Mass Spectrometry Imaging of Coniine and Other Hemlock Alkaloids after On-Tissue Derivatization Reveals Distinct Alkaloid Distributions in the Plant.

Specialized metabolites play important roles in plants and can, for example, protect plants from predators or pathogens. Alkaloids, due to their pronounced biological activity on higher animals, are one of the most intriguing groups of specialized metabolites, and many of them are known as plant defense compounds. Poison hemlock, Conium maculatum, is well-known for its high content of piperidine alkaloids, of which coniine is the most famous. The distribution, localization, and diversity of these compounds in C. maculatum tissues have not yet been studied in detail. The hemlock alkaloids are low molecular weight compounds with relatively high volatility. They are thus difficult to analyze on-tissue by MALDI mass spectrometry imaging due to delocalization, which occurs even when using an atmospheric pressure ion source. In this manuscript, we describe an on-tissue derivatization method that allows the subsequent determination of the spatial distribution of hemlock alkaloids in different plant tissues by mass spectrometry imaging. Coniferyl aldehyde was found to be a suitable reagent for derivatization of the secondary amine alkaloids. The imaging analysis revealed that even chemically closely related hemlock alkaloids are discretely distributed in different plant tissues. Additionally, we detected a yet undescribed hemlock alkaloid in Conium maculatum seeds.

Oxime-Linked Peptide-Daunomycin Conjugates as Good Tools for Selection of Suitable Homing Devices in Targeted Tumor Therapy: An Overview.

Chemotherapy is still one of the main therapeutic approaches in cancer therapy. Nevertheless, its poor selectivity causes severe toxic side effects that, together with the development of drug resistance in tumor cells, results in a limitation for its application. Tumor-targeted drug delivery is a possible choice to overcome these drawbacks. As well as monoclonal antibodies, peptides are promising targeting moieties for drug delivery. However, the development of peptide-drug conjugates (PDCs) is still a big challenge. The main reason is that the conjugates have to be stable in circulation, but the drug or its active metabolite should be released efficiently in the tumor cells. For this purpose, suitable linker systems are needed that connect the drug molecule with the homing peptide. The applied linker systems are commonly categorized as cleavable and non-cleavable linkers. Both the groups possess advantages and disadvantages that are summarized briefly in this manuscript. Moreover, in this review paper, we highlight the benefit of oxime-linked anthracycline-peptide conjugates in the development of PDCs. For instance, straightforward synthesis as well as a conjugation reaction proceed in excellent yields, and the autofluorescence of anthracyclines provides a good tool to select the appropriate homing peptides. Furthermore, we demonstrate that these conjugates can be used properly in in vivo studies. The results indicate that the oxime-linked PDCs are potential candidates for targeted tumor therapy.

Comparative reassessment of AcrB efflux inhibitors reveals differential impact of specific pump mutations on the activity of potent compounds.

Multidrug resistance poses global challenges, particularly with regard to Gram-negative bacterial infections. In view of the lack of new antibiotics, drug enhancers, such as efflux pump inhibitors (EPIs), have increasingly come into focus. A number of chemically diverse agents have been reported to inhibit AcrB, the main multidrug transporter in Escherichia coli, and homologs in other Gram-negative bacteria. However, due to the often varying methodologies used for their characterization, results remain difficult to compare. In this study, using a defined selection of antibiotics known to be efflux substrates, we reevaluated 38 published compounds for their in vitro EPI activity. When examined in an E. coli strain with stable wild-type AcrB overexpression, we found 17 compounds showing at least fourfold enhancing potency with more than 2 out of 10 test drugs (belonging to eight antibiotic classes). Pyranopyridines (MBX series) were confirmed as the most potent inhibitors among agents reported so far. A new and surprising finding was that their activity, unlike that of the pyridylpiperazine EPI BDM88855, was highly susceptible to the AcrB double-mutation G141D_N282Y, which had previously been shown to diminish drug enhancing of 1-(1-naphthylmethyl)piperazine in a predominantly substrate-specific manner. Conversely, transmembrane region mutation V411A, while eliminating the drug potentiating of the BDM compound, did not decrease the activity of the MBX EPIs. Besides comparative reassessment of the potency of reported EPIs, the study demonstrated the usefulness of mutagenesis approaches providing tools for an initial discrimination of EPIs regarding their mode of function.IMPORTANCEInfections with difficult-to-treat multidrug-resistant bacteria pose an urgent global threat in view of the stagnating development of new antimicrobial substances. Efflux pumps in Gram-negative pathogens are known to substantially contribute to multidrug resistance making them promising targets for chemotherapeutic interventions to restore the efficacy of conventional antibiotics. In the present study, the in vitro activity of previously reported efflux pump inhibitors was reassessed using standardized conditions. Relevant drug sensitizing activity could be proven for almost half of the tested compounds. Further characterization of potent inhibitors was achieved by investigating the impact of specific efflux pump mutations. A double-mutation previously known to decrease the activity of the arylpiperazine 1-(1-naphthylmethyl)piperazine also impaired that of the highly efficient pyranopyridine efflux pump inhibitors. Our findings provide direct comparability of reported efflux pump inhibitors and contribute to the elucidation of their mode of action.

