Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 1.

The alarming reduction in drug effectiveness against bacterial infections has created an urgent need for the development of new antibacterial agents that circumvent bacterial resistance mechanisms. We report here a series of DNA gyrase and topoisomerase IV inhibitors that demonstrate potent activity against a range of Gram-positive and selected Gram-negative organisms, including clinically-relevant and drug-resistant strains. In part 1, we present a detailed structure activity relationship (SAR) analysis that led to the discovery of our previously disclosed compound, REDX05931, which has a minimum inhibitory concentration (MIC) of 0.06 µg mL-1 against fluoroquinolone-resistant Staphylococcus aureus. Although in vitro hERG and CYP inhibition precluded further development, it validates a rational design approach to address this urgent unmet medical need and provides a scaffold for further optimisation, which is presented in part 2.

Rational design, synthesis and testing of novel tricyclic topoisomerase inhibitors for the treatment of bacterial infections part 2.

Building on our previously-reported novel tricyclic topoisomerase inhibitors (NTTIs), we disclose the discovery of REDX07965, which has an MIC90 of 0.5 µg mL-1 against Staphylococcus aureus, favourable in vitro pharmacokinetic properties, selectivity versus human topoisomerase II and an acceptable toxicity profile. The results herein validate a rational design approach to address the urgent unmet medical need for novel antibiotics.

The effects of backgrounding system on growing and finishing performance and carcass characteristics of beef steers.

The objective of this 2-yr study was to evaluate growing and finishing performance as well as carcass characteristics of spring-born steers backgrounded on 3 different systems, using feedstuffs readily available in the Midwest: 1) grazing corn residue and being supplemented with dried distillers plus solubles at 2.68 kg DM/steer 6 d/wk (RESIDUE), 2) grazing a late summer-planted oat-brassica forage mix (CCROP), or 3) being fed a corn silage-based diet in a drylot (DRYLOT). Steers ( = 715) were stratified by BW (278 kg ± 23 in yr 1 and 291 kg ± 91 in yr 2) and assigned to treatment and replicate (4 replications per treatment per yr). Steers assigned to DRYLOT were fed a corn silage-based diet for 54 d in yr 1 and 52 d in yr 2 before being transitioned to the finishing diet. Steers assigned to RESIDUE and those assigned to CCROP grazed 65 d in yr 1 and 66 d in yr 2 and then were fed a corn silage-based diet for 21 d in yr 1 and 33 d in yr 2 before being transitioned to the finishing diet. During backgrounding, the ADG (SEM 0.022) of steers assigned to DRYLOT (1.48 kg/d) was greater ( < 0.01) than that of steers assigned to both CCROP (1.05 kg/d) and RESIDUE (0.87 kg/d) and ADG of steers assigned to CCROP was greater ( < 0.01) than that of steers assigned to RESIDUE. At the start of the finishing period, BW of steers assigned to CCROP (381 kg) was greater ( < 0.01, SEM 2.5) than that of steers assigned to DRYLOT (361 kg) and RESIDUE (366 kg). The finishing period lasted 160 d for all treatments. Both 12th-rib fat ( = 0.89) and calculated yield grade ( = 0.39) did not differ among treatments. Finishing G:F of steers assigned to DRYLOT (0.162 kg/kg) was greater ( < 0.01, SEM 0.0015) than that of steers assigned to RESIDUE (0.153 kg/kg) and CCROP (0.153 kg/kg), which did not differ ( = 0.79). In yr 1, HCW of steers assigned to CCROP (402 kg) was greater ( < 0.01, SEM 2.1) than that of steers assigned to both RESIDUE (389 kg) and DRYLOT (391 kg), which did not differ ( = 0.40). This difference in HCW is most likely a result of differences in BW at the start of the finishing phase in yr 1. However in yr 2, HCW of steers assigned to CCROP (400 kg) and RESIDUE (397 kg) did not differ ( = 0.26, SEM 2.1) but were greater ( < 0.01) than that of steers assigned to DRYLOT (367 kg), despite the fact that steers assigned to RESIDUE entered the finishing phase at a lighter BW than steers assigned to CCROP. Marbling was greater ( = 0.01, SEM 3.9) for steers assigned to DRYLOT (429) than for steers assigned to RESIDUE (414), although steers assigned to CCROP (424) were not different ( ≥ 0.10) from steers assigned to DRYLOT or RESIDUE. When cost and price scenarios from the last 5 yr were conducted, no treatment appeared to be consistently superior in terms of cost of gain or net return. Therefore, all 3 systems appear to be viable options for producers.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor.

