Loss of a mycobacterial gene encoding a reductase leads to an altered cell wall containing beta-oxo-mycolic acid analogs and accumulation of ketones

Mycolic acids are essential components of the mycobacterial cell wall. In this study, we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The DeltaMSMEG4722 strain synthesized alpha-alkyl, beta-oxo intermediates of mycolic acids, which were found esterified to cell wall arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base treatment, their presence was established by prior reduction of the beta-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of alpha-alkyl, beta-oxo fatty acid precursors of mycolic acids.     

Concise synthesis of an arabinofuranose hexasaccharide present in the cell wall of Mycobacterium tuberculosis

Mycobacterium cell wall consists of three major polysaccharide portions and arabinofuranose (Araf) is present in two of the major portions, arabinogalactan (AG) and lipoarabinomannan (LAM). A peculiar Araf hexasaccharide possessing two β-linked Araf units are present in both AG and LAM polysaccharides. Herein, we report an efficient and concise synthesis of this Araf hexasaccharide using single starting 3,5-TIPDS protected Araf thioglycoside precursor. Double β-glycosylation was achieved using strained cyclic 2-p-methoxybenzyl-3,5-TIPDS Araf thioglycoside donor.     

Substrate specificity of Rv3378c, an enzyme from Mycobacterium tuberculosis, and the inhibitory activity of the bicyclic diterpenoids against macrophage phagocytosis

The Rv3378c gene product from Mycobacterium tuberculosis encodes a diterpene synthase to produce tuberculosinol (3), 13R-isotuberculosinol (4a), and 13S-isotuberculosinol (4b) from tuberculosinyl diphosphate (2). The product distribution ratios are 1 : 1 for 3 to 4 and 1 : 3 for 4a to 4b. The substrate specificity of the Rv3378c-encoded enzyme was examined. The 3 labdadienyl diphosphates, copalyl diphosphate (CDP) (7), ent-CDP (8), and syn-CDP (9), underwent the conversion reaction, with good yields (67-78%). Copalol (23) and manool (24) were produced from 7, ent-copalol (25) and ent-manool (26) from 8, and syn-copalol (27) and vitexifolin A (28) from 9. The ratio of 23 to 24 was 40 : 27, that of 25:26 was 22 : 50, and that of 27:28 was 16 : 62. Analysis on a GC-MS chromatograph equipped with a chiral column revealed that 24, 26, and 28 consisted of a mixture of 13R- (a) and 13S-stereoisomers (b) in the following ratio: ca. 1 : 1 for 24a to 24b, ca. 1 : 5 for 26a to 26b, and ca. 1 : 19 for 28a to 28b. The structures of these products indicate that the reactions of the 3 CDPs proceeded in the same fashion as that of 2. This is the first report on the enzymatic synthesis of natural diterpenes manool, ent-manool, and vitexifolin A. Both Rv3377c and Rv3378c genes are found in virulent Mycobacterium species, but not in avirulent species. We found that 3 and 4 inhibited the phagocytosis of opsonized zymosan particles by human macrophage-like cells. Interestingly, the inhibitory activity was synergistically increased by the coexistence of 3 and 4b. Other labdane-related diterpenes, 13-16 and 23-28, had little or no inhibitory activity. This synergistic inhibition by 3 and 4 may provide further advantage to the impairment of phagocyte function, which might contribute to pathogenicity of M. tuberculosis.     

Four isomers of c(96) fullerene structurally proven as c(96) cl(22) and c(96) cl(24)

Direct proof of the cage connectivities of four isomers of C(96) , the highest isolable empty fullerene, has been achieved. C(96) fractions, which were isolated from fullerene soot by recycling HPLC, were chlorinated and the resulting single crystals of C(96) Cl(22) and C(96) Cl(24) were studied by X-ray diffraction using synchrotron radiation. D(2) -C(96) (183)Cl(24) (see structure; gray C, green Cl) was obtained in two crystalline modifications.     

