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.

Inhibitors of the Elastase LasB for the Treatment of Pseudomonas aeruginosa Lung Infections.

Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa are emerging worldwide as a major threat to human health. Conventional antibiotic monotherapy suffers from rapid resistance development, underlining urgent need for novel treatment concepts. Here, we report on a nontraditional approach to combat P. aeruginosa-derived infections by targeting its main virulence factor, the elastase LasB. We discovered a new chemical class of phosphonates with an outstanding in vitro ADMET and PK profile, auspicious activity both in vitro and in vivo. We established the mode of action through a cocrystal structure of our lead compound with LasB and in several in vitro and ex vivo models. The proof of concept of a combination of our pathoblocker with levofloxacin in a murine neutropenic lung infection model and the reduction of LasB protein levels in blood as a proof of target engagement demonstrate the great potential for use as an adjunctive treatment of lung infections in humans.

Modeling the Effect of Hydrophobicity on the Passive Permeation of Solutes across a Bacterial Model Membrane.

Passive diffusion across biomembranes is an important mechanism of permeation for multiple drugs, including antibiotics. However, this process is frequently neglected while studying drug uptake and, in our view, warrants further investigation. Here, we apply molecular dynamics simulations to investigate the impact of changes in molecular hydrophobicity on the permeability of a series of inhibitors of the quorum sensing of Pseudomonas aeruginosa, previously discovered by us, across a membrane model. Overall, we show that permeation across this membrane model does not correlate with the molecule's hydrophobicity. We demonstrate that using a simple model for permeation, based on the difference between the maximum and minimum of the free energy profile, outperforms the inhomogeneous solubility-diffusion model, yielding a permeability ranking that better agrees with the experimental results, especially for hydrophobic permeants. The calculated differences in permeability could not explain differences in in bacterio activity. Nevertheless, substantial differences in molecular orientation along the permeation pathway correlate with the in bacterio activity, emphasizing the importance of analyzing, at an atomistic level, the permeation pathway of these solutes.

Structure-Based Design of α-Substituted Mercaptoacetamides as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa.

Antivirulence therapy has become a widely applicable method for fighting infections caused by multidrug-resistant bacteria. Among the many virulence factors produced by the Gram-negative bacterium Pseudomonas aeruginosa, elastase (LasB) stands out as an important target as it plays a pivotal role in the invasion of the host tissue and evasion of the immune response. In this work, we explored the recently reported LasB inhibitor class of α-benzyl-N-aryl mercaptoacetamides by exploiting the crystal structure of one of the compounds. Our exploration yielded inhibitors that maintained inhibitory activity, selectivity, and increased hydrophilicity. These inhibitors were found to reduce the pathogenicity of the bacteria and to maintain the integrity of lung and skin cells in the diseased state. Furthermore, two most promising compounds increased the survival rate of Galleria mellonella larvae treated with P. aeruginosa culture supernatant.

N-Aryl Mercaptopropionamides as Broad-Spectrum Inhibitors of Metallo-ß-Lactamases.

Drug-resistant pathogens pose a global challenge to public health as they cause diseases that are extremely difficult to cure. Metallo-ß-lactamases (MBLs) are a diverse set of zinc-containing enzymes that catalyze the hydrolysis of ß-lactam drugs, including carbapenems, which are considered as the last resort to fight severe infections. To restore the activity of current ß-lactam antibiotics and to offer an orthogonal strategy to the discovery of new antibiotics, we have identified a series of polar N-aryl mercaptopropionamide derivatives as potent inhibitors of several class B1 MBLs. We have identified a hit structure with high selectivity restoring the effect of imipenem and reducing minimum inhibitory concentration (MIC) values up to 256-fold in resistant isolates from Escherichia coli. Furthermore, the combination of imipenem with our inhibitor showed in vivo efficacy in a Galleria mellonella model, increasing the survival rate of infected larvae by up to 31%.

Metabolic Profiling of S-praziquantel: Structure Elucidation Using the Crystalline Sponge Method in Combination with Mass Spectrometry and Nuclear Magnetic Resonance.

