Antimicrobial and Resistance-Modifying Activities of LY2183240 Regioisomers

The rapid development of antimicrobial resistance over the past three decades represents a critical public health threat and urgent challenge. In this context, in order to establish lead compounds or identify novel modes of action one approach is to re-investigate drugs that affect eukaryotic processes for antimicrobial activity, particularly if their drug targets have homologies with bacterial proteins. In this study, regioisomers of LY2183240, a potent inhibitor of anandamide transport and fatty acid amide hydrolase were selected after a small screening study. The 2,5-LY2183240 regioisomer was shown to possess potent antimicrobial activity selective towards certain Gram-positive bacteria, which included Staphylococcus aureus and Bacillus subtilis, but not Enterococcus faecalis or Streptococcus pneumoniae. Conversely, the 1,5-LY2183240-regioisomer had no anti-bacterial activity strongly implicating the position of the carbamoyl on the tetrazole in the structure-activity relationship of the molecule. Investigation of the mechanism of antimicrobial activity suggested that while 2,5- LY2183240 had bacteriostatic activity this was probably not due to inhibition of protein or teichoic acid synthesis. Nevertheless, this activity may be related to the inhibition of bacterial fatty acid synthesis. Supporting this hypothesis, addition of exogenous fatty acids within Tween 80 was able to compromise 2-5-LY2183240 anti-staphylococcal activity. Based on the spectrum of activity and known redundancies at each of the steps in the fatty acid synthesis pathway, the most likely target was deduced to be FabI. However, characterization of a 2,5-LY2183240- resistant mutant revealed no alteration to the deduced amino acid sequence of FabI or relevant changes to FabI protein expression, an observation confirmed by analysis of the fabI promoter region and western blot studies. Since resistance to 2,5- LY2183240 could be mediated through non-target related factors such as drug efflux or drug entry into the cell, FabI could not be ruled out as a potential target. In addition, whole cell protein profiling revealed the 2,5-LY2183240-mutant to have differences in the expressions of several proteins, suggesting resistance may occur through a more global effect. Due to the promiscuous nature of LY2183240, the possibility of having multiple targets in S. aureus that collectively exhibit a bacteriostatic effect cannot be ruled out. This study also showed that the LY2183240 regioisomers are specific inhibitors of class C β-lactamases with optimum inhibitory activity dependent upon the position of the carbonyl on the tetrazole heterocyclic group (Ki values of 1.8 and 2.45 μM for 1,5- and 2,5-LY2183240, respectively). Molecular modelling suggested that LY2183240 regioisomers bind within the catalytic site of class C β-lactamases, interacting with some of the same residues, including Tyr150, Lys315 and Thr316, used by the substrate nitrocefin and β-lactamase inhibitors. The results substantiate the competitive inhibition model determined by enzyme kinetic studies. Furthermore, mass spectrometry data revealed non-covalent interactions between AmpC β- lactamase and LY2183240 regioisomers, which together with the non-reversible inhibitory activity, may indicate that both regioisomers present a high affinity towards this cephalosporinase and dissociate very slowly from the enzyme. LY2183240 may prove useful as a chemical scaffold for the development of novel and highly selective inhibitors of class C β-lactamases

Cobalt complexes modulate plasmid conjugation in <i>Escherichia coli</i> and <i style="">Klebsiella pneumoniae</i>

Antimicrobial resistance genes (ARG), such as extended-spectrum β-lactamase (ESBL) and carbapenemase genes, are commonly carried on plasmids. Plasmids can transmit between bacteria, disseminate globally, and cause clinically important resistance. Therefore, targeting plasmids could reduce ARG prevalence, and restore the efficacy of existing antibiotics. Cobalt complexes possess diverse biological activities, including antimicrobial and anticancer properties. However, their effect on plasmid conjugation has not been explored yet. Here, we assessed the effect of four previously characterised bis(N-picolinamido)cobalt(II) complexes lacking antibacterial activity on plasmid conjugation in Escherichia coli and Klebsiella pneumoniae. Antimicrobial susceptibility testing of these cobalt complexes confirmed the lack of antibacterial activity in E. coli and K. pneumoniae. Liquid broth and solid agar conjugation assays were used to screen the activity of the complexes on four archetypical plasmids in E. coli J53. The cobalt complexes significantly reduced the conjugation of RP4, R6K, and R388 plasmids, but not pKM101, on solid agar in E. coli J53. Owing to their promising activity, the impact of cobalt complexes was tested on the conjugation of fluorescently tagged extended-spectrum β-lactamase encoding pCTgfp plasmid in E. coli and carbapenemase encoding pKpQILgfp plasmid in K. pneumoniae, using flow cytometry. The complexes significantly reduced the conjugation of pKpQILgfp in K. pneumoniae but had no impact on pCTgfp conjugation in E. coli. The cobalt complexes did not have plasmid-curing activity, suggesting that they target conjugation rather than plasmid stability. To our knowledge, this is the first study to report reduced conjugation of clinically relevant plasmids with cobalt complexes. These cobalt complexes are not cytotoxic towards mammalian cells and are not antibacterial, therefore they could be optimised and employed as inhibitors of plasmid conjugation.</p

Supplementary material for the article: Cvijetić, I. N.; Verbić, T. Ž.; Ernesto de Resende, P.; Stapleton, P.; Gibbons, S.; Juranić, I. O.; Drakulić, B. J.; Zloh, M. Design, Synthesis and Biological Evaluation of Novel Aryldiketo Acids with Enhanced Antibacterial Activity against Multidrug Resistant Bacterial Strains. European Journal of Medicinal Chemistry 2018, 143, 1474–1488. https://doi.org/10.1016/j.ejmech.2017.10.045

