Yes / Bacteria sense their own population size, tune the expression of responding genes, and behave accordingly to environmental stimuli by secreting signaling molecules. This phenomenon is termed as quorum sensing (QS). By exogenously manipulating the signal transduction bacterial population behaviors could be controlled, which may be done through quorum quenching (QQ). QS related regulatory networks have been proven their involvement in regulating many virulence determinants in pathogenic bacteria in the course of infections. Interfering with QS signaling system could be a novel strategy against bacterial infections and therefore requires more understanding of their fundamental mechanisms. Here we review the development of studies specifically on the inhibition of production of N-acyl-homoserine lactone (AHL), a common proteobacterial QS signal. The opportunistic pathogen, Pseudomonas aeruginosa, equips the alkylquinolone (AQ)-mediated QS which also plays crucial roles in its pathogenicity. The studies in QQ targeting on AQ are also discussed. / University of Malaya High Impact Research Grants (UMC/625/1/HIR/MOHE/CHAN/01, A-000001-50001,and UMC/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

La synthèse tout-en-un de 4-quinolones et d'indoxyles a été réalisée par couplage de Sonogashira carbonylant suivi d'une cyclisation. Une étude en catalyse homogène a révélé que les catalyseurs présents contrôlaient la sélectivité vers l'un ou l'autre des composés. Si la première étape est pallado-catalysée, l'étape de cyclisation est catalysée par des nucléophiles organiques. Ainsi, des 4-quinolones ont été préparées par multi-catalyse {[Pd]+amine} et des indoxyles par catalyse tandem {[Pd]/phosphine}. Les systèmes catalytiques ont été hétérogénéisés par fonctionnalisation de silices mésoporeuses de type SBA. Deux stratégies ont été utilisées pour contrôler la localisation de la fonctionnalité. Différents complexes de Pd ont été intégrés dans les pores du matériau par greffage post-synthétique ou dans les murs par synthèse directe. Plusieurs amines et une phosphine ont été immobilisées par greffage post-synthétique donnant ainsi des catalyseurs nucléophiles fonctionnalisés dans leurs pores. Les activités catalytiques de ces matériaux ont été évaluées dans la synthèse de la 2-phényl-4-quinolone et du 2-benzylidène-indoxyle. La première a été préparée dans de bons rendements et le système {[Pd]@SBA+amine@SBA} est recyclable sur 3 cycles. Tous les matériaux ont montré une lixiviation du Pd mais leur utilisation permet de diminuer la contamination en Pd du produit final comparé à un complexe homogène. L'indoxyle a été obtenu avec un système semi-hétérogène {[Pd]@SBA/PPh3}, l'utilisation de la phosphine greffée conduisant à la transformation de l'indoxyle vers le 2-benzylindole. La formation d'α-céto-amides en catalyse hétérogène via double carbonylation a aussi été étudiée / The one-pot selective synthesis of 4-quinolones and indoxyls was achieved through a carbonylative Sonogashira coupling followed by cyclization. A study in homogeneous catalysis revealed that the nature of catalysts in presence controlled the selectivity toward each compounds. Whereas the first coupling step is palladium catalyzed, the cyclizations require organic nucleophilic species. Thus, 4-quinolones were obtained by one-pot multi-catalysis {[Pd]+amine} and indoxyls by one-pot tandem catalysis {[Pd]/phosphine}. The catalytic systems were heterogenized by functionalizing mesostructured SBA silicas. Two strategies were employed aiming at a control of the localization of the functionality. Different Pd complexes were integrated in the pores of the material by post-synthesis grafting or incorporated into the walls via direct synthesis. Different amines and a phosphine were immobilized by post-synthesis grafting affording hybrid materials containing amine or phosphine catalysts in their pores. Catalytic activities of those materials were evaluated in the reaction affording 2-phenyl-4-quinolone and 2-benzylidene-indoxyl. The former was obtained in good yields and the heterogeneous catalytic system {[Pd]@SBA+amine@SBA} was recyclable over 3 runs. All hybrid materials showed Pd leaching but their uses still enables to decrease the Pd contamination of the final product compared to homogeneous complexes. The indoxyle compound was obtained in a semi-heterogeneous system {[Pd]@SBA/PPh3}, the use of the grafted phosphine providing transformation of the indoxyle toward 2-benzylindole. Formation of α-keto-amids by heterogeneously catalyzed double carbonylation was also studied

