New approaches for the identification of antivirulence agents based on LsrK inhibition : from assay development to screening campaigns

To overcame the spread of bacterial resistance to traditional antbiotics, great interest has arisen towards antivirulence agents, compounds targeting virulence factors. In fact, as there is no link between growth and virulence, antivirulence agents are considered less prone to promote resistance development. In this context, quorum sensing (QS), a communication strategy among bacteria which regulates several bacterial functions including virulence, has been widely investigated for the development of QS inhibitors with the aim of limiting bacterial virulence. This study describes the development of a new assay for the discovery of inhibitors targeting LsrK, a key kinase for autoinducer 2 (AI-2) mediated QS establishment in enteric bacteria. LsrK in fact phosphorylates the AI-2 which, only in the phosphorylated form, can bind to the LsrR repressor and enhance the response to QS signals via activation of the lsr operon. The new assay was used for the screening of three different compound libraries. The best hits from the three campaigns were harpagoside and rosolic acid, presented in Study I, also active in cell-based AI-2 mediated QS assay. Additionally, the active compounds found in Study II and III provided interesting information about the catalytic site of LsrK. To facilitate the confirmation of hits selected by target-based assay and to offer a new tool for the rapid identification of QS inhibitors, Study IV describes the design, optimization and application of a new bioreporter strain, emitting luminescence as response to AI-2 mediated QS activation. The assay was used to test a set of 91 compounds selected to target the ATP binding site of LsrK. The same set of compounds was also tested in the target-based LsrK inhibition assay. The combined results led to the identification of 6 compounds, active in both assays, which thus may decrease response to QS by inhibiting LsrK. Additionally, 18 compounds were active only in the cell-based assay implying that they target other components of the pathway. These findings broaden our knowledge on LsrK and may be used as scaffolds to design compounds with improved properties. Furthermore, the AI-2 mediated QS interference assay represents an additional tool for the identification of QS inhibitors alone or in combination with target-based assays.Bakteerien lisääntyvä mikrobilääkeresistenssi on herättänyt suurta mielenkiintoa uudentyyppisten ehkäisykeinojen kuten bakteerien virulenssitekijöihin kohdistuvien yhdisteiden tutkimiseen. Koska bakteerien lisääntymisen ja virulenssin välillä ei ole suoraa yhteyttä, virulenssiin vaikuttavien yhdisteiden ei katsota johtavan resistenssin kehittymiseen. Bakteeripopulaatiossa tapahtuva laumaviestintä (engl. quorum sensing) säätelee mm. useiden virulenssitekijöiden ilmenemistä ja näinollen tämän laumaviestinnän häirintä on yksi tutkituista strategioista virulenssin rajoittamiseksi. Tässä tutkimuksessa keskityttiin useiden enteeristen bakteerien signaalinvälityksessä toimivaan kinaasientsyymiin LsrK ja tämän kinaasin toimintaa estävien yhdisteiden etsimiseen ja niiden jatkotutkimuksiin. LsrK katalysoi signaalimolekyyli autoinducer-2:n (AI-2) fosforylaatiota, joka johtaa lopulta laumaviestintävasteen aktivoitumiseen bakteerisolussa. LsrK-entsyymiä estävien yhdisteiden seulontaa varten kehitettiin tässä työssä uusi menetelmä, jota käytettiin kolmen erilaisen yhdistekokoelman seulontaan. Tärkeimpiä löydöksiä olivat ensimmäisessä osatutkimuksessa löydetyt LsrK-entsyymin toimintaa estävät harpagosidi ja rosolihappo, jotka todettiin aktiiviksi myös solupohjaisessa AI-2-signaaliketjun aktivoitumista mittaavassa menetelmässä. Näiden lisäksi toisessa ja kolmannessa osatutkimuksessa saatiin uutta tietoa esimerkiksi LsrK:n katalyyttisesta osasta. Kohdeperustaisen menetelmän avulla tunnistettujen aktiivisten yhdisteiden jatkotutkimuksia varten sekä uudenlaiseksi työkaluksi laumaviestintää häiritsevien yhdisteiden tunnistamiseksi, neljännessä osatutkimuksessa kehitettiin uusi bioreportterikanta. Tässä bioreportterikannassa AI-2-välitteisen signaaliketjun aktivoituminen saa aikaan luminesenssisignaalin, jota voidaan mitata. Kehitetyn menetelmän avulla testattiin 91 yhdistettä, joiden arveltiin sitoutuvan LsrK:n ATP:n sitoutumiskohtaan. Sama yhdistesarja tutkittiin myös LsrK:n aktiivisuutta mittaavalla menetelmällä. Tutkimuksessa tunnistettiin kuusi yhdistettä, jotka osoittivat aktiivisuutta sekä kohdeperustaisessa että bioreportterimenetelmässä ja joiden signaalinvälityksen häirintäkyky saattaa näinollen perustua LsrK:n toiminnan estämiseen. Lisäksi tunnistettiin 18 yhdistettä, jotka osoittivat aktiivisuutta vain bioreportterimenetelmässä, mikä viittaa siihen, että niiden vaikutus kohdistuu signaalinvälitysreitin muihin osatekijöihin. Tässä väitöskirjatyössä kehitetyt uudet menetelmät edistävät merkittävästi uusien AI-2- välitteistä laumaviestintää estävien yhdisteiden seulontaa. Tutkimuksessa tunnistettiin useita LsrK-entsyymin toimintaa ja AI-2-välitteistä viestintää estäviä yhdisteitä, joita voidaan jatkossa hyödyntää aihioina bakteerien virulenssia estäviä yhdisteitä kehitettäessä. Näitä yhdisteitä voidaan käyttää myös työkaluina bakteerien laumaviestintää tutkittaessa