A Screening of Antimalarials Extends the Range of Known Escherichia coli AcrB Efflux Substrates and Reveals Two Candidates with Antimicrobial Drug-Enhancing Activity.

Efflux by resistance nodulation cell division transporters, such as AcrAB-TolC in Escherichia coli, substantially contributes to the development of Gram-negative multidrug resistance. Therefore, the finding of compounds that counteract efflux is an urgent goal in the fight against infectious diseases. Previously, an efflux inhibitory activity of the antimalarials mefloquine and artesunate was reported. In this study, we have investigated further antimalarials regarding efflux by AcrB, the pumping part of AcrAB-TolC, and their drug-enhancing potency in E. coli. We show that 10 of the 24 drugs tested are substrates of the multidrug efflux pump AcrB. Among them, tafenoquine and proguanil, when used at subinhibitory concentrations, caused an at least 4- and up to 24-fold enhancement in susceptibility to 6 and 14 antimicrobial agents, respectively. Both antimalarials are able to increase the intracellular accumulation of Hoechst 33342, with proguanil showing similar effectiveness as the efflux inhibitor 1-(1-naphthylmethyl)piperazine. In the case of proguanil, AcrB-dependent efflux inhibition could also be demonstrated in a real-time efflux assay. In addition to presenting new AcrB substrates, our study reveals two previously unknown efflux inhibitors among antimalarials. Particularly proguanil appears as a promising candidate and its chemical scaffold might be further optimized for repurposing as antimicrobial drug enhancer.

Development and Biochemical Characterization of Self-Immolative Linker Containing GnRH-III-Drug Conjugates.

The human gonadotropin releasing hormone (GnRH-I) and its sea lamprey analogue GnRH-III specifically bind to GnRH receptors on cancer cells and can be used as targeting moieties for targeted tumor therapy. Considering that the selective release of drugs in cancer cells is of high relevance, we were encouraged to develop cleavable, self-immolative GnRH-III-drug conjugates which consist of a p-aminobenzyloxycarbonlyl (PABC) spacer between a cathepsin B-cleavable dipeptide (Val-Ala, Val-Cit) and the classical anticancer drugs daunorubicin (Dau) and paclitaxel (PTX). Alongside these compounds, non-cleavable GnRH-III-drug conjugates were also synthesized, and all compounds were analyzed for their antiproliferative activity. The cleavable GnRH-III bioconjugates revealed a growth inhibitory effect on GnRH receptor-expressing A2780 ovarian cancer cells, while their activity was reduced on Panc-1 pancreatic cancer cells exhibiting a lower GnRH receptor level. Moreover, the antiproliferative activity of the non-cleavable counterparts was strongly reduced. Additionally, the efficient cleavage of the Val-Ala linker and the subsequent release of the drugs could be verified by lysosomal degradation studies, while radioligand binding studies ensured that the GnRH-III-drug conjugates bound to the GnRH receptor with high affinity. Our results underline the high value of GnRH-III-based homing devices and the application of cathepsin B-cleavable linker systems for the development of small molecule drug conjugates (SMDCs).

Protein engineering of a stable and potent anti-inflammatory IL-37-Fc fusion with enhanced therapeutic potential.

Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.

Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate.

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

Limited Multidrug Resistance Efflux Pump Overexpression among Multidrug-Resistant Escherichia coli Strains of ST131.