Praziquantel (PZQ) is the drug of choice for treatment of the neglected tropical disease schistosomiasis. Although the drug has been extensively used over several decades and its metabolism well studied (several oxidative metabolites are known from literature), the knowledge of the complete structure of some of its metabolites remains elusive. Conventional techniques, such as nuclear magnetic resonance or liquid chromatography mass spectrometry were used in the past to investigate phase I and phase II metabolites of PZQ. These techniques are either limited to provide the complete molecular structure (liquid chromatography mass spectrometry) or require large amount of sample material (NMR), which are not always available when in vitro systems are used for investigation of the metabolites. In this study, we describe new structures of S-PZQ metabolites generated in vitro from human liver microsomes using the crystalline sponge method. After chromatographic separation and purification of the oxidative metabolites, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis was conducted to narrow down the position of oxidation to a certain part of the molecule. To determine the exact position of hydroxylation, singe-crystal X-ray diffraction analysis of the crystalline sponges and absorbed analyte was used to identify the structure of S-PZQ and its metabolites. The crystalline sponge method allowed for complete structure elucidation of the known metabolites S-trans-4'-hydroxy-PZQ (M1), S-cis-4'-hydroxy-PZQ (M2) and S-/R-11b-hydroxy-PZQ (M6) as well as the unknown metabolites S-9-hydroxy-PZQ (M3) and S-7-hydroxy-S-PZQ (M4). For comparison of structural elucidation techniques, one metabolite (M3) was additionally analyzed using NMR. SIGNIFICANCE STATEMENT: The information content of the metabolic pathway of praziquantel is still limited. The crystalline sponge method allowed the complete structural elucidation of three known and two unknown metabolites of S-praziquantel, using only trace amounts of analyte material, as demonstrated in this study.

Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors.

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a sub-micromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates.

17ß-Hydroxysteroid Dehydrogenase Type 1 Inhibition: A Potential Treatment Option for Non-Small Cell Lung Cancer.

In the face of the clinical challenge posed by non-small cell lung cancer (NSCLC), the present need for new therapeutic approaches is genuine. Up to now, no proof existed that 17ß-hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is a viable target for treating this disease. Synthesis of a rationally designed library of 2,5-disubstituted furan derivatives followed by biological screening led to the discovery of 17ß-HSD1 inhibitor 1, capable of fully inhibiting human NSCLC Calu-1 cell proliferation. Its pharmacological profile renders it eligible for further in vivo studies. The very high selectivity of 1 over 17ß-HSD2 was investigated, revealing a rational approach for the design of selective inhibitors. 17ß-HSD1 and 1 hold promise in fighting NSCLC.

Crystalline sponge affinity screening: A fast tool for soaking condition optimization without the need of X-ray diffraction analysis.

Structural elucidation of small molecules only available in low quantity (nanogram) is one of the big advantages of the crystalline sponge method. The optimization of various soaking parameters is crucial for effective analyte absorption and repetitive positioning in the pores of the crystal. Time-consuming X-ray diffraction measurements are necessary for data collection and confirmation of successful guest inclusion. In this work, we report a screening method to select optimal soaking conditions without the need of single-crystal X-ray diffraction analysis for individual compounds and mixtures. 14 substances were chosen as test compounds. Parallel guest soaking of individual compounds and mixtures was conducted using various soaking conditions. After evaporation of solvent, excessive material was removed, and guest molecules released through dissolution of the framework. Liquid chromatography-tandem mass spectrometry allowed the estimation of analyte trapped in the pores and the selection of optimal soaking condition dependent on the highest amount of analyte to crystal size (affinity factor). The tool allowed subsequent crystallographic analysis of ten compounds with minimal experiment time. Additionally, a study to examine the lower limit of detection of the crystalline sponge method was conducted. Determination of two target analytes was possible using only 5 ng of sample. Our study shows the potential of an affinity screening to prioritize soaking parameters by estimation of the guest concentration in a single crystal for one or multiple target compounds within a short period of time.

Structure-Activity Relationship and Mode-Of-Action Studies Highlight 1-(4-Biphenylylmethyl)-1H-imidazole-Derived Small Molecules as Potent CYP121 Inhibitors.