The goal of our research endeavor was to successfully employ modern lead discovery and optimization strategies towards the development and identification of compounds possessing antimalarial activity. Preliminary data from in vitro screening at the Walter Reed Army Institute of Research identified several chemotypes including 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones to have potent antimalarial activities. Multiple synthetic routes were devised and implemented which enabled the rapid preparation and isolation of over 400 structurally diverse 4(1H)-quinolones and 1,2,3,4-tetrahydroacridones. Our research towards discovering and optimizing antimalarials was inspired from the severe impact malaria has had on our planet especially on impoverished countries. There are over 300 million cases annually and over one million deaths. The staggering mortality rates combined with the global emergence of chemical resistance that the parasite Plasmodium falciparum has developed towards many of the common antimalarials compelled us to extend our research efforts to this growing problem. The need for identifying and developing new antimalarial drugs is very important. However, our approach focuses on the optimization of historic antimalarials such as endochin, floxacrine, or ICI 56,780 which possess liabilities such as lack of poor solubility, poor in vivo activity or lingering toxicity issues. Through these optimization efforts using both SAR and structure-property relationship (SPR) studies, a more suitable candidate was developed that had superior physicochemical properties. Our drug design approach included not only the identification of liabilities of historic compounds but also the synthesis and optimization of numerous analogs guided by SAR. All compounds were tested in vitro for antimalarial activity and characterized in parallel for physicochemical properties such as solubility, permeability, and logD7.4. Insights from both the antimalarial activity as well as the physicochemical properties determined which analogs would be advanced in the design process. Based on our early investigations, 6-chloro-7-methoxy-3-phenyl-4(1H)-quinolone emerged as a promising hit. Compared to endochin, which possesses EC50s of 8.6 nM and 46.6 nM against drug resistant strains W2 and TM90C2B, and a solubility of less than 2 µM, 6-chloro-7-methoxy-3-pheny-4(1H)-quinolone was superior with a 4-fold improvement in solubility (6 µM) as well as slightly improved antimalarial activity (EC50s of 26.2 nM and 15.3 nM against W2 and TM90C2B, respectively). Unfortunately, this compound failed to reduce parasitemia levels in P. berghei infected mice. Hit-to-lead optimization lead to the discovery of 6-chloro-7-methoxy-2-methyl-3-o-tolyl-4(1H)-quinolone which was shown to reduce parasitemia levels by 41% at day 6 post-exposure (PE) in P. berghei infected mice at a 50 mg/kg dose. The observed in vivo activity of 6-chloro-7-methoxy-2-methyl-3-o-tolyl-4(1H)-quinolone was believed to relate to the 3-fold increase in solubility (19 µM) over the 3-phenyl-susbtituted analogue. Continuation of SAR and SPR studies identified additional 4(1H)-quinolones suggesting that the microsomal stability of the compounds is as important for in vivo efficacy as the aqueous solubility. Several of the analogs that showed minimal degradation in human microsomal stability studies demonstrated increased in vivo activity in the ranges of 72-98% parasitemia reductions on day 6PE in P. berghei infected mice at 50 mg/kg. These results helped refine the final SAR and SPR optimization identifying a compound with radical curative activity in mice (99% parasitemia reductions on day 6PE in P. berghei infected mice at 50 mg/kg with five out of five mice surviving beyond 30 days). Theses studies not only highlight the effectiveness of detailed SAR and SPR strategies used in drug discovery programs, but they also showcase the importance of re-evaluating historic antimalarials and exploiting their shortcomings. These studies have opened the doors to several possibilities regarding the 4(1H)-quinolone scaffold optimization for future antimalarial development. Several of the compounds described in this work are currently being subjected to stringent head-to-head comparative studies to determine the analog best suited for pre-clinical trials.

La synthèse tout-en-un de 4-quinolones et d'indoxyles a été réalisée par couplage de Sonogashira carbonylant suivi d'une cyclisation. Une étude en catalyse homogène a révélé que les catalyseurs présents contrôlaient la sélectivité vers l'un ou l'autre des composés. Si la première étape est pallado-catalysée, l'étape de cyclisation est catalysée par des nucléophiles organiques. Ainsi, des 4-quinolones ont été préparées par multi-catalyse {[Pd]+amine} et des indoxyles par catalyse tandem {[Pd]/phosphine}. Les systèmes catalytiques ont été hétérogénéisés par fonctionnalisation de silices mésoporeuses de type SBA. Deux stratégies ont été utilisées pour contrôler la localisation de la fonctionnalité. Différents complexes de Pd ont été intégrés dans les pores du matériau par greffage post-synthétique ou dans les murs par synthèse directe. Plusieurs amines et une phosphine ont été immobilisées par greffage post-synthétique donnant ainsi des catalyseurs nucléophiles fonctionnalisés dans leurs pores. Les activités catalytiques de ces matériaux ont été évaluées dans la synthèse de la 2-phényl-4-quinolone et du 2-benzylidène-indoxyle. La première a été préparée dans de bons rendements et le système {[Pd]@SBA+amine@SBA} est recyclable sur 3 cycles. Tous les matériaux ont montré une lixiviation du Pd mais leur utilisation permet de diminuer la contamination en Pd du produit final comparé à un complexe homogène. L'indoxyle a été obtenu avec un système semi-hétérogène {[Pd]@SBA/PPh3}, l'utilisation de la phosphine greffée conduisant à la transformation de l'indoxyle vers le 2-benzylindole. La formation d'α-céto-amides en catalyse hétérogène via double carbonylation a aussi été étudiée

