A Rhodanine Derivative CCR-11 Inhibits Bacterial Proliferation by Inhibiting the Assembly and GTPase Activity of FtsZ

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (～15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.     

Structure elucidation of Sch 538415, a novel acyl carrier protein synthase inhibitor from a microorganism

A novel acyl carrier protein synthase inhibitor, Sch 538415 (1), was isolated from an unidentified bacterial microbe. Structure elucidation of 1 was accomplished based on analysis of spectroscopic data including UV, MS and 2D-NMR spectra. Compound 1 exhibited inhibitory activity in the acyl carrier protein synthase (AcpS) assay with an IC(50) value of 4.19 microM and showed antibacterial activity against Staphylococcus aureus in the agar diffusion assay.     

Synthesis and antibacterial activity of peptide deformylase inhibitors

Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.     

4'-phosphopantetheine transfer in primary and secondary metabolism of Bacillus subtilis

4'-Phosphopantetheine transferases (PPTases) transfer the 4'-phosphopantetheine moiety of coenzyme A onto a conserved serine residue of acyl carrier proteins (ACPs) of fatty acid and polyketide synthases as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases. This posttranslational modification converts ACPs and PCPs from their inactive apo into the active holo form. We have investigated the 4'-phosphopantetheinylation reaction in Bacillus subtilis, an organism containing in total 43 ACPs and PCPs but only two PPTases, the acyl carrier protein synthase AcpS of primary metabolism and Sfp, a PPTase of secondary metabolism associated with the nonribosomal peptide synthetase for the peptide antibiotic surfactin. We identified and cloned ydcB encoding AcpS from B. subtilis, which complemented an Escherichia coli acps disruption mutant. B. subtilis AcpS and its substrate ACP were biochemically characterized. AcpS also modified the d-alanyl carrier protein but failed to recognize PCP and an acyl carrier protein of secondary metabolism discovered in this study, designated AcpK, that was not identified by the Bacillus genome project. On the other hand, Sfp was able to modify in vitro all acyl carrier proteins tested. We thereby extend the reported broad specificity of this enzyme to the homologous ACP. This in vitro cross-interaction between primary and secondary metabolism was confirmed under physiological in vivo conditions by the construction of a ydcB deletion in a B. subtilis sfp(+) strain. The genes coding for Sfp and its homolog Gsp from Bacillus brevis could also complement the E. coli acps disruption. These results call into question the essential role of AcpS in strains that contain a Sfp-like PPTase and consequently the suitability of AcpS as a microbial target in such strains.     

Effect of berberine on Escherichia coli, Bacillus subtilis, and their mixtures as determined by isothermal microcalorimetry

The strong toxicity of pathogenic bacteria has resulted in high levels of morbidity and mortality in the general population. Developing effective antibacterial agents with high efficacy and long activity is in great demand. In this study, the microcalorimetric technique based on heat output of bacterial metabolism was applied to evaluate the effect of berberine on Escherichia coli, Bacillus subtilis, individually and in a mixture of both using a multi-channel microcalorimeter. The differences in shape of the power-time fingerprints and thermokinetic parameters of microorganism growth were compared. The results revealed that low concentration (20?μg/mL) of berberine began to inhibit the growth of E. coli and mixed microorganisms, while promoting the growth of B. subtilis; high concentration of berberine (over 100?μg/mL) inhibited B. subtilis. The endurance of E. coli to berberine was obviously lower than B. subtilis, and E. coli could decrease the endurance of B. subtilis to berberine. The sequence of half-inhibitory concentration (IC(50)) of berberine was: B. subtilis (952.37?μg/mL)?>?mixed microorganisms (682.47?μg/mL)?>?E. coli (581.69?μg/mL). Berberine might be a good selection of antibacterial agent used in the future. The microcalorimetric method should be strongly suggested in screening novel antibacterial agents for fighting against pathogenic bacteria.     