Gram-negative bacteria partly rely on efflux pumps to facilitate growth under stressful conditions and to increase resistance to a wide variety of commonly used drugs. In recent years, Escherichia coli sequence type 131 (ST131) has emerged as a major cause of extraintestinal infection frequently exhibiting a multidrug resistance (MDR) phenotype. The contribution of efflux to MDR in emerging E. coli MDR clones, however, is not well studied. We characterized strains from an international collection of clinical MDR E. coli isolates by MIC testing with and without the addition of the AcrAB-TolC efflux inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). MIC data for 6 antimicrobial agents and their reversion by NMP were analyzed by principal-component analysis (PCA). PCA revealed a group of 17 MDR E. coli isolates (n = 34) exhibiting increased susceptibility to treatment with NMP, suggesting an enhanced contribution of efflux pumps to antimicrobial resistance in these strains (termed enhanced efflux phenotype [EEP] strains). Only 1/17 EEP strains versus 12/17 non-EEP MDR strains belonged to the ST131 clonal group. Whole-genome sequencing revealed marked differences in efflux-related genes between EEP and control strains, with the majority of notable amino acid substitutions occurring in AcrR, MarR, and SoxR. Quantitative reverse transcription-PCR (qRT-PCR) of multiple efflux-related genes showed significant overexpression of the AcrAB-TolC system in EEP strains, whereas in the remaining strains, we found enhanced expression of alternative efflux proteins. We conclude that a proportion of MDR E. coli strains exhibit an EEP, which is linked to an overexpression of the AcrAB-TolC efflux pump and a distinct array of genomic variations. Members of ST131, although highly successful, are less likely to exhibit the EEP.

New Topoisomerase Inhibitors: Evaluating the Potency of Gepotidacin and Zoliflodacin in Fluoroquinolone-Resistant Escherichia coli upon tolC Inactivation and Differentiating Their Efflux Pump Substrate Nature.

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND (resistance-nodulation-cell division)-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux-bypassing drugs and provide AcrB targets with critical relevance for this purpose.

Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen.

Objectives: This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance. Methods: A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated. Results: Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3. Conclusions: Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

Improved In Vivo Anti-Tumor and Anti-Metastatic Effect of GnRH-III-Daunorubicin Analogs on Colorectal and Breast Carcinoma Bearing Mice.

Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety for drug delivery systems. The anti-tumor activity of the previously developed GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] conjugate and the novel synthesized GnRH-III-[2ΔHis,3d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] conjugate, containing the anti-cancer drug daunorubicin, were evaluated. Here, we demonstrate that both GnRH-III-Dau conjugates possess an efficient growth inhibitory effect on more than 20 cancer cell lines, whereby the biological activity is strongly connected to the expression of gonadotropin-releasing hormone receptors (GnRH-R). The novel conjugate showed a higher in vitro anti-proliferative activity and a higher uptake capacity. Moreover, the treatment with GnRH-III-Dau conjugates cause a significant in vivo tumor growth and metastases inhibitory effect in three different orthotopic models, including 4T1 mice and MDA-MB-231 human breast carcinoma, as well as HT-29 human colorectal cancer bearing BALB/s and SCID mice, while toxic side-effects were substantially reduced in comparison to the treatment with the free drug. These findings illustrate that our novel lead compound is a highly promising candidate for targeted tumor therapy in both colon cancer and metastatic breast cancer.

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives in Escherichia coli with Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways.

A major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives in Escherichia coli was confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

Proof of an Outer Membrane Target of the Efflux Inhibitor Phe-Arg-ß-Naphthylamide from Random Mutagenesis.

Phe-Arg-ß-naphthylamide (PAßN) has been characterized as an efflux pump inhibitor (EPI) acting on the major multidrug resistance efflux transporters of Gram-negative bacteria, such as AcrB in Eschericha coli. In the present study, in vitro random mutagenesis was used to evolve resistance to the sensitizing activity of PAßN with the aim of elucidating its mechanism of action. A strain was obtained that was phenotypically similar to a previously reported mutant from a serial selection approach that had no efflux-associated mutations. We could confirm that acrB mutations in the new mutant were unrelated to PAßN resistance. The next-generation sequencing of the two mutants revealed loss-of-function mutations in lpxM. An engineered lpxM knockout strain showed up to 16-fold decreased PAßN activity with large lipophilic drugs, while its efflux capacity, as well as the efficacy of other EPIs, remained unchanged. LpxM is responsible for the last acylation step in lipopolysaccharide (LPS) synthesis, and lpxM deficiency has been shown to result in penta-acylated instead of hexa-acylated lipid A. Modeling the two lipid A types revealed steric conformational changes due to underacylation. The findings provide evidence of a target site of PAßN in the LPS layer, and prove membrane activity contributing to its drug-sensitizing potency.