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05-0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05-0.001) and restored Akt phosphorylation in retinal pericytes. CONCLUSIONS/INTERPRETATION: Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.

Linkage analysis of candidate genes as susceptibility loci for osteoarthritis-suggestive linkage of COL9A1 to female hip osteoarthritis.

OBJECTIVE: To examine 11 candidate genes as susceptibility loci for osteoarthritis (OA). METHODS: A total of 481 families have been ascertained in which at least two siblings have had joint replacement surgery of the hip, or knee, or hip and knee for idiopathic OA. Each candidate gene was targeted using one or more intragenic or closely linked microsatellite marker. The linkage data were analysed unstratified and following stratification by sex and by joint replaced (hip or knee). RESULTS: The analyses revealed suggestive linkage of the type IX collagen gene COL9A1 (6q12-q13) to a subset of 132 families that contained affected females who were concordant for hip OA (female-hip) with a P-value of 0.00053 and logarithm of the odds (LOD) score of 2.33 [corrected P-value of 0. 0016, corrected LOD score of 1.85]. CONCLUSIONS: COL9A1 may therefore be a susceptibility locus for female hip OA. In addition, there was weak evidence of linkage to HLA/COL11A2 (6p21.3) in female hip OA with a corrected P-value of 0.016.

Linkage analysis of chromosome 2q in osteoarthritis.

BACKGROUND: In independent linkage studies chromosome 2q11-q24 and chromosome 2q23-35 have previously been implicated as regions potentially harbouring susceptibility loci for osteoarthritis (OA). OBJECTIVE: To test chromosome 2q for linkage to idiopathic osteoarthritis. METHODS: Using a cohort of 481 OA families that each contained at least one affected sibling pair with severe end-stage disease (ascertained by hip or knee joint replacement surgery), we conducted a linkage analysis of chromosome 2q using 16 polymorphic microsatellite markers at an average spacing of one marker every 8.5 cM. RESULTS: Our results provide suggestive evidence for a locus at 2q31 with a maximum multipoint logarithm of the odds score (MLS) of 1.22 which increased to 2.19 in those families concordant for hip-only disease (n = 311). This suggestive linkage was greater in male-hip families (MLS = 1.57, n = 71) than in female-hip families (MLS = 0.71, n = 132). CONCLUSIONS: Chromosome 2q is likely to contain at least one susceptibility locus for OA.

Osteoarthritis-susceptibility locus on chromosome 11q, detected by linkage.

We present a two-stage genomewide scan for osteoarthritis-susceptibility loci, using 481 families that each contain at least one affected sibling pair. The first stage, with 272 microsatellite markers and 297 families, involved a sparse map covering 23 chromosomes at intervals of approximately 15 cM. Sixteen markers that showed evidence of linkage at nominal P

Inhibition of human topoisomerase II by anti-neoplastic benzazolo[3,2-alpha]quinolinium chlorides.

Previously we reported [20] that there is no correlation between the cytotoxic activity of four new structural analogs of the antitumor DNA intercalator 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ-2) and their interaction with DNA. In the present study, we present evidence suggesting that the molecular basis for the anti-proliferative activity of these drugs is the inhibition of topoisomerase II. The NBQ-2 derivatives inhibited the relaxation of supercoiled DNA plasmid pRYG mediated by purified human topoisomerase II. Inhibition of the decatenation of kinetoplast DNA mediated by partially purified topoisomerase II extracted from the human histiocytic lymphoma U937 (a cell line previously shown to be sensitive to the drugs) was also caused by these drugs. The potency of the benzazolo[3,2-a]quinolinium drugs against topoisomerase II in vitro was the following: 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-59) > 4-chlorobenzothiazolo[3,2-a]quinolinium chloride (NBQ-76) > 7-ethyl-3-nitrobenzimidazolo[3,2-a]quinolinium chloride (NBQ-48) > 7-benzyl-3-nitrobenzimidazolol[3,2-a]quinolinium chloride (NBQ-38). This rank of potency for topoisomerase II inhibition correlated very well with the cytotoxicity elicited by these drugs. Furthermore, significant levels of topoisomerase II/DNA cleavage complex induced by these drugs in vivo were detected when U937 cells were treated with NBQ-59 and NBQ-76 whereas NBQ-38 and NBQ-48 produced negligible amounts of the cleavage complex. Our results strongly suggest that topoisomerase II is the major cellular target of this family of compounds.

