Discovery and optimization of thiazole-based quorum sensing inhibitors as potent blockers of Pseudomonas aeruginosa pathogenicity.

Pseudomonas aeruginosa causes life-threatening infections especially in hospitalized patients and shows an increasing resistance to established antibiotics. A process known as quorum sensing (QS) enables the pathogen to collectively adapt to various environmental conditions. Disrupting this cell-to-cell communication machinery by small-molecular entities leads to a blockade of bacterial pathogenicity. We aim to devise QS inhibitors acting on the PA-specific PQS QS system via the signal-molecule receptor and transcriptional regulator PqsR (MvfR). In this manuscript, we describe the further optimization of PqsR inverse agonists by broadening the structural space of a previously described triazole-bearing lead compound and arriving at highly potent thiazole derivatives with activities against P. aeruginosa virulence factor pyocyanin in the nanomolar range. All new derivatives were profiled regarding biological activity as well as in vitro ADMET parameters. Additionally, we assessed safety-pharmacology characteristics of the two most promising compounds both bearing a 3-chloro-4-isopropoxyphenyl motive. Demonstrating an overall favorable profile, our new PqsR inverse agonists represent a valuable addition as optimized lead compounds, enabling preclinical development of P. aeruginosa-specific pathoblockers.

Nanoparticles in liposomes: a platform for increased antibiotic selectivity in multidrug resistant bacteria in respiratory tract infections.

Antibiotic resistance is a cause of serious illness and death, originating often from insufficient permeability into gram-negative bacteria. Nanoparticles (NP) can increase antibiotic delivery in bacterial cells, however, may as well increase internalization in mammalian cells and toxicity. In this work, NP in liposome (NP-Lip) formulations were used to enhance the selectivity of the antibiotics (3C and tobramycin) and quorum sensing inhibitor (HIPS-1635) towards Pseudomonas aeruginosa by fusing with bacterial outer membranes and reducing uptake in mammalian cells due to their larger size. Poly (lactic-co-glycolic) acid NPs were prepared using emulsion solvent evaporation and incorporated in larger liposomes. Cytotoxicity and uptake studies were conducted on two lung cell lines, Calu-3 and H460. NP-Lip showed lower toxicity and uptake in both cell lines. Then formulations were investigated for suitability for oral inhalation. The deposition of NP and NP-Lip in the lungs was assessed by next generation impactor and corresponded to 75% and 45% deposition in the terminal bronchi and the alveoli respectively. Colloidal stability and mucus-interaction studies were conducted. NP-Lip showed higher diffusion through mucus compared to NPs with the use of nanoparticle tracking analyzer. Moreover, the permeation of delivery systems across a liquid-liquid interface epithelial barrier model of Calu-3 cells indicated that NP-Lip could cause less systemic toxicity upon in-vivo like administration by aerosol deposition. Monoculture and Pseudomonas aeruginosa biofilm with Calu-3 cells co-culture experiments were conducted, NP-Lip achieved highest toxicity towards bacterial biofilms and least toxicity % of the Calu-3 cells. Therefore, the NP- liposomal platform offers a promising approach for enhancing antibiotic selectivity and treating pulmonary infections.

Development and evaluation of 2,4-disubstituted-5-aryl pyrimidine derivatives as antibacterial agents.

Designing novel candidates as potential antibacterial scaffolds has become crucial due to the lack of new antibiotics entering the market and the persistent rise in multidrug resistance. Here, we describe a new class of potent antibacterial agents based on a 5-aryl-N2,N4-dibutylpyrimidine-2,4-diamine scaffold. Structural optimization focused on the 5-aryl moiety and the bioisosteric replacement of the side chain linker atom. Screening of the synthesized compounds focused on a panel of bacterial strains, including gram-positive Staphylococcus aureus strains (Newman MSSA, methicillin- and vancomycin-resistant), and the gram-negative Escherichia coli (ΔAcrB strain). Several compounds showed broad-spectrum antibacterial activity with compound 12, bearing a 4-chlorophenyl substituent, being the most potent among this series of compounds. This frontrunner compound revealed a minimum inhibitory concentration (MIC) value of 1 µg/mL against the S. aureus strain (Mu50 methicillin-resistant S. aureus/vancomycin-intermediate S. aureus) and an MIC of 2 µg/mL against other tested strains. The most potent derivatives were further tested against a wider panel of bacteria and evaluated for their cytotoxicity, revealing further potent activities toward Streptococcus pneumoniae, Enterococcus faecium, and Enterococcus faecalis. To explore the mode of action, compound 12 was tested in a macromolecule inhibition assay. The obtained data were supported by the safety profile of compound 12, which possessed an IC50 of 12.3 µg/mL against HepG2 cells. The current results hold good potential for a new class of extended-spectrum antibacterial agents.

