A New PqsR Inverse Agonist Potentiates Tobramycin Efficacy to Eradicate Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa (PA) infections can be notoriously difficult to treat and are often accompanied by the development of antimicrobial resistance (AMR). Quorum sensing inhibitors (QSI) acting on PqsR (MvfR) - a crucial transcriptional regulator serving major functions in PA virulence - can enhance antibiotic efficacy and eventually prevent the AMR. An integrated drug discovery campaign including design, medicinal chemistry-driven hit-to-lead optimization and in-depth biological profiling of a new QSI generation is reported. The QSI possess excellent activity in inhibiting pyocyanin production and PqsR reporter-gene with IC50 values as low as 200 and 11 × 10-9 m, respectively. Drug metabolism and pharmacokinetics (DMPK) as well as safety pharmacology studies especially highlight the promising translational properties of the lead QSI for pulmonary applications. Moreover, target engagement of the lead QSI is shown in a PA mucoid lung infection mouse model. Beyond that, a significant synergistic effect of a QSI-tobramycin (Tob) combination against PA biofilms using a tailor-made squalene-derived nanoparticle (NP) formulation, which enhance the minimum biofilm eradicating concentration (MBEC) of Tob more than 32-fold is demonstrated. The novel lead QSI and the accompanying NP formulation highlight the potential of adjunctive pathoblocker-mediated therapy against PA infections opening up avenues for preclinical development.

Flexible Fragment Growing Boosts Potency of Quorum-Sensing Inhibitors against Pseudomonas aeruginosa Virulence.

Hit-to-lead optimization is a critical phase in drug discovery. Herein, we report on the fragment-based discovery and optimization of 2-aminopyridine derivatives as a novel lead-like structure for the treatment of the dangerous opportunistic pathogen Pseudomonas aeruginosa. We pursue an innovative treatment strategy by interfering with the Pseudomonas quinolone signal (PQS) quorum sensing (QS) system leading to an abolishment of bacterial pathogenicity. Our compounds act on the PQS receptor (PqsR), a key transcription factor controlling the expression of various pathogenicity determinants. In this target-driven approach, we made use of biophysical screening via surface plasmon resonance (SPR) followed by isothermal titration calorimetry (ITC)-enabled enthalpic efficiency (EE) evaluation. Hit optimization then involved growth vector identification and exploitation. Astonishingly, the latter was successfully achieved by introducing flexible linkers rather than rigid motifs leading to a boost in activity on the target receptor and anti-virulence potency.

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.

Targeted Endocrine Therapy: Design, Synthesis, and Proof-of-Principle of 17ß-Hydroxysteroid Dehydrogenase Type 2 Inhibitors in Bone Fracture Healing.

Current therapies of steroid hormone-dependent diseases predominantly alter steroid hormone concentrations (or their actions) in plasma, in target and nontarget tissues alike, rather than in target organs only. Targeted therapy through the inhibition of steroidogenic enzymes may pose an attractive alternative with much less side effects. Here, we describe the design of a nanomolar potent 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) inhibitor (compound 15) and successful targeted intracrine therapy in a mouse bone fracture model. Blockade of 17ß-HSD2 in bone is thought to increase intracellular estradiol (E2) and testosterone (T), which thereby inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts, respectively. Administration of compound 15 in the mouse fracture model strongly increases the mechanical stability of the healing fractured bone because of a larger periosteal callus with newly formed bone without changing the plasma E2 and T concentrations. Steroidogenic 17ß-HSD2 inhibition thus enables targeted intracrine therapy.

Human airway mucus alters susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, but not colistin.