Erythro-1-(benzothiazol-2-yl)-1,2-dibromo-2-(2-chloro-5-nitrophenyl)ethane.

The title compound, C15H9Br2CIN2O2S, was synthesized by electrophilic addition of bromine to 2-(2-chloro-5-nitrostyryl)benzothiazole. The molecule consists of benzothiazole and 2-chloro-5-nitrophenyl rings linked by a 1,2-dibromoethane moiety. The dihedral angle between the benzothiazole and phenyl rings is 8.2(9) degrees. The benzothiazole ring is planar with a mean deviation of 0.168(7)A. The Br1--C8 and Br2--C9 bond distances are 1.972(5) and 2.007(6)A, respectively.

N-(o-tolyl)-2-bromo-4,5-dimethoxybenzamide.

The molecular structure of the title compound, C16H16BrNO3, consists of 2-bromo-4,5-dimethoxy-benzene and o-methylphenyl rings linked by an amide group. The two methoxy groups are almost coplanar with the phenyl ring.

Evidence for serum immunoglobulin G (IgG) antibody responses in Macaca fascicularis identified by monoclonal antibodies to human IgG subclasses.

This investigation determined the capacity of murine monoclonal antibodies directed to human immunoglobulin G (IgG) subclasses to identify molecules with conserved epitopes in the serum of the nonhuman primate, Macaca fascicularis. We subsequently utilized this cross-reactivity to document the characteristics of IgG subclass antibody responses in M. fascicularis to parenteral immunization with intact oral microorganisms, antigens from oral microorganisms, and finally a defined protein toxin, tetanus toxoid. The IgG response in nonhuman primates immunized with tetanus toxoid showed a 40-fold and 110-fold increase after primary and secondary immunizations, respectively. The major IgG subclass responses were IgG1 and IgG3, with little, though significant, responses in the IgG4 and IgG2 subclasses. Seventy-five to 94% of the natural IgG antibody in nonhuman primate sera to Porphyromonas gingivalis, Prevotella intermedia and Campylobacter rectus was IgG1. IgG2 and IgG3 predominated to Bacteroides fragilis, IgG4 to Actinomyces viscosus and an equal distribution among the subclasses was noted in response to Fusobacterium nucleatum. Parenteral immunization of nonhuman primates with intact P. gingivalis elicited primarily IgG3 and IgG4, while the post-immunization IgG response to P. intermedia was largely IgG1. Nonhuman primates were also parenterally immunized with cell envelope antigens of P. gingivalis, P. intermedia, or a combination of cell envelope antigen from C. rectus and F. nucleatum and cell wall antigens of A. viscosus. The greatest IgG antibody response seen post-immunization was reactive with anti-human IgG1 for all of these antigens except to C. rectus which bound nonhuman primate antibody reactive with anti-human IgG2. It appears that the bacteria and their products exhibit unique differences in their induction of serum IgG subclass antibody responses. The characteristics of their immunogenicity as detected by the nonhuman primate may contribute to the ability of the immune responses to effectively interact with these pathogens.

(E,Z)-2-(2-chloro-5-nitrostyryl)-1-(1-propenyl)benzimidazole.

The title compound, C18H14ClN3O2, was synthesized by the condensation of 2-chloro-5-nitrobenzaldehyde with 2-methyl-1-propenylbenzimidazole, and the molecule comprises a 2-chloro-5-nitrobenzene and a 1-(Z)-propenylbenzimidazole. The two aromatic moieties are conjugated through the vinyl group. The dihedral angle between the two rings is 1.4(6) degrees. The propenyl group lies out of the benzimidazole plane with a dihedral angle of 112.9(9) degrees.