Hit optimization by dynamic combinatorial chemistry on Streptococcus pneumoniae energy-coupling factor transporter ECF-PanT.

Herein, we present the first application of target-directed dynamic combinatorial chemistry (tdDCC) to the whole complex of the highly dynamic transmembrane, energy-coupling factor (ECF) transporter ECF-PanT in Streptococcus pneumoniae. In addition, we successfully employed the tdDCC technique as a hit-identification and -optimization strategy that led to the identification of optimized ECF inhibitors with improved activity. We characterized the best compounds regarding cytotoxicity and performed computational modeling studies on the crystal structure of ECF-PanT to rationalize their binding mode. Notably, docking studies showed that the acylhydrazone linker is able to maintain the crucial interactions.

Towards Translation of PqsR Inverse Agonists: From In Vitro Efficacy Optimization to In Vivo Proof-of-Principle.

Pseudomonas aeruginosa (PA) is an opportunistic human pathogen, which is involved in a wide range of dangerous infections. It develops alarming resistances toward antibiotic treatment. Therefore, alternative strategies, which suppress pathogenicity or synergize with antibiotic treatments are in great need to combat these infections more effectively. One promising approach is to disarm the bacteria by interfering with their quorum sensing (QS) system, which regulates the release of various virulence factors as well as biofilm formation. Herein, this work reports the rational design, optimization, and in-depth profiling of a new class of Pseudomonas quinolone signaling receptor (PqsR) inverse agonists. The resulting frontrunner compound features a pyrimidine-based scaffold, high in vitro and in vivo efficacy, favorable pharmacokinetics as well as clean safety pharmacology characteristics, which provide the basis for potential pulmonary as well as systemic routes of administration. An X-ray crystal structure in complex with PqsR facilitated further structure-guided lead optimization. The compound demonstrates potent pyocyanin suppression, synergizes with aminoglycoside antibiotic tobramycin against PA biofilms, and is active against a panel of clinical isolates from bronchiectasis patients. Importantly, this in vitro effect translated into in vivo efficacy in a neutropenic thigh infection model in mice providing a proof-of-principle for adjunctive treatment scenarios.

Development of (4-Phenylamino)quinazoline Alkylthiourea Derivatives as Novel NF-κB Inhibitors.

For many inflammatory diseases, new effective drugs with fewer side effects are needed. While it appears promising to target the activation of the central pro-inflammatory transcription factor NF-κB, many previously discovered agents suffered from cytotoxicity. In this study, new alkylthiourea quinazoline derivatives were developed that selectively inhibit the activation of NF-κB in macrophage-like THP-1 cells while showing low general cytotoxicity. One of the best compounds, 19, strongly inhibited the production of IL-6 (IC50 = 0.84 µM) and, less potently, of TNFα (IC50 = 4.0 µM); in comparison, the reference compound, caffeic acid phenethyl ester (CAPE), showed IC50s of 1.1 and 11.4 µM, respectively. Interestingly, 19 was found to block the translocation of the NF-κB dimer to the nucleus, although its release from the IκB complex was unaffected. Furthermore, 19 suppressed the phosphorylation of NF-κB-p65 at Ser468 but not at Ser536; however, 19 did not inhibit any kinase involved in NF-κB activation. The only partial suppression of p65 phosphorylation might be associated with fewer side effects. Since several compounds selectively induced cell death in activated macrophage-like THP-1 cells, they might be particularly effective in various inflammatory diseases that are exacerbated by excess activated macrophages, such as arteriosclerosis and autoimmune diseases.

From EGFR kinase inhibitors to anti-inflammatory drugs: Optimization and biological evaluation of (4-(phenylamino)quinazolinyl)-phenylthiourea derivatives as novel NF-κB inhibitors.