Enhanced In Vitro Antitumor Activity of GnRH-III-Daunorubicin Bioconjugates Influenced by Sequence Modification.

Receptors for gonadotropin releasing hormone (GnRH) are highly expressed in various human cancers including breast, ovarian, endometrial, prostate and colorectal cancer. Ligands like human GnRH-I or the sea lamprey analogue GnRH-III represent a promising approach for the development of efficient drug delivery systems for targeted tumor therapy. Here, we report on the synthesis and cytostatic effect of 14 oxime bond-linked daunorubicin GnRH-III conjugates containing a variety of unnatural amino acids within the peptide sequence. All compounds demonstrated a reduced cell viability in vitro on estrogen receptor α (ERα) positive and ERα negative cancer cells. The best candidate revealed an increased cancer cell growth inhibitory effect compared to our lead-compound GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)]. Flow cytometry and fluorescence microscopy studies showed that the cellular uptake of the novel conjugate is substantially improved leading to an accelerated delivery of the drug to its site of action. However, the release of the active drug-metabolite by lysosomal enzymes was not negatively affected by amino acid substitution, while the compound provided a high stability in human blood plasma. Receptor binding studies were carried out to ensure a high binding affinity of the new compound for the GnRH-receptor. It was demonstrated that GnRH-III-[²ΔHis,³d-Tic,4Lys(Bu),8Lys(Dau=Aoa)] is a highly potent and promising anticancer drug delivery system for targeted tumor therapy.

Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin-GnRH-III conjugates developed for targeted drug delivery.

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin-GnRH-III conjugate (GnRH-III-[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin-GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.

Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate.

Drug efflux by resistance-nodulation-cell division (RND)-type transporters, such as AcrAB-TolC of Escherichia coli, is an important resistance mechanism in Gram-negative bacteria; however, its contribution to multidrug resistance (MDR) in clinical isolates is poorly defined. We inactivated acrB of a sequence type 131 E. coli human isolate that showed high-level MDR, but had no mutations within the known efflux-associated local or global regulators. The resistance profile of the acrB deletion mutant revealed significantly increased susceptibility to drugs from seven antibiotic classes, including agents usually inactive against Gram-negative bacteria, notably the new oxazolidinone, tedizolid (512-fold enhanced susceptibility). AcrB deficiency reduced, but did not abolish, the efflux of dyes, which indicates the activity of at least one more efflux transporter. The findings demonstrate the efficacy of AcrAB-TolC-mediated broad-spectrum drug efflux, including agents primarily developed for Gram-positive pathogens, in a clinical isolate representative of a globally-emerging lineage. The results illustrate the need to develop molecules modified to impede their transport by AcrAB-TolC and its homologues and new efflux inhibitors.

Evidence of a Substrate-Discriminating Entrance Channel in the Lower Porter Domain of the Multidrug Resistance Efflux Pump AcrB.

Efflux pumps of the resistance nodulation cell division (RND) transporter family, such as AcrB of Escherichia coli, play an important role in the development of multidrug resistance, but the molecular basis for their substrate promiscuity is not yet completely understood. From a collection of highly clarithromycin-resistant AcrB periplasmic domain mutants derived from in vitro random mutagenesis, we identified variants with an unusually altered drug resistance pattern characterized by increased susceptibility to many drugs of lower molecular weight, including fluoroquinolones, tetracyclines, and oxazolidinones, but unchanged or increased resistance to drugs of higher molecular weight, including macrolides. Sequencing of 14 such "divergent resistance" phenotype mutants and 15 control mutants showed that this unusual phenotype was associated with mutations at residues I38 and I671 predominantly to phenylalanine and threonine, respectively, both conferring a similar susceptibility pattern. Reconstructed I38F and I671T single mutants as well as an engineered I38F I671T double mutant with proved efflux competence revealed an equivalent phenotype with enhanced or unchanged resistance to many large AcrB substrates but increased susceptibility to several lower-molecular-weight drugs known to bind within the distal binding pocket. The two isoleucines located in close vicinity to each other in the lower porter domain of AcrB beneath the bottom of the proximal binding pocket may be part of a preferential small-drug entrance pathway that is compromised by the mutations. This finding supports recent indications of distinct entrance channels used by compounds with different physicochemical properties, of which molecular size appears to play a prominent role.