1-(1,3-Benzothiazol-2-yl)-2-(2-bromo-5-nitrophenyl)ethanone.

The title compound, C15H9BrN2O3S, was isolated as an unexpected product from the reaction of the anion of sodium 2-(1,3-benzothiazolyl)ethanonitrile with alpha,2-dibromo-5-nitrotoluene. Its structure features a benzothiazole fragment and a bromo- and nitro-substituted phenyl ring linked by a methyl ketone group. The dihedral angle between the benzothiazole and phenyl rings is 103.7 (2) degrees. The benzothiazole fragment is planar, with a maximum deviation of 0.021 (2) A. The nitro group is slightly rotated out of the phenyl-ring plane, with a O(2)-N(2)-C(14)-C(15) torsion angle of 16.4(7) degrees.

Bis[(E)-2-(2,4-dichloro-5-nitrostyryl)-1,3-benzothiazole] hydrate.

The title compound, bis[(E)-2-(2,4-dichloro-5-nitrostyryl)-1,3-benzothiazole] hydrate, 2C15H8Cl2N2O2S.-H2O, was obtained from the condensation of 2-methyl-benzothiazole with 2,4-dichloro-5-nitrobenzaldehyde. Single-crystal X-ray analysis showed that the asymmetric unit contains two crystallographically unique but structurally similar molecules. The dihedral angle between the benzothiazole fragment and the phenyl ring is 6.1 (4) degrees in molecule A and 19.5 (4) degrees in molecule B.

DNA binding-independent anti-proliferative action of benzazolo[3,2-alpha]quinolinium DNA intercalators.

The proposed mechanism of action of the antineoplastic drug 3-nitrobenzothiazolo[3,2-alpha]quinolinium chloride (NBQ-2) involves its interaction with DNA by intercalation and inhibition of topoisomerase II activity by arresting the enzyme in a covalent cleavage complex. In an attempt to identify some structural determinants for activity and develop a molecular structure/cytotoxicity correlation, four new structural analogs of the antitumor NBQ-2 were prepared and their cytotoxic activity and DNA binding properties were investigated. The cytotoxic activity was evaluated against six different human tumor cell lines: U937, K-562, HL-60, HT-29, HeLa, and A431. The results showed that these new drugs elicit pronounced cytotoxic effects against U937, K-562, HL-60 and A431 while HeLa and HT-29 were less sensitive to the new drugs. This apparent selectivity was different to that of m-AMSA, a drug currently used for cancer treatment. Since the interaction of NBQ-2 to DNA by intercalation has been proposed as the initial step leading to its antineoplastic activity, DNA binding and changes in DNA contour length induced by the new NBQ-2 structural analogs were also investigated using calf thymus and human DNA. The drug, 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-alpha]quinolinium chloride (NBQ-59) was the most cytotoxic agent of the analog series (IC50 = 16 microM for HL-60 cells), however, it demonstrated the weakest binding to DNA (Kint = 0.9 x 10[5] M-1 for calf thymus DNA). NBQ-59 was also found to be a poor intercalator into the DNA double helix. Therefore, our results suggest that DNA binding is not the primary mechanism of drug action for this family of compounds. In addition structural determinants important for cytotoxicity of the benzazolo quinolinium chlorides were suggested by our results. In particular, the nitro group in the 3 position does not seem to be necessary for bioactivity, while substitutions in the benzazolo moiety have striking effects on the biological activity of the drugs.

Alterations in nitric oxide synthase activity, superoxide anion generation, and platelet aggregation in systemic hypertension, and effects of celiprolol.