The transcription factor NF-κB is a pivotal mediator of chronic inflammatory and autoimmune diseases. Based on our previously published dual EGFR/NF-κB inhibitors, we designed and synthesized new thiourea quinazoline derivatives that retained only the NF-κB inhibitory activity. Several congeners displayed a strong suppression of NF-κB activity in a reporter gene assay, yet low cytotoxicity, and were further evaluated in differentiated macrophage-like THP-1 cells. The compounds exhibited a strong inhibition of IL-6 and, less potently, of TNFα release, which was accompanied by a selective induction of macrophage cell death. The mode of action was investigated with a selected inhibitor, 18, revealing that the translocation of p65/RelA to the nucleus but not its release from the IκB complex was inhibited. Eventually, 18 was identified as the first small molecule inhibitor affecting only the phosphorylation of p65-Ser468 but not of Ser536, which may be causally related to the retention of NF-κB in the cytoplasm. Altogether, our novel NF-κB inhibitors seem applicable for the suppression of cytokine release and the additional selective depletion of activated macrophages in various inflammatory diseases.

Structure-Guided Optimization of Small-Molecule Folate Uptake Inhibitors Targeting the Energy-Coupling Factor Transporters.

Here, we report on a potent class of substituted ureidothiophenes targeting energy-coupling factor (ECF) transporters, an unexplored target that is not addressed by any antibiotic in the market. Since the ECF module is crucial for the vitamin transport mechanism, the prevention of substrate uptake should ultimately lead to cell death. By utilizing a combination of virtual and functional whole-cell screening of our in-house library, the membrane-bound protein mediated uptake of folate could be effectively inhibited. Structure-based optimization of our hit yielded low-micromolar inhibitors, whereby the most active compounds showed in addition potent antimicrobial activities against a panel of clinically relevant Gram-positive pathogens without significant cytotoxic effects.

Design and Synthesis of Novel Bis-Imidazolyl Phenyl Butadiyne Derivatives as HCV NS5A Inhibitors.

In today's global plan to completely eradicate hepatitis C virus (HCV), the essential list of medications used for HCV treatment are direct-acting antivirals (DAAs), as interferon-sparing regimens have become the standard-of-care (SOC) treatment. HCV nonstructural protein 5A (NS5A) inhibitors are a very common component of these regimens. Food and Drug Administration (FDA)-approved NS5A inhibitors, although very potent, do not have the same potency against all eight genotypes of HCV. Therefore, this study aims to synthesize NS5A inhibitor analogues with high potency pan-genotypic activity and high metabolic stability. Starting from an NS5A inhibitor scaffold previously identified by our research group, we made several modifications. Two series of compounds were created to test the effect of changing the length and spatial conformation (para-para vs. meta-meta-positioned bis-imidazole-proline-carbamate), replacing amide groups in the linker with imidazole groups, as well as different end-cap compositions and sizes. The frontrunner inhibits genotype 1b (Con1) replicon, with an EC50 value in the picomolar range, and showed high genotypic coverage with nanomolar range EC50 values against four more genotypes. This together with its high metabolic stability (t½ > 120 min) makes it a potential preclinical candidate.

NanoTafla Nanocomposite as a Novel Low-Cost and Eco-Friendly Sorbent for Strontium and Europium Ions.

Now the wide use of nanooxides is attributed to their remarkable collection of properties. Nanocomposites have an impressive variety of important applications. A thermal decomposition approach provides a more optimistic method for nanocrystal synthesis due to the low cost, high efficiency, and expectations for large-scale production. Therefore, in this study a new eco-friendly nanooxide composite with sorption characteristics for europium (Eu(III)) and strontium (Sr(II)) was synthesized by a one-step thermal treatment process using earth-abundant tafla clay as a starting material to prepare a modified tafla (M-Taf) nanocomposite. The synthesized nancomposite was characterized by different techniques before and after sorption processes. Different factors that affected the sorption behavior of Eu(III) and Sr(II) in aqueous media by the M-Taf nanocomposite were studied. The results obtained illustrated that the kinetics of sorption of Eu(III) and Sr(II) by the M-Taf nanocomposite are obeyed according to the pseudo-second order and controlled by a Langmuir isotherm model with maximum sorption capacities (Q max) of 25.5 and 23.36 mg/g for Eu(III) and Sr(II), respectively. Also, this novel low-cost and eco-friendly sorbent has promising properties and can be used to separate and retain some radionuclides in different applications.

Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Modification of the benzenoid part.

Bacterial resistance to currently used antibiotics demands the development of novel antibacterial agents with good safety margins and sufficient efficacy against multi-drug resistant isolates. We have previously described the synthesis of N-butyl-2-(butylthio)quinazolin-4-amine (I) as an optimized hit with broad-spectrum antibacterial activity and low cytotoxicity. In addition, we have identified a potential growing vector for this series of compounds. Herein, we describe further hit optimization which includes systematic diversifications of both the benzenoid part and the substituents at position 6 and 7 of compound I. Growing of the molecule beside the core modifications yielded several compounds with remarkable anti(myco)bacterial activity against a panel of pathogenic bacteria, including drug-resistant strains. Compound 12 showed a 2-4 fold improvement in activity than I against S. aureus Newman, S. pneumoniae DSM-20566 and E. faecalis DSM-20478. The compounds also showed a good safety profile towards human HepG2 cells.