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Native species of the flora from South Brazil are the aim of the research of the Núcleo de Pesquisa de Produtos Naturais of Federal University of Santa Maria, Brazil. One of the most studied families is the Sterculiaceae and, in that family, there is the Waltheria douradinha Saint Hilaire specie, study purpose of this master degree work. W. douradinha, popularly known as douradinha do campo , is used to treat lung infections, cystitis, chronic ulcer and to wash syphilitic wounds. Secondary metabolites isolated from the bark of the roots from this specie, among the amide (O)-metiltembamide, three known quinolone alkaloids, Antidesmone, Waltherione-A and Waltherione-B, two cyclopeptide alkaloids also known, Waltherine-A and Chamaedrine, and a new quinolone alkaloid VG8. The structures of these substances are determined, principally, by spectroscopics methods, like Nuclear Magnetic Resonance 1H and 13 C (COSY, HMQC e HMBC), mass spectrometry and by the comparison with melting point and literature data. The alkaloids Waltherione-A (WA) and Waltherione-B (WB) are crystallized and analyzed by X-Ray Diffraction to determine their relative stereochemistries. The extracts obtained from several parts of W. douradinha, the fractions obtained from the division of the crude extract of the root and the pure alkaloids, Waltherine-A, VG8, WA, WB and their derivatives were submitted to microbiologic assays (fungus and Gram-positive and Gram-negative bacteria) by bioautography method and by Minimum Inhibitory Concentration (MIC). The alkaloid VG8 was the most active upon the tested microorganisms, with a positive result of inhibition with 0.5 μg by bioautography assay and with 50 μg mL-1 by MIC assay. / Espécies nativas da flora do Sul do Brasil são alvo de pesquisa do Núcleo de Pesquisa de Produtos Naturais da Universidade Federal de Santa Maria. Uma das famílias mais estudadas é a Sterculiaceae e, dentro desta, encontra-se a espécie Waltheria douradinha Saint Hilaire, objetivo de estudo deste trabalho de mestrado. W. douradinha, conhecida popularmente como douradinha do campo , é utilizada no tratamento de afecções pulmonares, cistite, úlceras crônicas e também para lavar feridas de origem sifilítica. Metabólitos secundários foram isolados da casca da raiz desta espécie, dentre eles a amida (O)-metiltembamida, três alcalóides quinolônicos já conhecidos, Antidesmona, Waltheriona-A e Waltheriona-B, dois alcalóides ciclopeptídicos também já conhecidos, Waltherina-A e Chamaedrina, e um novo alcalóide quinolônico VG8. As estruturas destas substâncias foram determinadas, principalmente, por métodos espectroscópicos, como Ressonância Magnética Nuclear 1H e 13 C (COSY, HMQC e HMBC), espectrometria de massas e através da comparação dos dados obtidos a partir do ponto de fusão e dados da literatura. Os alcalóides Waltheriona-A (WA) e Waltheriona-B (WB) foram cristalizados e analisados por Difração de Raios-X a fim de se confirmar suas estereoquímicas relativas. Os extratos obtidos das várias partes da espécie, as frações obtidas do particionamento do extrato bruto da raiz do material vegetal e os alcalóides puros, Waltherina-A, VG8, WA, WB e seus derivados foram submetidos a ensaios Waltheria douradinha, alcalóides quinolônicos e ciclopeptídicos, atividade antimicrobiana.

QepA is a plasmid-mediated efflux pump found in some strains of Escherichia coli, in which it significantly elevates the resistance against quinolones. The protein has similarities with 14-TMS major facilitator superfamily transporters and is situated in the inner membrane of the bacteria. It was acquired by horizontal gene transfer and integrated into a now inactivated class 1 integron, also harbouring several other antibiotic resistance genes such as rmtB and blaTEM-1. QepA alone is not sufficient to raise the resistance level over the clinical breakpoint and is in clinical isolates therefore associated with other quinolone antibiotic resistance genes or quinolone target point mutations. The mechanisms regulating qepA expression are not yet understood. Therefore, in this study the qepA gene was amplified from an E. coli clinical isolate and, together with its upstream promotor sequence, was inserted into the E. coli chromosome. It was shown that qepA gene expression can be induced by exposure to 0.5-fold MIC concentrations of ciprofloxacin, trimethoprim and other DNA damaging antimicrobials. The deletion of a LexA binding site situated after a PcW promotor, which was predicted to drive qepA expression, did not alter this induction behaviour. Nested deletions of up to 200 nts downstream sequence of the PcW promotor, led to the identification of a sequence region required for expression induction. This study showed that qepA expression is induced by environmental factors leading to DNA damage and further identified a previously unknown DNA sequence required for expression regulation.