CYP121 of Mycobacterium tuberculosis (Mtb) is an essential target for the development of novel potent drugs against tuberculosis (TB). Besides known antifungal azoles, further compounds of the azole class were recently identified as CYP121 inhibitors with antimycobacterial activity. Herein, we report the screening of a similarity-oriented library based on the former hit compound, the evaluation of affinity toward CYP121, and activity against M. bovis BCG. The results enabled a comprehensive SAR study, which was extended through the synthesis of promising compounds and led to the identification of favorable features for affinity and/or activity and hit compounds with 2.7-fold improved potency. Mode of action studies show that the hit compounds inhibit substrate conversion and highlighted CYP121 as the main antimycobacterial target of our compounds. Exemplified complex crystal structures of CYP121 with three inhibitors reveal a common binding site. Engaging in both hydrophobic interactions as well as hydrogen bonding to the sixth iron ligand, our compounds block a solvent channel leading to the active site heme. Additionally, we report the first CYP inhibitors that are able to reduce the intracellular replication of M. bovis BCG in macrophages, emphasizing their potential as future drug candidates against TB.

From Celecoxib to a Novel Class of Phosphodiesterase 5 Inhibitors: Trisubstituted Pyrazolines as Novel Phosphodiesterase 5 Inhibitors with Extremely High Potency and Phosphodiesterase Isozyme Selectivity.

A ligand-based approach involving systematic modifications of a trisubstituted pyrazoline scaffold derived from the COX2 inhibitor, celecoxib, was used to develop novel PDE5 inhibitors. Novel pyrazolines were identified with potent PDE5 inhibitory activity lacking COX2 inhibitory activity. Compound d12 was the most potent with an IC50 of 1 nM, which was three times more potent than sildenafil and more selective with a selectivity index of >10,000-fold against all other PDE isozymes. Sildenafil inhibited the full-length and catalytic fragment of PDE5, while compound d12 only inhibited the full-length enzyme, suggesting a mechanism of enzyme inhibition distinct from sildenafil. The PDE5 inhibitory activity of compound d12 was confirmed in cells using a cGMP biosensor assay. Oral administration of compound d12 achieved plasma levels >1000-fold higher than IC50 values and showed no discernable toxicity after repeated dosing. These results reveal a novel strategy to inhibit PDE5 with unprecedented potency and isozyme selectivity.

Phosphonate as a Stable Zinc-Binding Group for "Pathoblocker" Inhibitors of Clostridial Collagenase H (ColH).

Microbial infections are a significant threat to public health, and resistance is on the rise, so new antibiotics with novel modes of action are urgently needed. The extracellular zinc metalloprotease collagenase H (ColH) from Clostridium histolyticum is a virulence factor that catalyses tissue damage, leading to improved host invasion and colonisation. Besides the major role of ColH in pathogenicity, its extracellular localisation makes it a highly attractive target for the development of new antivirulence agents. Previously, we had found that a highly selective and potent thiol prodrug (with a hydrolytically cleavable thiocarbamate unit) provided efficient ColH inhibition. We now report the synthesis and biological evaluation of a range of zinc-binding group (ZBG) variants of this thiol-derived inhibitor, with the mercapto unit being replaced by other zinc ligands. Among these, an analogue with a phosphonate motif as ZBG showed promising activity against ColH, an improved selectivity profile, and significantly higher stability than the thiol reference compound, thus making it an attractive candidate for future drug development.

Homology modeling meets site-directed mutagenesis: An ideal combination to elucidate the topology of 17ß-HSD2.

17ß-Hydroxysteroid dehydrogenase type 2 (17ß-HSD2) catalyzes the conversion of highly active estrogens and androgens into their less active forms using NAD+ as cofactor. Substrate and cofactor specificities of 17ß-HSD2 have been reported and potent 17ß-HSD2 inhibitors have been discovered in a ligand-based approach. However, the molecular basis and the amino acids involved in the enzymatic functionality are poorly understood, as no crystal structure of the membrane-associated 17ß-HSD2 exists. The functional properties of only few amino acids are known. The lack of topological information impedes structure-based drug design studies and limits the design of biochemical experiments. The aim of this work was the determination of the 17ß-HSD2 topology. For this, the first homology model of 17ß-HSD2 in complex with NAD+ and 17ß-estradiol was built, using a multi-fragment "patchwork" approach. To confirm the quality of the model, fifteen selected amino acids were exchanged one by one using site directed mutagenesis. The mutants' functional behavior demonstrated that the generated model was of very good quality and allowed the identification of several key amino acids involved in either ligand or internal structure stabilization. The final model is an optimal basis for further experiments like, for example, lead optimization.

A hydrogel-based in vitro assay for the fast prediction of antibiotic accumulation in Gram-negative bacteria.

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria.

Discovery of trisubstituted pyrazolines as a novel scaffold for the development of selective phosphodiesterase 5 inhibitors.