A decrease in endothelium-derived relaxing factor or nitric oxide has been proposed as a potential mechanism of increased vascular resistance in hypertension. An increase in the generation of superoxide anions, which degrade nitric oxide and induce platelet aggregation, may also compromise regional blood flow in hypertension. Recent studies show that human neutrophils generate nitric oxide, which has a similar biologic profile to the endothelium-derived relaxing factor. This study measured nitric oxide synthase activity and superoxide generation in human neutrophils and platelet aggregation in patients with essential hypertension. Nitric oxide synthase activity, measured as conversion of 3H-L-arginine to 3H-L-citrulline, in peripheral blood neutrophils was decreased in hypertensive subjects (percent conversion of 3H-L-arginine: 4.2 +/- 0.5 vs 9.0 +/- 3.0 in control subjects; p < 0.01). Neutrophil superoxide anion generation, measured as conversion of ferricytochrome C to ferrocytochrome C, in response to phorbol-12-myristate 13-acetate (100 ng/ml) was higher in hypertensive subjects (17.5 +/- 8.1 vs 13.2 +/- 3.0 nmoles/10(6) cells/10 minutes in control subjects; p < 0.05). Patients were treated with a selective beta blocker, celiprolol, for 8 weeks. Supine blood pressure decreased from 177/103 mm Hg (mean +/- SD 18/7) to 160/92 mm Hg (mean +/- 10/5; p < 0.02), while heart rate was unchanged (73 +/- 11 vs 69 +/- 10 beats/min). Epinphrine and adenosine diphosphate-induced platelet aggregation was also increased in hypertensive subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Some dream mechanisms in Finnegans Wake.

This paper does not purport to offer a psychoanalytic reading of Finnegans Wake, but rather to demonstrate how, in recreating the mechanisms of the dream, Joyce's masterpiece offers to psychoanalysis a uniquely rich opportunity to explore the shadowy play of the dream in the permanence of a work of art. In particular, the manner in which Finnegans Wake tells its story through the distortions of dream narrative while bodying forth its protagonists in the substitutions of dream-identity are explored, with the help of certain Freudian and Lacanian concepts of unconscious structure.

Reductive activation of benzazolo[3,2-a]-quinolinium chlorides.

Initial ferricytochrome c (Cyt(III)c) reduction rates occurring in aerobic or anaerobic solutions containing either 3-nitrobenzothiazolo[3,2-a]-(NBQCl), 1-ethyl-3-nitrobenzimidazolo[3,2-a]-(ENBIQCl), 7-ethylbenzimidazolo[3,2-a]quinolinium chloride (EHBIQCL), or nitrofurantoin (NFT) and xanthine/xanthine oxidase were measured. Maximum rates in nitrogen-saturated solutions follow the order NFT > NBQCL > ENBIQCL > EHBIQCL. These rates correlate linearly with the half-wave reduction potentials (E1/2) of these compounds. With the exception of EHBIQCl, smaller rates of Cyt(III)c reduction were obtained in air-saturated than in nitrogen-saturated solutions at the quinolinium salt concentrations used. Larger concentrations of superoxide dismutase (SOD) are needed for 50% inhibition of the Cyt(III)c reduction reaction for heterocyclic compounds with larger E1/2 values. Thus, measurement of the portion of the Cyt(III)c reduction rate under air that is inhibited by SOD does not account solely for the production of superoxide. These observations suggest that NBQCL, ENBIQCl, and less probably EHBIQCl may interfere with mitochondrial energy metabolism or induce DNA damage through reduced intermediates.

Absence of mutagenicity of the antitumor drug 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ) in the germ cells of Drosophila melanogaster males.

The antitumor drug, 3-nitrobenzothiazolo[3,2-a]quinolinium chloride (NBQ) was tested for genotoxicity with the sex-linked recessive lethal test by feeding Drosophila melanogaster males. Although toxic to adults, the drug tested negative at the concentrations studied.

Case eight. The acquisition of a community hospital by a religiously sponsored healthcare system.

St. Martin's for many years enjoyed a friendly and supportive relationship with a small community hospital nearby even though it was the leading provider of healthcare in the other's market place. In the highly competitive environment reported in the case, it was clear that the smaller hospital would have to move aggressively to increase its market share, consider closure or merge with one of the larger competing hospitals. St. Martin's administration knew that it needed to decide whether to attempt to align itself with Community in some fashion or to compete aggressively enough with Community to so dilute Community's market that it could no longer function. One of these actions was required to avoid serious erosion of St. Martin's own patient base. The decision was made to pursue acquisition. This case presents the successful implementation of a carefully crafted and realized strategy. It provides the opportunity to consider multiple "what ifs" in terms of the alternative strategies not chosen.