Possible involvement of lymphocyte activating factor (LAF) as an endogenous pyrogen in fever induced by the cell wall skeleton of Nocardia rubra (N-CWS)

We investigated whether interleukin-1 (IL-1) acts as an endogenous pyrogen (EP) on the fever caused by the cell wall skeleton of Nocardia rubra (N-CWS) in guinea pigs. IL-1 activity was expressed as potency of lymphocyte activating factor (LAF). When guinea pig peritoneal macrophages were pulse-stimulated with N-CWS (1-100 micrograms/ml), dose-dependent LAF activity was detected in the supernatants after culture for 4 h. Gel filtration of the culture supernatants on Sephadex G-200 showed that the fractions with LAF activity were not the same as those with cytotoxic activity for L-929 cells, which was measured as an index of tumor necrosis factor (TNF) in parallel with LAF activity. Pyretic activity was detected both in the fractions with LAF activity and in those with cytotoxic activity for L-929 cells. Furthermore, when these macrophages were pulse-stimulated again, this time with the supernatant obtained from macrophages previously pulse-stimulated with N-CWS, LAF and cytotoxic activity for L-929 cells continued to be released from the macrophages. We suggest that IL-1 might be a possible EP in the process of fever elicited by N-CWS, and that such an EP stimulates the macrophages to release further IL-1 or TNF. The resultant long-lasting fever would thus be caused by the continuous release of an EP.     

Poly(Methyl Methacrylate) as a matrix for immobilization of lipase

Poly(methyl methacrylate) (PMMA) was found to be suitable for the immobilization of lipase fromCandida rugosa. The best result based on hydrolytic activity was obtained by adsorption of the purified unbuffered enzyme solution onto PMMA beads without any modification of the beads. Prolonged exposure of the protein to the beads increased its adsorption but the expressed activity decreased after 1 h of exposure. The magnitude of the immobilized activity also varied with the size of the beads. Immobilization of the lipase shifted its optimal reaction temperature from 37 to 45°C. The immobilized enzyme is also more stable than the free enzyme in solution. The operational half-life of the immobilized lipase packed in a column and assayed in a closed system is 40 d.     

Possible involvement of a tumor necrosis factor (TNF)-like mediator as an endogenous pyrogen in fever induction by Nocardia rubra cell wall skeleton (N-CWS)

Tumor necrosis factor (TNF), a cytokine produced in macrophages, also acts as an endogenous pyrogen (EP). To investigate whether TNF has a role in the fever induced by Nocardia rubra cell wall skeleton (N-CWS), the relationship between fever and TNF production was studied in guinea pigs. N-CWS injected i.v. to guinea pigs caused biphasic fever and had L-929 cell-killing activity which resembled that of TNF in the sera 30 min before the first phase of fever appeared. In vitro, L-929 cell-killing activity was demonstrated in the culture supernatant of guinea pig peritoneal macrophages pretreated with N-CWS, and the activity increased dependently on N-CWS concentration or culture duration. When the supernatant of the macrophages was fractionated by gel filtration and each fraction was assayed for fever-inducing and L-929 cell-killing activities, the fraction with the cell-killing activity also induced fever with characteristics similar to that by i.v. injection of N-CWS in guinea pigs. These results suggest that TNF acts as an EP on the fever induced by N-CWS in guinea pigs.     

In Silico Identification of Possible Inhibitors for Protein Kinase B (PknB) of Mycobacterium tuberculosis

With tuberculosis still being one of leading causes of death in the world and the emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), researchers have been seeking to find further therapeutic strategies or more specific molecular targets. PknB is one of the 11 Ser/Thr protein kinases of Mtb and is responsible for phosphorylation-mediated signaling, mainly involved in cell wall synthesis, cell division and metabolism. With the amount of structural information available and the great interest in protein kinases, PknB has become an attractive target for drug development. This work describes the optimization and application of an in silico computational protocol to find new PknB inhibitors. This multi-level computational approach combines protein–ligand docking, structure-based virtual screening, molecular dynamics simulations and free energy calculations. The optimized protocol was applied to screen a large dataset containing 129,650 molecules, obtained from the ZINC/FDA-Approved database, Mu.Ta.Lig Virtual Chemotheca and Chimiothèque Nationale. It was observed that the most promising compounds selected occupy the adenine-binding pocket in PknB, and the main interacting residues are Leu17, Val26, Tyr94 and Met155. Only one of the compounds was able to move the active site residues into an open conformation. It was also observed that the P-loop and magnesium position loops change according to the characteristics of the ligand. This protocol led to the identification of six compounds for further experimental testing while also providing additional structural information for the design of more specific and more effective derivatives.     