Synthesis and antimicrobial activity of tetrodecamycin partial structures

An efficient synthetic approach to the core structure 5 of the novel polyketide antibiotic tetrodecamycin (1) was developed. This approach features the acid-catalyzed cyclization of a tert-butyldimethylsilyl protected methyl alpha-(gamma-hydroxyacyl) tetronate, leading to the novel tricyclic ring skeleton exhibited by 5, and an efficient strategy for the parallel introduction of the cis-diol and exo-methylene function. In addition to 5, diastereomer 26, analogue 6 and several derivatives (16, 27-29) were prepared and evaluated for their antibacterial activities against Staphylococcus aureus (including MRSA) and Enterococcus faecalis and for their cytotoxic activities against human leukemia cell lines (HL-60, Jurkat T-cells). While compound 5 did not inhibit the growth of the Gram-positive pathogens (MICs >128 microg mL(-1)), analogue 6 and 2-naphthoyl derivative 27 showed promising antibacterial activities with MICs of 4-16 microg mL(-1). Remarkably, the antibacterial activity of these compounds was paralleled by cytotoxicity (IC(50) 10-23 microM). The reactive exo-methylene moiety was shown to be crucial, but not sufficient by its own, for both the antibacterial and the cytotoxic activities.     

The novel pyrimidine and purine derivatives of l-ascorbic acid: synthesis, one- and two-dimensional 1H and 13C NMR study, cytostatic and antiviral evaluation

The syntheses of the novel C-5 substituted pyrimidine derivatives of l-ascorbic acid containing free hydroxy groups at C-2' (6-10) or C-2' and C-3' (11-15) positions of the lactone ring are described. Debenzylation of the 6-chloro- and 6-(N-pyrrolyl)purine derivatives of 2,3-O,O-dibenzyl-l-ascorbic acid (16 and 17) gave the new compounds containing hydroxy groups at C-2' (18) and C-2' and C-3' (19 and 20). Z- and E-configuration of the C4'C5' double bond and position of the lactone ring of the compounds 6-9 were deduced from their one- and two-dimensional (1)H and (13)C NMR spectra and connectivities in NOESY and HMBC spectra. Compounds 15 and 18 showed the best inhibitory activities of all evaluated compounds in the series. The compound 15 containing 5-(trifluoromethyl)uracil showed marked inhibitory activity against all human malignant cell lines (IC(50): 5.6-12.8 microM) except on human T-lymphocytes. Besides, this compound influenced the cell cycle by increasing the cell population in G2/M phase and induced apoptosis in SW 620 and MiaPaCa-2 cells. The compound 18 containing 6-chloropurine ring expressed the most pronounced inhibitory activities against HeLa (IC(50): 6.8 microM) and MiaPaCa-2 cells (IC(50): 6.5 microM). The compound 20 with 6-(N-pyrrolyl)purine moiety showed the best differential inhibitory effect against MCF-7 cells (IC(50): 35.9 microM).     

Cytotoxic constituents from Brazilian red propolis and their structure-activity relationship

Several classes of flavonoids [flavanoids (1-10), flavonol (11), isoflavones (12-18), isoflavanones (19-22), isoflavans (23-26), chalcones (27-30), auronol (31), pterocarpans (32-37), 2-arylbenzofuran (38), and neoflavonoid (39)] and lignans (40-42) isolated from the MeOH extract of Brazilian red propolis were investigated for their cytotoxic activity against a panel of six different cancer cell lines including murine colon 26-L5 carcinoma, murine B16-BL6 melanoma, murine Lewis lung carcinoma, human lung A549 adenocarcinoma, human cervix HeLa adenocarcinoma, and human HT-1080 fibrosarcoma cell lines. Based on the observed results, structure-activity relationships were discussed. Among the tested compounds, 7-hydroxy-6-methoxyflavanone (3) exhibited the most potent activity against B16-BL6 (IC(50), 6.66microM), LLC (IC(50), 9.29microM), A549 (IC(50), 8.63microM), and HT-1080 (IC(50), 7.94microM) cancer cell lines, and mucronulatol (26) against LLC (IC(50), 8.38microM) and A549 (IC(50), 9.9microM) cancer cell lines. These activity data were comparable to those of the clinically used anticancer drugs, 5-fluorouracil and doxorubicin, against the tested cell lines, suggesting that 3 and 26 are the good candidates for future anticancer drug development.     