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition

Targeting Quorum Sensing: High-Throughput Screening to Identify Novel LsrK Inhibitors

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 ≤ 10 µM confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition

Butyrylcholinesterase and Acetylcholinesterase polymorphisms in Multiple Sclerosis patients: Implication in peripheral inflammation

Multiple Sclerosis (MS) is an autoimmune disease, having not fully understood aetiology, and both genetic and environmental factors contribute to the pathogenesis of the disease. The cholinergic system has been indicated as a mediator of neuro-immune interactions, as well as an internal regulator of immune responses. The aim of the present research was to assess the associations between BChE and AChE genetic variations and serum cholinergic and inflammatory profiles in 102 Relapsing Remitting-MS patients and 117 healthy controls. An increased frequency of the BChE K-allele in MS patients as compared to controls was found. In addition, data showed that patients had higher BChE enzymatic activity, which is increased by the presence of the polymorphic allele and reduced amounts of circulating ACh. AChE polymorphism was significantly associated to reduced activity in both patients and controls. We propose that serum BChE and AChE activity may be used as a secondary markers to assess the role of non-neuronal cholinergic system in regulating peripheral inflammation via ACh regulation. This pilot study shed light on the role of the non-neuronal cholinergic system in immune cells to better understand MS pathogenesis. The cross-talk between the periphery and the CNS could have a new undescribed crucial role for MS, regarded as a systemic disease

2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents

2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents

A new cell-based AI-2-mediated quorum sensing interference assay in screening of LsrK-targeted inhibitors

Quorum sensing (QS), a bacterial communication strategy, has been recognized as one of the control mechanisms of virulence in bacteria. Thus, targeting QS offers an interesting opportunity to impair bacterial pathogenicity and develop antivirulence agents. Aiming to enhance the discovery of QS inhibitors, we developed a bioreporter Escherichia coli JW5505 pET-Plsrlux and set up a cell-based assay for identifying inhibitors of autoinducer-2 (AI-2)-mediated QS. A comparative study on the performance of target- versus cell-based assays was performed, and 91 compounds selected with the potential to target the ATP binding pocket of LsrK, a key enzyme in AI-2 processing, were tested in an LsrK inhibition assay, providing 36 hits. The same set of compounds was tested by the AI-2-mediated QS interference assay, resulting in 24 active compounds. Among those, six were also found to be active against LsrK, whereas 18 might target other components of the pathway. Thus, this AI-2-mediated QS interference cell-based assay is an effective tool for complementing target-based assays, yet also stands as an independent assay for primary screening.Peer reviewe

DPD-inspired discovery of novel LsrK kinase inhibitors: an opportunity to fight antimicrobial resistance

Antibiotic resistance is posing a continuous threat to global public health and represents a huge burden for society as a whole. In the past decade, the interference with bacterial quorum sensing (QS) (i.e., cell cell communication) mechanisms has extensively been investigated as a valid therapeutic approach in the pursuit of a next generation of antimicrobials. (S)-4,5-Dihydroxy-2,3-pentanedione, commonly known as (S)-DPD, a small signaling molecule that modulates QS in both Gram-negative and Gram-positive bacteria, is phosphorylated by LsrK, and the resulting phospho-DPD activates QS. We designed and prepared a small library of DPD derivatives, characterized by five different scaffolds, and evaluated their LsrK inhibition in the context of QS interference. SAR studies highlighted the pyrazole moiety as an essential structural element for LsrK inhibition. Particularly, four compounds were found to be micromolar LsrK inhibitors (IC50 ranging between 100 mu M and 500 mu M) encouraging further exploration of novel analogues as potential new antimicrobials.Peer reviewe

High prevalence of BRCA1 deletions in BRCAPRO-positive patients with high carrier probability.

Mutation screening of the BRCA1 and BRCA2 genes in probands with familial breast/ovarian cancer has been greatly improved by the multiplex ligation-dependent probe amplification (MLPA) assay able to evidence gene rearrangements not detectable by standard screening methods. However, no criteria for selection of cases to be submitted to the MLPA test have been reported yet. We used the BRCAPro software for the selection of familial breast/ovarian cancer probands investigated with the MLPA approach after negative BRCA1/2 conventional mutation screening. One hundred and seventy-seven probands were investigated for germline BRCA1/2 mutations after assessment of genetic risk using BRCAPro. Probands were classified as BRCAPro positive (n = 67) when the carrier probability (CP) was >10% and as BRCAPro negative (n = 110), when the CP was <10%. Conventional mutational analyses of the BRCA1/2 genes and, in one case, of p53 identified 22 pathogenetic germline mutations, 12 in BRCA1, 9 in BRCA2 and 1 in p53, in 22/177 (12.4%) probands. All the mutations except one were detected in BRCAPro-positive patients. In the 46 BRCAPro-positive cases that resulted negative by BRCA1/2 mutation, screening analysis of rearrangements within BRCA1/2 by MLPA was carried out. Three patients with a very high CP showed BRCA1 deletions, consisting of deletions of exons 1–2 in two probands and of exon 24 in the third proband. In one case, the exons 1–2 deletion was shown to cosegregate with disease in the family. No BRCA2 rearrangements were detected, but one patient showed the 1100delC of the CHEK2 gene, whose probe is present in the BRCA2 kit. In our series, the highest carrier detection rate of mutation screening plus MLPA analysis (52.3%) was in patients with a BRCAPro CP >50%