Targeting the IspD Enzyme in the MEP Pathway: Identification of a Novel Fragment Class.

The enzymes of the 2-C-methylerythritol-d-erythritol 4-phosphate (MEP) pathway (MEP pathway or non-mevalonate pathway) are responsible for the synthesis of universal precursors of the large and structurally diverse family of isoprenoids. This pathway is absent in humans, but present in many pathogenic organisms and plants, making it an attractive source of drug targets. Here, we present a high-throughput screening approach that led to the discovery of a novel fragment hit active against the third enzyme of the MEP pathway, PfIspD. A systematic SAR investigation afforded a novel chemical structure with a balanced activity-stability profile (16). Using a homology model of PfIspD, we proposed a putative binding mode for our newly identified inhibitors that sets the stage for structure-guided optimization.

Sorbent extraction behavior of cesium and strontium from nitric acid solutions using a new high thermal stability material.

In this study a new low-cost carbonaceous material was prepared from husks of opuntia-ficus-indica as a starting material (precursor) which was accomplished by chemical activation route using H3PO4 impregnation. The material has been identified by different analytical tools. The sorption performance of Cs(I) and Sr(II) from HNO3 solutions was examined through batch system. Variations of the distribution coefficients (Kd) as a function of HNO3 concentration in the range 0.001-5.0 M were presented. Some of separation probabilities were suggested. The results attained signals that the Sr(II) selectivity is higher than that of Cs(I) at high molarities. The retention capacity (qe) of Cs(I) and Sr(II) ions increased with growing temperature. The capacities at 0.001 M HNO3 are 34 and 108 mg/g for Cs(I) and Sr(II), respectively. Whereas, at 2.0 M HNO3 capacities were about 4 and 37 mg/g for each of Cs(I) and Sr (II), respectively. This studies demonstrates that the prepared carbonaceous sorbent is an economically effective sorbent for retention of Cs(I) and Sr(II) species from HNO3 solutions. Cs(I) and Sr(II) removal potential was tested from simulated low- and intermediate-level radioactive waste samples.

Redesigning of the cap conformation and symmetry of the diphenylethyne core to yield highly potent pan-genotypic NS5A inhibitors with high potency and high resistance barrier.

Herein, we report the discovery of several NS5A inhibitors with potency against HCV genotype 1b in the picomolar range. Compounds (15, 33) were of extremely high potency against HCV genotype 1b (EC50 ≈ 1 pM), improved activity against genotype 3a (GT 3a) and good metabolic stability. We studied the impact of changing the cap conformation relative to the diphenylethyne core and/or compound symmetry on both potency and metabolic stability. The analogs obtained exhibited improved potency against HCV genotypes 1a, 1b, 3a and 4a compared to the clinically approved candidate daclatasvir with EC50 values in the low picomolar range and SI50s > 7 orders of magnitude. Compound 15, a symmetrically m-, m'-substituted diphenyl ethyne analog, was 150-fold more potent than daclatasvir against GT 3a, while compound 33, an asymmetrically m-, p-substituted diphenyl ethyne analog, was 35-fold more potent than daclatasvir against GT 3a. In addition, compound 15 exhibited a higher resistance barrier than daclatasvir against genotype 1b.

Novel 2,4-disubstituted quinazoline analogs as antibacterial agents with improved cytotoxicity profile: Optimization of the 2,4-substituents.

The emergence of bacterial resistance has triggered a multitude of efforts to develop new antibacterial agents. There are many compounds in literature that were reported as potent antibacterial agents, however, they lacked the required safety to mammalian cells or no clear picture about their toxicity profile was presented. Inspired by discovered hit from our in-house library and by previously reported 2,4-diaminosubstituted quinazolines, we describe the design and synthesis of novel 2,4-disubstituted-thioquinazolines (3-13 and 36), 2-thio-4-amino substituted quinazolines (14-33) and 6-substituted 2,4-diamonsubstituted quinazolines (37-39). The synthesized compounds showed potent antibacterial activity against a panel of Gram-positive, efflux deficient E.coli and Mycobacterium smegmatis. The panel also involved resistant strains including methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis and vancomycin-resistant Enterococcus faecium, in addition to Mycobacterium smegmatis. The newly synthesized compounds revealed MIC values against the tested strains ranging from 1 to 64 µg/mL with a good safety profile. Most of the 2-thio-4-amino substituted-quinazolines showed significant antimycobacterial activity with the variations at position 2 and 4 offering additional antibacterial activity against the different strains. Compared to previously reported 2,4-diaminosubstituted quinazolines, the bioisosteric replacement of the 2-amino with sulfur offered a successful approach to keep the high antibacterial potency while substantially improving safety profile as indicated by the reduced activity on different cell lines and a lack of hemolytic activity.

