCL-81), J774A.1 (ATCC^® TIB-67), and MRC-5 (ATCC^® CCL-171) cell lines. The data suggest that the incorporation of the complexes into NLS improved the inhibitory action against Mtb by 52-, 27-, and 4.7-fold and the SI values by 173-, 43-, and 7-fold for the compounds 1, 2 and 3, respectively. The incorporation of the complexes 1, 2 and 3 into the NLS also resulted in a significant decrease of toxicity towards an alternative model (Artemia salina L.). These findings suggest that the NLS may be considered as a platform for incorporation of metallic complexes aimed at the treatment of TB.     

Development of Potent Inhibitors of the Mycobacterium tuberculosis Virulence Factor Zmp1 and Evaluation of Their Effect on Mycobacterial Survival inside Macrophages

The enzyme Zmp1 is a zinc‐containing peptidase that plays a critical role in the pathogenicity of Mycobacterium tuberculosis. Herein we describe the identification of a small set of Zmp1 inhibitors based on a novel 8‐hydroxyquinoline‐2‐hydroxamate scaffold. Among the synthesized compounds, N‐(benzyloxy)‐8‐hydroxyquinoline‐2‐carboxamide ( 1 c ) was found to be the most potent Zmp1 inhibitor known to date, and its binding mode was analyzed both by kinetics studies and molecular modeling, identifying critical interactions of 1 c with the zinc ion and residues in the active site. The effect of 1 c on intracellular Mycobacterium survival was assayed in J774 murine macrophages infected with M. tuberculosis H37Rv or M. bovis BCG and human monocyte‐derived macrophages infected with M. tuberculosis H37Rv. Cytotoxicity and genotoxicity were also assessed. Overall, inhibitor 1 c displays interesting in vitro antitubercular properties worthy of further investigation.     

Biological Evaluation of Potent Triclosan‐Derived Inhibitors of the Enoyl–Acyl Carrier Protein Reductase InhA in Drug‐Sensitive and Drug‐Resistant Strains of Mycobacterium tuberculosis

New triclosan (TRC) analogues were evaluated for their activity against the enoyl–acyl carrier protein reductase InhA in Mycobacterium tuberculosis (Mtb). TRC is a well‐known inhibitor of InhA, and specific modifications to its positions 5 and 4′ afforded 27 derivatives; of these compounds, seven derivatives showed improved potency over that of TRC. These analogues were active against both drug‐susceptible and drug‐resistant Mtb strains. The most active compound in this series, 4‐(n‐butyl)‐1,2,3‐triazolyl TRC derivative 3 , had an MIC value of 0.6 μg mL^?1 (1.5 μM ) against wild‐type Mtb. At a concentration equal to its MIC, this compound inhibited purified InhA by 98 %, and showed an IC₅₀ value of 90 nM . Compound 3 and the 5‐methylisoxazole‐modified TRC 14 were able to inhibit the biosynthesis of mycolic acids. Furthermore, mc²4914, an Mtb strain overexpressing inhA, was found to be less susceptible to compounds 3 and 14 , supporting the notion that InhA is the likely molecular target of the TRC derivatives presented herein.     

Structure‐Guided Design of Thiazolidine Derivatives as Mycobacterium tuberculosis Pantothenate Synthetase Inhibitors

The pantothenate biosynthetic pathway is essential for the persistent growth and virulence of Mycobacterium tuberculosis (Mtb) and one of the enzymes in the pathway, pantothenate synthetase (PS, EC: 6.3.2.1), encoded by the panC gene, has become an appropriate target for new therapeutics to treat tuberculosis. Herein, we report nanomolar thiazolidine inhibitors of Mtb PS developed by a rational inhibitor design approach. The thiazolidine compounds were discovered by using energy‐based pharmacophore modelling and subsequent in vitro screening, which resulted in compounds with a half maximal inhibitory concentration (IC₅₀) value of (1.12±0.12) μM . These compounds were subsequently optimised by a combination of modelling and synthetic chemistry. Hit expansion of the lead by chemical synthesis led to an improved inhibitor with an IC₅₀ value of 350 nM and an Mtb minimum inhibitory concentration (MIC) of 1.55 μM . Some of these compounds also showed good activity against dormant Mtb cells.     

Fueling Open‐Source Drug Discovery: 177 Small‐Molecule Leads against Tuberculosis

With the aim of fuelling open‐source, translational, early‐stage drug discovery activities, the results of the recently completed antimycobacterial phenotypic screening campaign against Mycobacterium bovis BCG with hit confirmation in M. tuberculosis H37Rv were made publicly accessible. A set of 177 potent non‐cytotoxic H37Rv hits was identified and will be made available to maximize the potential impact of the compounds toward a chemical genetics/proteomics exercise, while at the same time providing a plethora of potential starting points for new synthetic lead‐generation activities. Two additional drug‐discovery‐relevant datasets are included: a) a drug‐like property analysis reflecting the latest lead‐like guidelines and b) an early lead‐generation package of the most promising hits within the clusters identified.     