Objectives: In the context of cystic fibrosis, Pseudomonas aeruginosa biofilms often develop in the vicinity of airway mucus, which acts as a protective physical barrier to inhaled matter. However, mucus can also adsorb small drug molecules administered as aerosols, including antibiotics, thereby reducing their bioavailability. The efficacy of antibiotics is typically assessed by determining the MIC using in vitro assays. This widespread technique, however, does not consider either bacterial biofilm formation or the influence of mucus, both of which may act as diffusion barriers, potentially limiting antibiotic efficacy. Methods: We grew P. aeruginosa biofilms in the presence or absence of human tracheal mucus and tested their susceptibility to tobramycin and colistin. Results: A significant reduction of tobramycin efficacy was observed when P. aeruginosa biofilms were grown in the presence of mucus compared with those grown in the absence of mucus. Diffusion of tobramycin through mucus was reduced; however, this reduction was more pronounced in biofilm/mucus mixtures, suggesting that biofilms in the presence of mucus respond differently to antibiotic treatment. In contrast, the influence of mucus on colistin efficacy was almost negligible and no differences in mucus permeability were observed. Conclusions: These findings underline the important role of mucus in the efficacy of anti-infective drugs.

Synthesis of 1,1'- and 2,2'-Bicarbazole Alkaloids by Iron(III)-Catalyzed Oxidative Coupling of 2- and 1-Hydroxycarbazoles.

We describe the synthesis of 1,1'- and 2,2'-bicarbazoles by oxidative homocoupling of 2- and 1-hydroxycarbazoles. The oxidative coupling using catalytic amounts of F16 PcFe can be applied to both groups of substrates. Although F16 PcFe generally provides the best yields for the synthesis of 1,1'-bicarbazoles, di-tert-butyl peroxide affords better results for the 2,2'-bicarbazoles. In our study, we have achieved the first syntheses of the biscarbalexines A-C, bisglybomine B, 2,2'-dihydroxy-7,7'-dimethoxy-3,3'-dimethyl-1,1'-bicarbazole, bispyrayafoline C, and bisisomahanine. The iron-catalyzed coupling of koenigine led to an improved synthesis of 8,8''-biskoenigine and afforded an unprecedented decacylic product. Oxidative coupling of 1-hydroxycarbazoles led to bisclausenol, and to the first total syntheses of bismurrayafoline B and D.

Quantitative Structure-Retention Relationships for Polycyclic Aromatic Hydrocarbons and their Oligoalkynyl-Substituted Derivatives.

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been carried out for a series of unsubstituted polycyclic aromatic hydrocarbons (PAHs) and the corresponding ethynyl, 1,3-butadiynyl, and 1,3,5-hexatriynyl derivatives. Theoretical values of the isotropic polarizability and several polarity descriptors have been computed for each compound by using semiempirical models and density functional theory (DFT), with the aim of evaluating linear functions as quantitative structure-retention relationships (QSRRs). The polarity has been described by using either the permanent electric dipole moment, the subpolarity, or a topological electronic index. Three types of partial atomic charges have been used to calculate the subpolarity and a topological index. The choice of the theoretical model, of the polarity descriptor, and of the partial atomic charges is discussed and the resulting QSRRs are compared. Calculating the retention times from the polarizability and the topological electronic index (AM1, PM3, or DFT-B3LYP/6-31+G(d,p)) gives the best agreement with the experimental values.

Anti-tuberculosis activity and structure-activity relationships of oxygenated tricyclic carbazole alkaloids and synthetic derivatives.

A series of 49 oxygenated tricyclic carbazole derivatives has been tested for inhibition of the growth of Mycobacterium tuberculosis and a mammalian cell line (vero cells). From this series, twelve carbazoles showed a significant anti-TB activity. The four most active compounds were the naturally occurring carbazole alkaloids clauszoline-M (45), murrayaline-C (41), carbalexin-C (27), and the synthetic carbazole derivative 22 with MIC90 values ranging from 1.5 to 3.7µM. The active compounds were virtually nontoxic for the mammalian cell line in the concentration range up to 50µM.

Treatment of estrogen-dependent diseases: Design, synthesis and profiling of a selective 17ß-HSD1 inhibitor with sub-nanomolar IC50 for a proof-of-principle study.