A continuous sirtuin activity assay without any coupling to enzymatic or chemical reactions.

Sirtuins are NAD(+) dependent lysine deacylases involved in many regulatory processes such as control of metabolic pathways, DNA repair and stress response. Modulators of sirtuin activity are required as tools for uncovering the biological function of these enzymes and as potential therapeutic agents. Systematic discovery of such modulators is hampered by the lack of direct and continuous activity assays. The present study describes a novel continuous assay based on the increase of a fluorescence signal subsequent to sirtuin mediated removal of a fluorescent acyl chain from a modified TNFα-derived peptide. This substrate is well recognized by human sirtuins 1-6 and represents the best sirtuin 2 substrate described so far with a kcat/KM-value of 176 000 M(-1)s(-1). These extraordinary substrate properties allow the first determination of Ki-values for the specific Sirt2 inhibitory peptide S2iL5 (600 nM) and for the quasi-universal sirtuin inhibitor peptide thioxo myristoyl TNFα (80 nM).

Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes in a Combined Real-Time Influx and Efflux Assay.

In this study, we tested five compounds belonging to a novel series of piperazine arylideneimidazolones for the ability to inhibit the AcrAB-TolC efflux pump. The biphenylmethylene derivative (BM-19) and the fluorenylmethylene derivative (BM-38) were found to possess the strongest efflux pump inhibitor (EPI) activities in the AcrAB-TolC-overproducingEscherichia colistrain 3-AG100, whereas BM-9, BM-27, and BM-36 had no activity at concentrations of up to 50 µM in a Nile red efflux assay. MIC microdilution assays demonstrated that BM-19 at 1/4 MIC (intrinsic MIC, 200 µM) was able to reduce the MICs of levofloxacin, oxacillin, linezolid, and clarithromycin 8-fold. BM-38 at 1/4 MIC (intrinsic MIC, 100 µM) was able to reduce only the MICs of oxacillin and linezolid (2-fold). Both compounds markedly reduced the MIC of rifampin (BM-19, 32-fold; and BM-38, 4-fold), which is suggestive of permeabilization of the outer membrane as an additional mechanism of action. Nitrocefin hydrolysis assays demonstrated that in addition to their EPI activity, both compounds were in fact weak permeabilizers of the outer membrane. Moreover, it was found that BM-19, BM-27, BM-36, and BM-38 acted as near-infrared-emitting fluorescent membrane probes, which allowed for their use in a combined influx and efflux assay and thus for tracking of the transport of an EPI across the outer membrane by an efflux pump in real time. The EPIs BM-38 and BM-19 displayed the most rapid influx of all compounds, whereas BM-27, which did not act as an EPI, showed the slowest influx.

Random mutagenesis of the multidrug transporter AcrB from Escherichia coli for identification of putative target residues of efflux pump inhibitors.

Efflux is an important mechanism of bacterial multidrug resistance (MDR), and the inhibition of MDR pumps by efflux pump inhibitors (EPIs) could be a promising strategy to overcome MDR. 1-(1-Naphthylmethyl)-piperazine (NMP) and phenylalanine-arginine-ß-naphthylamide (PAßN) are model EPIs with activity in various Gram-negative bacteria expressing AcrB, the major efflux pump of Escherichia coli, or similar homologous pumps of the resistance-nodulation-cell division class. The aim of the present study was to generate E. coli AcrB mutants resistant to the inhibitory action of the two model EPIs and to identify putative EPI target residues in order to better understand mechanisms of pump inhibition. Using an in vitro random mutagenesis approach focusing on the periplasmic domain of AcrB, we identified the double mutation G141D N282Y, which substantially compromised the synergistic activity of NMP with linezolid, was associated with similar intracellular linezolid concentrations in the presence and absence of NMP, and did not impair the intrinsic MICs of various pump substrates and dye accumulation. We propose that these mutations near the outer face of the distal substrate binding pocket reduce NMP trapping. Other residues found to be relevant for efflux inhibition by NMP were G288 and A279, but mutations at these sites also changed the susceptibility to several pump substrates. Unlike with NMP, we were unable to generate AcrB periplasmic domain mutants with resistance or partial resistance to the EPI activity of PAßN, which is consistent with the modes of action of PAßN differing from those of NMP.