Den vanligaste cancerformen hos män är prostatacancer, med 10 000 nya dödsfall årligen. Vid prostatacancerdiagnostik utförs prostata biopsi. För att minska risken för komplikationer i samband med biopsi ges i Sverige en singeldos av antibiotikapreparatet Ciprofloxacin. Andelen bakterier som är resistenta mot ciprofloxacin har ökat. För detektion av genmutationer som orsakar antibiotikaresistens kan droplet digital PCR (ddPCR) användas. Det är en metod som ger en absolut kvantifiering av antalet DNA-sekvenser. Den är baserad på vatten olja-emulsionsdroppsystem.  Syftet med denna studie var att optimera och validera en digital droplet PCR för att detektera och kvantifiera mutationen S83L i gyrA genen från faecesprover, samt att jämföra digital droplet resultat från studieprover med odlingsresultat från resistensbestämning och ration mellan S83L allelen och vildtyps allelen i prover tagna före och efter biopsi. För att optimera och validera metoden användes prover tagna före och efter biopsi från nio patienter som genomgått en transrektal prostatabiopsi och fått en dos antibiotika profylax i samband med ingreppet.  Den optimala annealingtemperaturen bedömdes vara 60°C och den optimala  primer- och probekoncentrationen bestämdes till 1,2 µM respektive 0,4µM. Dessa koncentrationer gav lägst antal falskt positiva droppar. Den minsta detektionsnivån för S83L gyrA (EC40) var 160 kopior/ml och för vildtyps gyrA (EC108) var det 78 kopior/ml. Resultatet från valideringen visade att både vildtyps gyrA och S83L gyrA kunde detekteras och kvantifieras i rektalprover från samtliga patienter. / The most common form of cancer in men is prostate cancer, with 10,000 new deaths annually. In prostate cancer diagnosis, prostate biopsy is performed. To reduce the risk of complications in connection with biopsy, a single dose of the antibiotic drug Ciprofloxacin is given in Sweden. The proportion of bacteria that are resistant to ciprofloxacin has increased. For the detection of gene mutations that cause antibiotic resistance, droplet digital PCR (ddPCR) can be used. It is a method that provides an absolute quantification of a DNA sequence in a sample. It is based on water oil emulsion drop system.  The purpose of this study was to optimize and validate a digital droplet PCR to detect and quantify the S83L mutation in the gyrA gene from faecal samples and to compare digital droplet results from study samples with culture results from resistance determination and the ration between the S83L allele and the wild-type allele in samples taken before and after biopsy. To validate the method, samples taken before and after biopsy were used from nine patients who had undergone a transrectal prostate biopsy and received a dose of ciprofloxacin or trimethoprim in connection with the procedure.  The optimal annealing temperature was determined to be 60 °C and the optimal primer and probe concentrations were determined to be 1.2 µM and 0.4µM, respectively. These concentrations gave the lowest number of false positive droplets. The minimum detection level for S83L gyrA (EC40) was 160 copies/ml and for wild-type gyrA (EC108) it was 78 copies/ml. The results showed that both wild-type gyrA and S83L gyrA could be detected and quantified in rectal samples from all nine patients.

Yes / Chronic obstructive pulmonary disease (COPD) in humans, describes a group of lung conditions characterised by airflow limitation that is poorly reversible. The airflow limitation usually progresses slowly and is related to an abnormal inflammatory response of the lung to toxic particles. COPD is characterised by oxidative stress and an increased risk of lung carcinoma. The 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) is one of a number of mutagenic/carcinogenic heterocyclic amines found mainly in well-cooked meats which are thus part of the regular diet. Antioxidants are very important in order to protect the cells against oxidative damage. The aim of the present study was to assess the effects of IQ on the level of DNA damage and susceptibility to a potent mutagen in peripheral blood cells of COPD patients. DNA damage and the frequency of micronuclei (MNi) were evaluated using the Comet and micronucleus assays, respectively. Differential expressions of both mRNA and protein of the endogenous antioxidant enzyme catalase were evaluated with quantitative polymerase chain reaction (qPCR) and Western blot analysis, respectively. Furthermore, the effect of bulk and nano forms of quercetin and their combination with IQ were examined. Results of the present study clearly demonstrated that MNi frequency in the peripheral blood lymphocytes exhibited a positive correlation with the DNA damage as evident from the different Comet assay parameters. Increase of the endogenous antioxidant catalase also showed there was a stimulation of this enzyme system by IQ. Whereas, the endogenous antioxidant quercetin significantly reduced oxidative stress in COPD patients and healthy individuals. / Libyan Government

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.