Celecoxib, is a selective cyclooxygenase-2 (COX2) inhibitor with a 1,5-diaryl pyrazole scaffold. Celecoxib has a better safety profile compared to other COX2 inhibitors having side effects of systemic hypertension and thromboembolic complications. This may be partly attributed to an off-target activity involving phosphodiesterase 5 (PDE5) inhibition and the potentiation of NO/cGMP signalling allowing coronary vasodilation and aortic relaxation. Inspired by the structure of celecoxib, we synthesized a chemically diverse series of compounds containing a 1,3,5-trisubstituted pyrazoline scaffold to improve PDE5 inhibitory potency, while eliminating COX2 inhibitory activity. SAR studies for PDE5 inhibition revealed an essential role for a carboxylic acid functionality at the 1-phenyl and the importance of the non-planar pyrazoline core over the planar pyrazole with the 5-phenyl moiety tolerating a range of substituents. These modifications led to new PDE5 inhibitors with approximately 20-fold improved potency to inhibit PDE5 and no COX-2 inhibitory activity compared with celecoxib. PDE isozyme profiling of compound 11 revealed a favorable selectivity profile. These results suggest that trisubstituted pyrazolines provide a promising scaffold for further chemical optimization to identify novel PDE5 inhibitors with potential for less side effects compared with available PDE5 inhibitors used for the treatment of penile erectile dysfunction and pulmonary hypertension.

Micro-rheological properties of lung homogenates correlate with infection severity in a mouse model of Pseudomonas aeruginosa lung infection.

Lung infections caused by Pseudomonas aeruginosa pose a serious threat to patients suffering from, among others, cystic fibrosis, chronic obstructive pulmonary disease, or bronchiectasis, often leading to life-threatening complications. The establishment of a chronic infection is substantially related to communication between bacteria via quorum-sensing networks. In this study, we aimed to assess the role of quorum-sensing signaling molecules of the Pseudomonas quinolone signal (PQS) and to investigate the viscoelastic properties of lung tissue homogenates of PA-infected mice in a prolonged acute murine infection model. Therefore, a murine infection model was successfully established via intra-tracheal infection with alginate-supplemented Pseudomonas aeruginosa NH57388A. Rheological properties of lung homogenates were analyzed with multiple particle tracking (MPT) and quorum-sensing molecules were quantified with LC-MS/MS. Statistical analysis of bacterial load and quorum-sensing molecules showed a strong correlation between these biomarkers in infected lungs. This was accompanied by noticeable changes in the consistency of lung homogenates with increasing infection severity. Furthermore, viscoelastic properties of the lung homogenates strongly correlated with bacterial load and quorum sensing molecules. Considering the strong correlation between the viscoelasticity of lung homogenates and the aforementioned biomarkers, the viscoelastic properties of infected lungs might serve as reliable new biomarker for the evaluation of the severity of P. aeruginosa infections in murine models.

Finding New Molecular Targets of Familiar Natural Products Using In Silico Target Prediction.

Natural products comprise a rich reservoir for innovative drug leads and are a constant source of bioactive compounds. To find pharmacological targets for new or already known natural products using modern computer-aided methods is a current endeavor in drug discovery. Nature's treasures, however, could be used more effectively. Yet, reliable pipelines for the large-scale target prediction of natural products are still rare. We developed an in silico workflow consisting of four independent, stand-alone target prediction tools and evaluated its performance on dihydrochalcones (DHCs)-a well-known class of natural products. Thereby, we revealed four previously unreported protein targets for DHCs, namely 5-lipoxygenase, cyclooxygenase-1, 17ß-hydroxysteroid dehydrogenase 3, and aldo-keto reductase 1C3. Moreover, we provide a thorough strategy on how to perform computational target predictions and guidance on using the respective tools.

Evaluation of Bacterial RNA Polymerase Inhibitors in a Staphylococcus aureus-Based Wound Infection Model in SKH1 Mice.