A one-pot strategy for synthesis of 5-O-(alpha-D-arabinofuranosyl)-6-O-(beta-D-galactofuranosyl)-D-galactofuranose present in motif E of the Mycobacterium tuberculosis cell wall

5-O-(Alpha-D-arabinofuranosyl)-6-O-(beta-D-galactofuranosyl)-D-galactofuranose 6 present in motif E of the Macobacterium tuberculosis cell wall has been regio- and stereospecifically synthesized using 3-O-benzoyl-1,2-O-isopropylidine-alpha-D-galactofuranose (10) as the glycosyl acceptor by the trichloroacetamidate method in a one-pot manner. The diol glycosyl acceptor 10 was smoothly derived from 1,2:5,6-di-O-isopropylidene-alpha-D-galactofuranose (8) by 3-O-benzoylation and then selective 5,6-O-deacetonation. The preparation of 8 was greatly improved by increasing the ratio of DMF to acetone and using a solid-supported catalyst.     

Monoterpene-Containing Substituted Coumarins as Inhibitors of Respiratory Syncytial Virus (RSV) Replication

Respiratory syncytial virus (RSV) is a critical cause of infant mortality. However, there are no vaccines and adequate drugs for its treatment. We showed, for the first time, that O-linked coumarin–monoterpene conjugates are effective RSV inhibitors. The most potent compounds are active against both RSV serotypes, A and B. According to the results of the time-of-addition experiment, the conjugates act at the early stages of virus cycle. Based on molecular modelling data, RSV F protein may be considered as a possible target.     

Complex of tris(2-hydroxyethyl)amine with zinc bis(2-methylphenoxyacetate) as an inhibitor of the acid lipase of the aortic intima

The previously unknown ability of Zitrimin, the complex of tris(2-hydroxyethyl)amine with zinc(ii) bis(2-methylphenoxyacetate), to affect the activity of acid lipase of the aortic intima was studied. Daily administration of an aqueous solution of the tris(2-hydroxyethyl)amine complex with zinc bis(2-methylphenoxyacetate) in a dose of 10 mg kg^?1 for 3 months to rabbilts with experimental atherosclerosis was found to decrease the cholesterol and total lipid levels in the aortic tissue and to decrease the degree of aortic damage with atherosclerotic plaques. According to the results of enzymatic analysis, development of atherosclerosis is accompanied by 68% increase in the activity of acid lipase in the intima of the aorta compared to the control.

     

Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure–activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.     

A new synthetic approach to mycobacterial cell wall α-(1→5)-d-arabinofuranosyl C-oligosaccharides

Three designed arabinofuranose building blocks allowed the diastereoselective synthesis of a C-disaccharide and a C-trisaccharide by Wittig olefination. The latter compound represents the first example of all-carbon linked arabinofuranotriose analogue.     

Homology modeling and molecular dynamic simulation of UDP-N-acetylmuramoyl-l-alanine-d-glutamate ligase (MurD) from Mycobacterium tuberculosis H37Rv using in silico approach

The present study aimed to identify the prospective inhibitors of MurD, a cytoplasmic enzyme that catalyzes the addition of d-glutamate to the UDP-N-acetylmuramoyl-l-alanine nucleotide precursor in Mycobacterium tuberculosis (MTB), using virtual screening, docking studies, pharmacokinetic analysis, Molecular Dynamic (MD) simulation, and Molecular Mechanics Generalized Born and Surface Area (MM-GBSA) analyses. The three dimensional (3D) structure was determined based on the homology technique using a template from Streptococcus agalactiae. The modeled structure had three binding sites, namely; substrate binding site (Val18, Thr19, Asp39, Asp40, Gly75, Asn147, Gln171 and His192), the ATP binding site (Gly123, Lys124, Thr125, Thr126, Glu166, Asp283, and Arg314) and the glutamic acid binding site (Arg382, Ser463, and Tyr470). These residues mentioned above play a critical role in the catalytic activity of the enzyme, and their inhibition could serve as a stumbling block to the normal function of the enzyme. A total of 10,344 obtained from virtual screened of Zinc and PubChem databases. These compounds further screened for Lipinski rule of five, docking studies and pharmacokinetic analysis. Four compounds with good binding energies (ZINC11881196 = −10.33 kcal/mol, ZINC12247644 = −8.90 kcal/mol, ZINC14995379 =−8.42 kcal/mol, and PubChem6185 = −8.20 kcal/mol), better than the binding energies of the ATP (−2.31 kcal/mol) and the ligand with known IC₅₀, Aminothiazole (−7.11 kcal/mol) were selected for the MD simulation and MM-GBSA analyses. The result of the analyses showed that all the four ligands formed a stable complex and had the binding free energies better than the binding energy of ATP. Therefore, these ligands considered as suitable prospective inhibitors of the MurD after experimental validation.     