Current endocrine therapeutics for the estrogen-dependent disease endometriosis often lead to considerable side-effects as they act by reducing estrogen action systemically. A more recent approach takes advantage of the fact that the weak estrogen estrone (E1) which is abundant in the plasma, is activated in the target cell to the highly estrogenic estradiol (E2) by 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1). 17ß-HSD1 is overexpressed in endometriosis and thus a promising target for the treatment of this disease, with the prospect of less target-associated side-effects. Potent inhibitors from the class of bicyclic substituted hydroxyphenylmethanones with sulfonamide moiety recently described by us suffered from high molecular weight and low selectivity over 17ßHSD2, the physiological adversary of 17ß-HSD1. We describe the structural optimizations leading to the discovery of (5-(3,5-dichloro-4-methoxyphenyl)thiophen-2-yl)(2,6-difluoro-3-hydroxyphenyl)methanone 20, which displayed a sub-nanomolar IC50 towards 17ß-HSD1 as well as high selectivity over the type 2 enzyme, the estrogen receptors α and ß and a range of hepatic CYP enzymes. The compound did neither show cellular toxicity, nor PXR activation nor mutagenicity in the AMES II assay. Additional favourable pharmacokinetic properties (rat) make 20 a suitable candidate for proof-of-principle studies using xenotransplanted immunodeficient rats.

Structure-Activity Relationships of 2-Sufonylpyrimidines as Quorum-Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa.

Drug-resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS-QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure-activity relationships of 2-sulfonylpyrimidines, which were previously identified as dual-target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.

Synthesis of Methylene-Bridged Biscarbazole Alkaloids by using an Ullmann-type Coupling: First Total Synthesis of Murrastifoline-C and Murrafoline-E.

We describe the total synthesis of methylene-bridged biscarbazole alkaloids by using a late-stage Ullmann-type coupling of fully functionalised carbazole subunits. The carbazole derivatives were synthesised via a sequence of palladium(0)- and palladium(II)-catalysed coupling reactions. Our approach has provided bismurrayafoline-A, bismurrayafolinol, chrestifolines B-D, and the first total synthesis of murrastifoline-C and murrafoline-E.

Exploring the chemical space of ureidothiophene-2-carboxylic acids as inhibitors of the quorum sensing enzyme PqsD from Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs a quorum sensing (QS) communication system that makes use of small diffusible molecules. Among other effects, the QS system coordinates the formation of biofilm which decisively contributes to difficulties in the therapy of Pseudomonas infections. The present work deals with the structure-activity exploration of ureidothiophene-2-carboxylic acids as inhibitors of PqsD, a key enzyme in the biosynthetic pathway of signal molecules in the Pseudomonas QS system. We describe an improvement of the inhibitory activity by successfully combining features from two different PqsD inhibitor classes. Furthermore the functional groups, which are responsible for the inhibitory potency, were identified. Moreover, the inability of the new inhibitors, to prevent signal molecule formation in whole cell assays, is discussed.

First total syntheses of chrestifoline-B and (±)-chrestifoline-C, and improved synthetic routes to bismurrayafoline-A, bismurrayafolinol and chrestifoline-D.

We describe an efficient synthesis of the methylene-bridged biscarbazole alkaloids bismurrayafoline-A, bismurrayafolinol and chrestifoline B-D using an Ullmann-type coupling at the benzylic position.

Novel approach to biscarbazole alkaloids via Ullmann coupling--synthesis of murrastifoline-A and bismurrayafoline-A.

Unprecedented Ullmann couplings of murrayafoline-A with either 6-bromo- or 4-bromocarbazole derivatives provide highly efficient synthetic routes to the biscarbazole alkaloids murrastifoline-A (6 steps, 66% overall yield) and bismurrayafoline-A (6 steps, 28% overall yield).

First total synthesis of the biscarbazole alkaloid oxydimurrayafoline.

We report the first total synthesis of oxydimurrayafoline via nucleophilic substitution at the benzylic position at C-3 of the carbazole framework.