Lysophospholipids from the Guangxi Sponge Spirastrella purpurea

Four known ( 1 – 4 ) and two new ( 5 and 6 ) lysophospholipids were isolated from the sponge Spirastrella purpurea from Weizhou Island, Guangxi Autonomous Region, China. The structures of the new compounds ( 5 and 6 ) were elucidated by detailed spectroscopic techniques, including 1D and 2D NMR (¹H and ¹³C NMR, ¹H–¹H COSY, HSQC, and HMBC) as well as mass spectrometry and optical rotation experiments. The known compounds ( 1 – 4 ) were identified by comparison of their spectroscopic data and specific optical rotation with those reported in the literature. The isolated compounds displayed various moderate in vitro antifungal activities against four fungi (Cryptococcus neoformans, Candida glabrata, Trichophyton rubrum, and Aspergillus fumigatus), whereas they displayed no neuroprotective activity against Aβ_25‐35‐induced SH‐SY5Y cell damage.     

Hydrodynamics of shallow,conical spouted beds

Experiments were conducted with shallow beds, in the presence of atomizing air injected into the base, under the following conditions: Particles — urea, sulphur coated urea, polyethylene, polyformaldehyde and polystyrene; particle diameters (d_p) – 2.1 to 2.8 mm; cone angle – 60°; cylindrical bed diameter (D) – 0.24 and 0.45 m; bed height (H) – 0.24 to 0.40 m; orifice diameter (d_i) – 21 to 35 mm; main spouting air (Q_s) ≤ 37 L(actual)/s; atomizing air (Q_a) ≤ 0.87 L(actual)/s. The minimum spouting velocity is well represented by: U_ms = 13.5 (2gH)^0.5 (d_p/D')^1.17 (D'_i/D')^0.372 (H/D')^?0.148 [(τ_p - τ)/τ]^0.289 where D' and D'_i denote the modified column and orifice diameter, respectively. Q_a, which affects D'_i, significantly influenced the air velocity in the spout, but not the pressure profiles in the annulus. Morgan and Littman's (1980) correlation could be adapted to predict the experimental pressure profiles in the annulus.     

DNA Interstrand Crosslinks by H‐pin Polyamide (S)‐seco‐CBI Conjugates

Although DNA interstrand crosslinking (ICL) agents are widely used as antitumor drugs, DNA sequence‐specific ICL agents are quite rare. In this study, H‐pin imidazole‐pyrrole polyamide 1‐(chloromethyl)‐2,3‐dihydro‐1H‐benzo[e]indol‐5‐ol (seco‐CBI) conjugates that produce sequence‐specific DNA ICLs were designed and synthesized. Conjugates with H‐pin polyamide and seco‐CBI moieties were constructed to recognize a 7 bp DNA sequence, and their reactivity and selectivity in DNA alkylation were evaluated by using high‐resolution denaturing gel electrophoresis and sequence‐specific plasmid cleavage. One conjugate ( 6 ), which contained a chiral (S)‐seco‐CBI, exhibited greater sequence‐specific ICL activity toward the target DNA sequence and was cytotoxic to a cancer cell line. Molecular modeling studies indicated that the greater activity of 6 resulted from the relative orientation of the cyclopropane group in the (S)‐CBI unit.     

Development of Cyclobutene‐ and Cyclobutane‐Functionalized Fatty Acids with Inhibitory Activity against Mycobacterium tuberculosis

Eleven fatty acid analogues incorporating four‐membered carbocycles (cyclobutenes, cyclobutanes, cyclobutanones, and cyclobutanols) were investigated for the ability to inhibit the growth of Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). A number of the analogues displayed inhibitory activity against both mycobacterial species in minimal media. Several of the molecules displayed potent levels of inhibition against Mtb, with MIC values equal to or below those observed with the anti‐tuberculosis drugs D ‐cycloserine and isoniazid. In contrast, two of the analogues that display the greatest activity against Mtb failed to inhibit E. coli growth under either set of conditions. Thus, the active molecules identified herein may provide the basis for the development of anti‐mycobacterial agents against Mtb.     

Preorganized macrocyclic receptors featuring endo-carboxylic acid Groups. Host synthesis and inclusion compounds with alcohol and amine guests

The synthesis and characterization of four new macrocyclic host compounds 1 – 4 having modified diphenylmethane units as bridging elements and two endo-orientated carboxylic acid groups attached to aromatic building blocks are described. The complexation properties of the macrocycles towards amines and alcohols are reported, showing that the ability to form convergent inclusion compounds depends on the type of the spacer element. For the dicarboxylic hosts 1 and 2 endo-complexation of guest molecules based on hydrogen bonding to the acid functions is proved using ¹H NMR investigation and X-ray crystal structure analysis.