Target repurposing unravels avermectins and derivatives as novel antibiotics inhibiting energy-coupling factor transporters (ECFTs).

Energy-coupling factor transporters (ECFTs) are membrane-bound ATP-binding cassette (ABC) transporters in prokaryotes that are found in pathogens against which novel antibiotics are urgently needed. To date, just 54 inhibitors of three molecular-structural classes with mostly weak inhibitory activity are known. Target repurposing is a strategy that transfers knowledge gained from a well-studied protein family to under-studied targets of phylogenetic relation. Forty-eight human ABC transporters are known that may harbor structural motifs similar to ECFTs to which particularly multitarget compounds may bind. We assessed 31 multitarget compounds which together target the entire druggable human ABC transporter proteome against ECFTs, of which nine showed inhibitory activity (hit rate 29.0%) and four demonstrated moderate to strong inhibition of an ECFT (IC50 values between 4.28 and 50.2 µM) as well as antibacterial activity against ECFT-expressing Streptococcus pneumoniae. Here, ivermectin was the most potent candidate (MIC95: 22.8 µM), and analysis of five ivermectin derivatives revealed moxidectin as one of the most potent ECFT-targeting antibacterial agents (IC50: 2.23 µM; MIC95: 2.91 µM). Distinct molecular-structural features of avermectins and derivatives as well as the differential biological response of the hit compounds in general provided first indications with respect to the structure-activity relationships and mode of action, respectively.

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.

Clean Synthetic Strategies to Biologically Active Molecules from Lignin: A Green Path to Drug Discovery.

Deriving active pharmaceutical agents from renewable resources is crucial to increasing the economic feasibility of modern biorefineries and promises to alleviate critical supply-chain dependencies in pharma manufacturing. Our multidisciplinary approach combines research in lignin-first biorefining, sustainable catalysis, and alternative solvents with bioactivity screening, an in vivo efficacy study, and a structural-similarity search. The resulting sustainable path to novel anti-infective, anti-inflammatory, and anticancer molecules enabled the rapid identification of frontrunners for key therapeutic indications, including an anti-infective against the priority pathogen Streptococcus pneumoniae with efficacy in vivo and promising plasma and metabolic stability. Our catalytic methods provided straightforward access, inspired by the innate structural features of lignin, to synthetically challenging biologically active molecules with the core structure of dopamine, namely, tetrahydroisoquinolines, quinazolinones, 3-arylindoles and the natural product tetrahydropapaveroline. Our diverse array of atom-economic transformations produces only harmless side products and uses benign reaction media, such as tunable deep eutectic solvents for modulating reactivity in challenging cyclization steps.

Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design.

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor.

Synthesis, biological evaluation, and molecular docking studies of aldotetronic acid-based LpxC inhibitors.

In order to develop novel inhibitors of the bacterial deacetylase LpxC bearing a substituent to target the UDP binding site of the enzyme, a series of aldotetronic acid-based hydroxamic acids was accessed in chiral pool syntheses starting from 4,6-O-benzylidene-d-glucose and l-arabinitol. The synthesized hydroxamic acids were tested for LpxC inhibitory activity in vitro, revealing benzyl ether 17a ((2S,3S)-4-(benzyloxy)-N,3-dihydroxy-2-[(4-{[4-(morpholinomethyl)phenyl]ethynyl}benzyl)oxy]butanamide) as the most potent LpxC inhibitor. This compound was additionally tested for antibacterial activity against a panel of clinically relevant Gram-negative bacteria, bacterial uptake, and susceptibility to efflux pumps. Molecular docking studies were performed to rationalize the observed structure-activity relationships.

An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay.

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure-activity relationships.

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.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases.

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.

Synthesis and biological evaluation of 3-tetrazolo steroidal analogs: Novel class of 5α-reductase inhibitors.

In the present study, a series of steroidal tetrazole derivatives of androstane and pregnane have been prepared in which the tetrazole moiety was appended at C-3 and 17a-aza locations. 3-Tetrazolo-3,5-androstadien-17-one (6), 3-tetrazolo-19-nor-3,5-androstadien-17-one (10), 3-tetrazolo-3,5-pregnadien-20-one (14), 17a-substituted 3-tetrazolo-17a-aza-D-homo-3,5-androstadien-17-one (26-31) and 3-(2-acetyltetrazolo)-17a-aza-d-homo-3,5-androstadien-17-one (32) were synthesized from dehydroepiandrosterone acetate (1) through multiple synthetic steps. Some of the synthesized compounds were evaluated for their in vitro 5α-reductase (5AR) inhibitory activity by measuring the conversion of [(3)H] androstenedione in human embryonic kidney (HEK) cells. In vivo 5α-reductase inhibitory activity also showed a significant reduction (p <0.05) in rat prostate weight. The most potent compound 14 showed 5AR-2 inhibition with IC50 being 15.6nM as compared to clinically used drug finasteride (40nM). There was also a significant inhibition of 5AR-1 with IC50 547nM compared to finasteride (453nM).

Inhalable Clarithromycin Microparticles for Treatment of Respiratory Infections.