NMR studies of buspirone (an anxiolytic drug) analogues

Conformations of piperazine rings in 8-{4-[4-(2-pyrimidyl)-1-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (buspir-one — 1) and its two analogues 8-{4-[4-(2-quinolinyl)-1-piperazinyl]butyl}-8-azaspiro[4.5]-decane-7,9-dione (kaspar — 2) and 4,4-dimethyl-1-{4-[4-(2-quinolinyl)-1-piperazinyl]butyl}-2,6-piperidinedione (mesmar — 3) (Fig. 1) have been studied with the aid of ¹H NMR and ¹³C NMR spectra. For free bases the two bands corresponding to piperazine hydrogen atoms in the spectra broaden considerably with a decrease in temperature to divide into four separate bands, indicating the presence of a dynamic exchange process. A similar dynamic process, but for higher temperatures, was observed for buspirone (1), kaspar (2) and mesmar (3) hydrochlorides. Proton and carbon atom resonance lines have been assigned with the aid of 2D COSY and 2D HETCOR two-dimensional spectra.     

The high-pressure segmented quadrupole collision cell (HP-SQCC) as an ion molecule reactor

A high-pressure 20-segment quadrupole collision cell (HP-SQCC), which replaces a collision cell in a modified triple-quadrupole mass spectrometer is investigated in this work as an ion-molecule reactor with an inherent heat source. The highest working pressure achievable is 20 mTorr. The 20 quadrupole segments permit superimposition of linear axial electric field over the conventional quadrupole field in the radial direction. The axial and radial fields are employed to control ion temperature. Heat is transferred to the reactants through ion frictional heating. The HP-SQCC utilizes a combination of several physicochemical phenomena and an attempt is made to examine a range of ion-molecule reactions. Due to a sufficiently large number of reactive collisions, the reactor is used to promote sequential exothermic ion-molecule reactions. To characterize the performance of the HP-SQCC, the various ion-molecule reactions between the fragment ions of ferrocene (Cp(2)Fe), cobaltocene (Cp(2)Co) and nitrogen, oxygen, water and carbon monoxide are investigated.     

Hydrazones of 4-(Trifluoromethyl)benzohydrazide as New Inhibitors of Acetyl- and Butyrylcholinesterase

Based on the broad spectrum of biological activity of hydrazide–hydrazones, trifluoromethyl compounds, and clinical usage of cholinesterase inhibitors, we investigated hydrazones obtained from 4-(trifluoromethyl)benzohydrazide and various benzaldehydes or aliphatic ketones as potential inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). They were evaluated using Ellman’s spectrophotometric method. The hydrazide–hydrazones produced a dual inhibition of both cholinesterase enzymes with IC₅₀ values of 46.8–137.7 µM and 19.1–881.1 µM for AChE and BuChE, respectively. The majority of the compounds were stronger inhibitors of AChE; four of them (2-bromobenzaldehyde, 3-(trifluoromethyl)benzaldehyde, cyclohexanone, and camphor-based 2o, 2p, 3c, and 3d, respectively) produced a balanced inhibition of the enzymes and only 2-chloro/trifluoromethyl benzylidene derivatives 2d and 2q were found to be more potent inhibitors of BuChE. 4-(Trifluoromethyl)-N’-[4-(trifluoromethyl)benzylidene]benzohydrazide 2l produced the strongest inhibition of AChE via mixed-type inhibition determined experimentally. Structure–activity relationships were identified. The compounds fit physicochemical space for targeting central nervous systems with no apparent cytotoxicity for eukaryotic cell line together. The study provides new insights into this CF₃-hydrazide–hydrazone scaffold.