Chronic wounds infected with pathogens such as Staphylococcus aureus represent a worldwide health concern, especially in patients with a compromised immune system. As antimicrobial resistance has become an immense global problem, novel antibiotics are urgently needed. One strategy to overcome this threatening situation is the search for drugs targeting novel binding sites on essential and validated enzymes such as the bacterial RNA polymerase (RNAP). In this work, we describe the establishment of an in vivo wound infection model based on the pathogen S. aureus and hairless Crl:SKH1-Hrhr (SKH1) mice. The model proved to be a valuable preclinical tool to study selected RNAP inhibitors after topical application. While rifampicin showed a reduction in the loss of body weight induced by the bacteria, an acceleration of wound healing kinetics, and a reduced number of colony forming units in the wound, the ureidothiophene-2-carboxylic acid 1 was inactive under in vivo conditions, probably due to strong plasma protein binding. The cocrystal structure of compound 1 with RNAP, that we hereby also present, will be of great value for applying appropriate structural modifications to further optimize the compound, especially in terms of plasma protein binding.

Synthesis, Optimization, Antifungal Activity, Selectivity, and CYP51 Binding of New 2-Aryl-3-azolyl-1-indolyl-propan-2-ols.

A series of 2-aryl-3-azolyl-1-indolyl-propan-2-ols was designed as new analogs of fluconazole (FLC) by replacing one of its two triazole moieties by an indole scaffold. Two different chemical approaches were then developed. The first one, in seven steps, involved the synthesis of the key intermediate 1-(1H-benzotriazol-1-yl)methyl-1H-indole and the final opening of oxiranes by imidazole or 1H-1,2,4-triazole. The second route allowed access to the target compounds in only three steps, this time with the ring opening by indole and analogs. Twenty azole derivatives were tested against Candida albicans and other Candida species. The enantiomers of the best anti-Candida compound, 2-(2,4-dichlorophenyl)-3-(1H-indol-1-yl)-1-(1H-1,2,4-triazol-1-yl)-propan-2-ol (8g), were analyzed by X-ray diffraction to determine their absolute configuration. The (-)-8g enantiomer (Minimum inhibitory concentration (MIC) = IC80 = 0.000256 µg/mL on C. albicans CA98001) was found with the S-absolute configuration. In contrast the (+)-8g enantiomer was found with the R-absolute configuration (MIC = 0.023 µg/mL on C. albicans CA98001). By comparison, the MIC value for FLC was determined as 0.020 µg/mL for the same clinical isolate. Additionally, molecular docking calculations and molecular dynamics simulations were carried out using a crystal structure of Candida albicans lanosterol 14α-demethylase (CaCYP51). The (-)-(S)-8g enantiomer aligned with the positioning of posaconazole within both the heme and access channel binding sites, which was consistent with its biological results. All target compounds have been also studied against human fetal lung fibroblast (MRC-5) cells. Finally, the selectivity of four compounds on a panel of human P450-dependent enzymes (CYP19, CYP17, CYP26A1, CYP11B1, and CYP11B2) was investigated.

Crystalline Sponges as a Sensitive and Fast Method for Metabolite Identification: Application to Gemfibrozil and its Phase I and II Metabolites.

Understanding the metabolism of new drug candidates is important during drug discovery and development, as circulating metabolites may contribute to efficacy or cause safety issues. In the early phase of drug discovery, human in vitro systems are used to investigate human relevant metabolism. Though conventional techniques are limited in their ability to provide complete molecular structures of metabolites (liquid chromatography mass spectrometry) or require a larger amount of material not available from in vitro incubation (nuclear magnetic resonance), we here report for the first time the use of the crystalline sponge method to identify phase I and phase II metabolites generated from in vitro liver microsomes or S9 fractions. Gemfibrozil was used as a test compound. Metabolites generated from incubation with microsomes or S9 fractions, were fractionated using online fraction collection. After chromatographic purification and fractionation of the generated metabolites, single crystal X-ray diffraction of crystalline sponges was used to identify the structure of gemfibrozil metabolites. This technique allowed for complete structure elucidation of 5'-CH2OH gemfibrozil (M1), 4'-OH gemfibrozil (M2), 5'-COOH gemfibrozil (M3), and the acyl glucuronide of gemfibrozil, 1-O-ß-glucuronide (M4), the first acyl glucuronide available in the Cambridge Crystallographic Data Centre. Our study shows that when optimal soaking is possible, crystalline sponges technology is a sensitive (nanogram amount) and fast (few days) method that can be applied early in drug discovery to identify the structure of pure metabolites from in vitro incubations. SIGNIFICANCE STATEMENT: Complete structure elucidation of human metabolites plays a critical role in early drug discovery. Low amounts of material (nanogram) are only available at this stage and insufficient for nuclear magnetic resonance analysis. The crystalline sponge method has the potential to close this gap, as demonstrated in this study.