Supplementary material for: [https://doi.org/10.1016/j.ejmech.2017.10.045]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2075

Synthesis and in silico modelling of the potential dual mechanistic activity of small cationic peptides potentiating the antibiotic novobiocin against susceptible and multi‐drug resistant escherichia coli

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3–9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs

Supplementary material for the article: Cvijetić, I. N.; Verbić, T. Ž.; Ernesto de Resende, P.; Stapleton, P.; Gibbons, S.; Juranić, I. O.; Drakulić, B. J.; Zloh, M. Design, Synthesis and Biological Evaluation of Novel Aryldiketo Acids with Enhanced Antibacterial Activity against Multidrug Resistant Bacterial Strains. European Journal of Medicinal Chemistry 2018, 143, 1474–1488. https://doi.org/10.1016/j.ejmech.2017.10.045

Supplementary material for: [https://doi.org/10.1016/j.ejmech.2017.10.045]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2075

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P &lt; 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Atividade de frações bioativas de erva-mate (ilex paraguariensis A. St. Hil.) sobre o metabolismo de ratos wistar submetidos à dieta hiperlipídica

Ilex paraguariensis A. St. Hil. é uma espécie nativa da América do Sul popularmente conhecida como mate ou erva-mate. Três principais classes de compostos bioativos estão presentes nas folhas: metilxantinas, polifenóis e saponinas, nas quais são atribuídas diversas atividades biológicas, incluindo efeitos no metabolismo lipídico. Do ponto de vista químico e biológico, essa espécie é bastante estudada. Em relação ao processo de obtenção, enriquecimento e purificação das frações do mate, porém, a pesquisa tecnológica revela-se restrita. Este trabalho teve como objetivo avaliar a atividade dos principais grupos de compostos do mate sobre o metabolismo de ratos Wistar submetidos à uma dieta hiperlipídica. A extração de polifenóis, metilxantinas e saponinas das folhas foi otimizado mediante um desenho experimental do tipo Box-Behnken, obtendo-se rendimentos máximos de 357, 41 e 15 mg/g de planta seca, respectivamente. Uma fração enriquecida em polifenóis e metilxantinas (MXPL) e outra enriquecida em saponinas (SFL) foram preparadas por fracionamento em fase sólida de resina polimérica, metanol e água em gradiente decrescente de polaridade. Os rendimentos para polifenóis, metilxantinas e saponinas foram 497, 88 e 245 mg/g de material seco, respectivamente. Vinte quatro ratos Wistar machos foram submetidos a uma dieta hiperlipídica e seis ratos submetidos à uma dieta padrão durante cinco semanas. Posteriormente, foram separados em dois grupos controles (dieta padrão e hiperlipídica), um grupo tratado com extrato de mate (YME), outro tratado com MXPL e outro tratado com SFL. Nenhum aumento significativo no peso corporal em ratos com dieta hiperlipídica foi observado, contudo, houve alterações evidentes em outros parâmetros relevantes relacionados ao metabolismo. Resultados referentes à redução de peso corporal, níveis plasmáticos de glicose e lipogênese se contrapõem a outros antes relatados na literatura, sugerindo uma relação com o modelo animal utilizado. Atividades observadas para o extrato e frações indicam claramente que essas podem ser diferentes e até opostas, levando acreditar que os principais compostos do mate possuam diferentes efeitos no metabolismo quando testados separadamente.Ilex paraguariensis A. St. Hil. is a South American native species commonly known as mate or yerba mate and is widely used as stimulant tea-like beverages. Methylxanthines, polyphenols and saponins are the main compounds present in its leaves. To these, are attributed several biological activities, including effects in lipid metabolism. Although there are a range of studies of mate, in the point of view of technological processing and biological testing of enriched and purified fractions revealed to be restricted. In this sense, the aim of this study is to evaluate the activity of the main compounds found in mate on the lipid metabolism of male Wistar rats fed with a high-fat diet. For this propose, it was obtained and optimized an extract of mate leaves (YME) by a Box-Behnken design, obtaining maximum yields of 357, 41 and 15 mg/g dry material, respectively. Subsequently, the main compounds were separated by solid phase extraction and fractionation using a polymer resin, methanol and water, in decreasing gradient polarity, achieving an enriched fraction of polyphenol and methylxanthines (MXPL) and an enriched fraction of saponins (SFL). Yields for polyphenols, saponins and methylxanthines were 497, 88 and 245 mg/g of dry material, respectively. Twenty four male Wistar rats were submitted to a high-fat diet and six to a standard diet, during five weeks and separated in five groups (standard diet (control) (n = 6); high-fat diet (n = 6); YME-treated group (n = 6); MXPL-treated group (n = 6); SFL-treated group (n = 6). No significant increase in rats body weight fed with high-fat diet was observed. However, there were evident alterations in fat other relevant parameters related to metabolism. Dissimilar results regarding the reduction of body weight, plasma levels of glucose and lipogenesis, for instance, are opposed to other studies, previously reported in the literature, suggesting that the animal model might be determinant in the evaluation of these activities. Likewise, effects observed in the crude extract and enriched fractions clearly indicate that these may be different, even opposite, leading to believe that the main compounds of mate have different effects on metabolism when are tested separately