Crystal structure of Streptococcus pneumoniae acyl carrier protein synthase: an essential enzyme in bacterial fatty acid biosynthesis

Acyl carrier protein synthase (AcpS) catalyzes the formation of holo-ACP, which mediates the essential transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and lipids in the cell. Thus, AcpS plays an important role in bacterial fatty acid and lipid biosynthesis, making it an attractive target for therapeutic intervention. We have determined, for the first time, the crystal structure of the Streptococcus pneumoniae AcpS and AcpS complexed with 3'5'-ADP, a product of AcpS, at 2.0 and 1.9 A resolution, respectively. The crystal structure reveals an alpha/beta fold and shows that AcpS assembles as a tightly packed functional trimer, with a non-crystallographic pseudo-symmetric 3-fold axis, which contains three active sites at the interface between protomers. Only two active sites are occupied by the ligand molecules. Although there is virtually no sequence similarity between the S.pneumoniae AcpS and the Bacillus subtilis Sfp transferase, a striking structural similarity between both enzymes was observed. These data provide a starting point for structure-based drug design efforts towards the identification of AcpS inhibitors with potent antibacterial activity.     

New delta8(14)-ketosterols with an oxygenated side chain

New analogues of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) with modified 17-chains [(22S,23S,24S)- and (22R,23R,24S)-3beta-hydroxy-24-methyl-22,23-oxido-5alpha-cholest-8(14)-en-15-ones and (22RS,23xi,24S)-24-methyl-5alpha-cholesta-3beta,22,23-triol-15-one] were synthesized from (22E,24S)-3beta-acetoxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one. The chiralities of their 22 and 23 centers were determined by NMR spectroscopy. The isomeric 22,23-epoxides effectively inhibited cholesterol biosynthesis in hepatoma Hep G2 cells (IC50 0.9 +/- 0.2 and 0.7 +/- 0.2 microM, respectively), and their activities significantly exceeded those of 15-ketosterol (IC50 4.0 +/- 0.5 microM), (22E,24S)-3beta-hydroxy-24-methyl-5alpha-cholesta-8(14),22-dien-15-one (IC50 3.1 +/- 0.4 microM), and the 3beta,22,23-triol synthesized (IC50 6.0 +/- 1.0 microM). The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Novel azolyl-(phenylmethyl)]aryl/heteroarylamines: potent CYP26 inhibitors and enhancers of all-trans retinoic acid activity in neuroblastoma cells

The synthesis and potent inhibitory activity of novel 4-[(imidazol-1-yl and triazol-1-yl)(phenyl)methyl]aryl-and heteroaryl amines versus a MCF-7 CYP26A1 cell assay is described. Biaryl imidazole ([4-(imidazol-1-yl-phenyl-methyl)-phenyl]-naphthalen-2-yl-amine (8), IC(50)=0.5 microM; [4-(imidazol-1-yl-phenyl-methyl)-phenyl]-indan-5-yl-amine (9), IC(50)=1.0 microM) and heteroaryl imidazole derivatives ((1H-benzoimidazol-2-yl)-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (15), IC(50)=2.5 microM; benzooxazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (16), IC(50)=0.9 microM; benzothiazol-2-yl-{4-[(5H-imidazol-1-yl)-phenyl-methyl]-phenyl}-amine (17), IC(50)=1.5 microM) were the most potent CYP26 inhibitors. Using a CYP26A1 homology model differences in activity were investigated. Incubation of SH-SY5Y human neuroblastoma cells with the imidazole aryl derivative 8, and the imidazole heteroaryl derivatives 16 and 17 potentiated the atRA-induced expression of CYP26B1. These data suggest that further structure-function studies leading to clinical development are warranted.     

Design, synthesis, and application of novel triclosan prodrugs as potential antimalarial and antibacterial agents

A number of new triclosan-conjugated analogs bearing biodegradable ester linkage have been synthesized, characterized and evaluated for their antimalarial and antibacterial activities. Many of these compounds exhibit good inhibition against Plasmodium falciparum and Escherichia coli. Among them tertiary amine containing triclosan-conjugated prodrug (5) inhibited both P. falciparum (IC(50); 0.62microM) and E. coli (IC(50); 0.26microM) at lower concentrations as compared to triclosan. Owing to the presence of a cleavable ester moiety, these new prodrugs are hydrolyzed under physiological conditions and parent molecule, triclosan, is released. Further, introduction of tertiary/quaternary functionality increases their cellular uptake. These properties impart them with higher potency to their antimalarial as well as antibacterial activities. The best compound among them 5 shows close to four-fold enhanced activities against P. falciparum and E. coli cultures as compared to triclosan.     