Expanding the Myxochelin Natural Product Family by Nicotinic Acid Containing Congeners.

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation, and structure elucidation of three new myxochelins N1-N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing, and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.

Removal of strontium radionuclides from liquid scintillation waste and environmental water samples.

Strontium-90 (t1/2 = 29 y) is one of the most concerned isotopes in both nuclear accidents and reprocessing of nuclear fuel. In this study, the removal of strontium using low cost and valuable Dowex-HCR-S/S (DHS) resin was achieved. The kinetic and equilibrium sorption studies have been investigated using batch technique. The results of kinetic studies showed that the pseudo-second-order kinetic model was found to correlate well with the experimental data. Equilibrium data were also analyzed by sorption isotherm models indicating that the monolayer capacity of Sr(II) at equilibrium is 400.0 mg/g. It was concluded that resin has an efficient sorption capacity compared to many sorbents. The thermodynamic parameters of the removal (ΔHo, ΔSo, and ΔGo) were also determined. The removal process was endothermic and spontaneous. The resin has been successfully applied for the removal of 85Sr from organic liquid scintillator waste and some environmental waters such as tap water, river water, sea water and ground water samples. The present work concludes that the low-cost and commercial DHS resin used under these conditions has a major possibility as an efficacious material for the removal of 90Sr from environmental and real radioactive wastewaters. It can therefore have a site in the treatment of radioactive liquid waste because it is of an affordable and commercially available retention material.

Cystobactamid 507: Concise Synthesis, Mode of Action, and Optimization toward More Potent Antibiotics.

Lack of new antibiotics and increasing antimicrobial resistance are among the main concerns of healthcare communities nowadays, and these concerns necessitate the search for novel antibacterial agents. Recently, we discovered the cystobactamids-a novel natural class of antibiotics with broad-spectrum antibacterial activity. In this work, we describe 1) a concise total synthesis of cystobactamid 507, 2) the identification of the bioactive conformation using noncovalently bonded rigid analogues, and 3) the first structure-activity relationship (SAR) study for cystobactamid 507 leading to new analogues with high metabolic stability, superior topoisomerase IIA inhibition, antibacterial activity and, importantly, stability toward the resistant factor AlbD. Deeper insight into the mode of action revealed that the cystobactamids employ DNA minor-groove binding as part of the drug-target interaction without showing significant intercalation. By designing a new analogue of cystobactamid 919-2, we finally demonstrated that these findings could be further exploited to obtain more potent hexapeptides against Gram-negative bacteria.

Development of potential preclinical candidates with promising in vitro ADME profile for the inhibition of type 1 and type 2 17ß-Hydroxysteroid dehydrogenases: Design, synthesis, and biological evaluation.

Estrogens are the major female sex steroid hormones, estradiol (E2) being the most potent form in humans. Disturbing the balance between E2 and its weakly active oxidized form estrone (E1) leads to diverse types of estrogen-dependent diseases such as endometriosis or osteoporosis. 17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) catalyzes the biosynthesis of E2 by reduction of E1 while the type 2 enzyme catalyzes the reverse reaction. Thus, 17ß-HSD1 and 17ß-HSD2 are attractive targets for treatment of estrogen-dependent diseases. Recently, we reported the first proof-of-principle study of a 17ß-HSD2 inhibitor in a bone fracture mouse model, using subcutaneous administration. In the present study, our aim was to improve the in vitro ADME profile of the most potent 17ß-HSD1 and 17ß-HSD2 inhibitors described so far. The optimized compounds show strong and selective inhibition of both the human enzymes and their murine orthologs. In addition, they display good metabolic stability in human liver microsomes (S9 fraction), low in vitro cytotoxicity as well as better aqueous solubility and physicochemical properties compared to the lead compounds. These achievements make the compounds eligible for testing in preclinical in vivo animal model studies on the effects of inhibition of 17ß-HSD1 and 17ß-HSD2.

Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high in vivo efficacy.

There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 µg mL-1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 µg mL-1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.