PURPOSE: The aim of this work was to develop clarithromycin microparticles (CLARI-MP) and evaluate their aerodynamic behavior, safety in bronchial cells and anti-bacterial efficacy. METHODS: Microparticles containing clarithromycin were prepared as dry powder carrier for inhalation, using leucine and chitosan. CLARI-MP were deposited on Calu-3 grown at air-interface condition, using the pharmaceutical aerosol deposition device on cell cultures (PADDOCC). Deposition efficacy, transport across the cells and cytotoxicity were determined. Anti-antibacterial effect was evaluated against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus. RESULTS: Microparticles were of spherical shape, smooth surface and size of about 765 nm. Aerosolization performance showed a fine particle fraction (FPF) of 73.3%, and a mass median aerodynamic diameter (MMAD) of 1.8 µm. Deposition on Calu-3 cells using the PADDOCC showed that 8.7 µg/cm(2) of deposited powder were transported to the basolateral compartment after 24 h. The safety of this formulation is supported by the integrity of the cellular epithelial barrier and absence of toxicity, and the antimicrobial activity demonstrated for Gram positive and Gram negative bacteria. CONCLUSIONS: The appropriate aerodynamic properties and the excellent deposition on Calu-3 cells indicate that clarithromycin microparticles are suitable for administration via pulmonary route and are efficient to inhibit bacteria proliferation.

New insights into the bacterial RNA polymerase inhibitor CBR703 as a starting point for optimization as an anti-infective agent.

CBR703 was reported to inhibit bacterial RNA polymerase (RNAP) and biofilm formation, considering it to be a good candidate for further optimization. While synthesized derivatives of CBR703 did not result in more-active RNAP inhibitors, we observed promising antibacterial activities. These again correlated with a significant cytotoxicity toward mammalian cells. Furthermore, we suspect the promising effects on biofilm formation to be artifacts. Consequently, this class of compounds can be considered unattractive as antibacterial agents.

A detective story in drug discovery: elucidation of a screening artifact reveals polymeric carboxylic acids as potent inhibitors of RNA polymerase.

Chasing the active impurity: In the validation of a screening hit it was discovered that a polymeric trace impurity was responsible for the biological activity. Such a side product can be formed with similar compounds. During the investigations it was discovered that the negatively charged macromolecule interacts very efficiently with the protein surface of E. coli RNAP via electrostatic interactions.

Exploring the Translational Gap of a Novel Class of Escherichia coli IspE Inhibitors.

Discovery of novel antibiotics needs multidisciplinary approaches to gain target enzyme and bacterial activities while aiming for selectivity over mammalian cells. Here, we report a multiparameter optimisation of a fragment-like hit that was identified through a structure-based virtual-screening campaign on Escherichia coli IspE crystal structure. Subsequent medicinal-chemistry design resulted in a novel class of E. coli IspE inhibitors, exhibiting activity also against the more pathogenic bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. While cytotoxicity remains a challenge for the series, it provides new insights on the molecular properties for balancing enzymatic target and bacterial activities simultaneously as well as new starting points for the development of IspE inhibitors with a predicted new mode of action.

Not Every Hit-Identification Technique Works on 1-Deoxy-d-Xylulose 5-Phosphate Synthase (DXPS): Making the Most of a Virtual Screening Campaign.

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.

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.

Contrast enhancement of the brain by folate-conjugated gadolinium-diethylenetriaminepentaacetic acid-human serum albumin nanoparticles by magnetic resonance imaging.

Different from regular small molecule contrast agents, nanoparticle-based contrast agents have a longer circulation time and can be modified with ligands to confer tissue-specific contrasting properties. We evaluated the tissue distribution of polymeric nanoparticles (NPs) prepared from human serum albumin (HSA), loaded with gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA) (Gd-HSA-NP), and coated with folic acid (FA) (Gd-HSA-NP-FA) in mice by magnetic resonance imaging (MRI). FA increases the affinity of the Gd-HSA-NP to FA receptor-expressing cells. Clinical 3 T MRI was used to evaluate the signal intensities in the different organs of mice injected with Gd-DTPA, Gd-HSA-NP, or Gd-HSA-NP-FA. Signal intensities were measured and standardized by calculating the signal to noise ratios. In general, the NP-based contrast agents provided stronger contrasting than Gd-DTPA. Gd-HSA-NP-FA provided a significant contrast enhancement (CE) in the brain (p  =  .0032), whereas Gd-DTPA or Gd-HSA-NP did not. All studied MRI contrast agents showed significant CE in the blood, kidney, and liver (p < .05). Gd-HSA-NP-FA elicited significantly higher CE in the blood than Gd-HSA-NP (p  =  .0069); Gd-HSA-NP and Gd-HSA-NP-FA did not show CE in skeletal muscle and gallbladder; Gd-HSA-NP, but not Gd-HSA-NP-FA, showed CE in the cardiac muscle. Gd-HSA-NP-FA has potential as an MRI contrast agent in the brain.

The Structures and Binding Modes of Small-Molecule Inhibitors of Pseudomonas aeruginosa Elastase LasB.

Elastase B (LasB) is a zinc metalloprotease and a crucial virulence factor of Pseudomonas aeruginosa. As the need for new strategies to fight antimicrobial resistance (AMR) constantly rises, this protein has become a key target in the development of novel antivirulence agents. The extensive knowledge of the structure of its active site, containing two subpockets and a zinc atom, led to various structure-based medicinal chemistry programs and the optimization of several chemical classes of inhibitors. This review provides a brief reminder of the structure of the active site and a summary of the disclosed P. aeruginosa LasB inhibitors. We specifically focused on the analysis of their binding modes with a detailed representation of them, hence giving an overview of the strategies aiming at targeting LasB by small molecules.

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.

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.