Potential anti-allergic ent-kaurene diterpenes from the bark of Suregada multiflora

Two ent-kaurene diterpenes, ent-16-kaurene-3beta,15beta,18-triol (1) and ent-3-oxo-16-kaurene-15beta,18-diol (2), were isolated from a dichloromethane extract of the bark of Suregada multiflora along with five known diterpenes:ent-16-kaurene-3beta,15beta-diol (3), abbeokutone (4), helioscopinolide A (5), helioscopinolide C (6) and helioscopinolide I (7). Their structures were elucidated on the basis of spectroscopic analysis. Compounds 1-7 possessed appreciable anti-allergic activities in RBL-2H3 cells model with IC50 values ranging from 22.5 to 42.2 microM.     

Conformationally restricted analogs of deoxynegamycin

Deoxynegamycin (1b) is a protein synthesis inhibitor with activity against Gram-negative (GN) bacteria. A series of conformationally restricted analogs were synthesized to probe its bioactive conformation. Indeed, some of the constrained analogs were found to be equal or better than deoxynegamycin in protein synthesis assay (1b, IC(50)=8.2 microM; 44, IC(50)=6.6 microM; 35e(2), IC(50)=1 microM). However, deoxynegamycin had the best in vitro whole cell antibacterial activity (Escherichia coli, MIC=4-16 microg/mL; Klebsiella pneumoniae, MIC=8 microg/mL) suggesting that other factors such as permeation may also be contributing to the overall whole cell activity. A new finding is that deoxynegamycin is efficacious in an E. coli murine septicemia model (ED(50)=4.8 mg/kg), providing further evidence of the favorable in vivo properties of this class of molecules.     

Synthesis and anticancer evaluation of bis(benzimidazoles), bis(benzoxazoles), and benzothiazoles

Four classes of UK-1 analogues were synthesized and their cytotoxicity testing against human A-549, BFTC-905, RD, MES-SA, and HeLa carcinoma cell lines was determined. The results revealed that UK-1 and four of these analogues (15-18) are potent against the cancer cell lines. In particular, compound 16 is more potent than UK-1 against A-549 and HeLa cell lines, and compounds 15, 17, and 18 selectively exhibit potent cytotoxic activity against the BFTV-905 cells (IC50 9.6 microM), A-549 cells (IC50 6.6 microM), and MES-SA cells (IC50 9.2 microM), respectively.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Potent and selective inhibitors of bacterial methionyl tRNA synthetase derived from an oxazolone-dipeptide scaffold

The preparation and structure-activity relationships (SARs) of potent and selective small molecule inhibitors of bacterial methionyl-tRNA synthetase (MetRS) derived from an oxazolone-dipeptide scaffold are described. Examples combine Staphylococcus aureus MetRS (SaMetRS) potency with selectivity over human MetRS. As a result of the SAR expansion compound 14a was identified, as a potent SaMetRS inhibitor (IC(50)=18 nM) having moderate inhibition of MetRS derived from Enterococci faecalis (IC(50)=3.51 microM).     

Modified norcantharidins; synthesis, protein phosphatases 1 and 2A inhibition, and anticancer activity

Fourteen modified norcantharidin analogues have been synthesised and screened for their ability to inhibit the serine/threonine protein phosphatases 1 and 2A. The most potent compounds found were 10 (PP1 IC(50)=13+/-5 microM; PP2A IC(50)=7+/-3 microM) and 16 (PP1 IC(50)=18+/-8 microM; PP2A IC(50)=3.2+/-0.4 microM). Overall, only analogues possessing at least one acidic residue at the former anhydride warhead displayed any PP1 or PP2A inhibitory action. The ability of these analogues to inhibit PP1 and PP2A correlates well with their observed anti-cancer activity against a panel of five cancer cell lines: A2780 (human ovarian carcinoma), G401 (human kidney carcinoma), HT29 (human colorectal carcinoma), H460 (human lung carcinoma) and L1210 (murine leukemia).     

Ability of 15-hydroxyeicosatrienoic acid (15-OH-20:3) to modulate macrophage arachidonic acid metabolism

Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production.