Ring-fused 2-pyridones effective against multidrug-resistant Gram-positive pathogens and synergistic with standard-of-care antibiotics.

The alarming rise of multidrug-resistant Gram-positive bacteria has precipitated a healthcare crisis, necessitating the development of new antimicrobial therapies. Here we describe a new class of antibiotics based on a ring-fused 2-pyridone backbone, which are active against vancomycin-resistant enterococci (VRE), a serious threat as classified by the Centers for Disease Control and Prevention, and other multidrug-resistant Gram-positive bacteria. Ring-fused 2-pyridone antibiotics have bacteriostatic activity against actively dividing exponential phase enterococcal cells and bactericidal activity against nondividing stationary phase enterococcal cells. The molecular mechanism of drug-induced killing of stationary phase cells mimics aspects of fratricide observed in enterococcal biofilms, where both are mediated by the Atn autolysin and the GelE protease. In addition, combinations of sublethal concentrations of ring-fused 2-pyridones and standard-of-care antibiotics, such as vancomycin, were found to synergize to kill clinical strains of VRE. Furthermore, a broad range of antibiotic resistant Gram-positive pathogens, including those responsible for the increasing incidence of antibiotic resistant healthcare-associated infections, are susceptible to this new class of 2-pyridone antibiotics. Given the broad antibacterial activities of ring-fused 2-pyridone compounds against Gram-positive (GmP) bacteria we term these compounds GmPcides, which hold promise in combating the rising tide of antibiotic resistant Gram-positive pathogens.

THCz: Small molecules with antimicrobial activity that block cell wall lipid intermediates.

Emerging antibiotic resistance demands identification of novel antibacterial compound classes. A bacterial whole-cell screen based on pneumococcal autolysin-mediated lysis induction was developed to identify potential bacterial cell wall synthesis inhibitors. A hit class comprising a 1-amino substituted tetrahydrocarbazole (THCz) scaffold, containing two essential amine groups, displayed bactericidal activity against a broad range of gram-positive and selected gram-negative pathogens in the low micromolar range. Mode of action studies revealed that THCz inhibit cell envelope synthesis by targeting undecaprenyl pyrophosphate-containing lipid intermediates and thus simultaneously inhibit peptidoglycan, teichoic acid, and polysaccharide capsule biosynthesis. Resistance did not readily develop in vitro, and the ease of synthesizing and modifying these small molecules, as compared to natural lipid II-binding antibiotics, makes THCz promising scaffolds for development of cell wall-targeting antimicrobials.

The Bactericidal Fatty Acid Mimetic 2CCA-1 Selectively Targets Pneumococcal Extracellular Polyunsaturated Fatty Acid Metabolism.

Streptococcus pneumoniae, a major cause of pneumonia, sepsis, and meningitis worldwide, has the nasopharynges of small children as its main ecological niche. Depletion of pneumococci from this niche would reduce the disease burden and could be achieved using small molecules with narrow-spectrum antibacterial activity. We identified the alkylated dicyclohexyl carboxylic acid 2CCA-1 as a potent inducer of autolysin-mediated lysis of S. pneumoniae, while having low activity against Staphylococcus aureus 2CCA-1-resistant strains were found to have inactivating mutations in fakB3, known to be required for uptake of host polyunsaturated fatty acids, as well as through inactivation of the transcriptional regulator gene fabT, vital for endogenous, de novo fatty acid synthesis regulation. Structure activity relationship exploration revealed that, besides the central dicyclohexyl group, the fatty acid-like structural features of 2CCA-1 were essential for its activity. The lysis-inducing activity of 2CCA-1 was considerably more potent than that of free fatty acids and required growing bacteria, suggesting that 2CCA-1 needs to be metabolized to exert its antimicrobial activity. Total lipid analysis of 2CCA-1 treated bacteria identified unique masses that were modeled to 2CCA-1 containing lysophosphatidic and phosphatidic acid in wild-type but not in fakB3 mutant bacteria. This suggests that 2CCA-1 is metabolized as a fatty acid via FakB3 and utilized as a phospholipid building block, leading to accumulation of toxic phospholipid species. Analysis of FabT-mediated fakB3 expression elucidates how the pneumococcus could ensure membrane homeostasis and concurrent economic use of host-derived fatty acids.IMPORTANCE Fatty acid biosynthesis is an attractive antibiotic target, as it affects the supply of membrane phospholipid building blocks. In Streptococcus pneumoniae, it is not sufficient to target only the endogenous fatty acid synthesis machinery, as uptake of host fatty acids may bypass this inhibition. Here, we describe a small-molecule compound, 2CCA-1, with potent bactericidal activity that upon interactions with the fatty acid binding protein FakB3, which is present in a limited number of Gram-positive species, becomes metabolized and incorporated as a toxic phospholipid species. Resistance to 2CCA-1 developed specifically in fakB3 and the regulatory gene fabT These mutants reveal a regulatory connection between the extracellular polyunsaturated fatty acid metabolism and endogenous fatty acid synthesis in S. pneumoniae, which could ensure balance between efficient scavenging of host polyunsaturated fatty acids and membrane homeostasis. The data might be useful in the identification of narrow-spectrum treatment strategies to selectively target members of the Lactobacillales such as S. pneumoniae.

Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine Fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-ß Fibrils.

A BF3·OEt2 catalyzed intramolecular Povarov reaction was used to synthesize 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with several O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid ß fibrils in vitro. Analogues substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.

Chemical disarming of isoniazid resistance in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) killed more people in 2017 than any other single infectious agent. This dangerous pathogen is able to withstand stresses imposed by the immune system and tolerate exposure to antibiotics, resulting in persistent infection. The global tuberculosis (TB) epidemic has been exacerbated by the emergence of mutant strains of Mtb that are resistant to frontline antibiotics. Thus, both phenotypic drug tolerance and genetic drug resistance are major obstacles to successful TB therapy. Using a chemical approach to identify compounds that block stress and drug tolerance, as opposed to traditional screens for compounds that kill Mtb, we identified a small molecule, C10, that blocks tolerance to oxidative stress, acid stress, and the frontline antibiotic isoniazid (INH). In addition, we found that C10 prevents the selection for INH-resistant mutants and restores INH sensitivity in otherwise INH-resistant Mtb strains harboring mutations in the katG gene, which encodes the enzyme that converts the prodrug INH to its active form. Through mechanistic studies, we discovered that C10 inhibits Mtb respiration, revealing a link between respiration homeostasis and INH sensitivity. Therefore, by using C10 to dissect Mtb persistence, we discovered that INH resistance is not absolute and can be reversed.

Methyl sulfonamide substituents improve the pharmacokinetic properties of bicyclic 2-pyridone based Chlamydia trachomatis inhibitors.

Chlamydia trachomatis infections are a global health problem and new approaches to treat C. trachomatis with drugs of high specificity would be valuable. A library of substituted ring fused 2-pyridones has been synthesized and evaluated for their ability to attenuate C. trachomatis infectivity. In vivo pharmacokinetic studies were performed, with the best candidates demonstrating that a C8-methylsulfonamide substituent improved pharmacokinetic properties important for oral administration. C8-Methyl sulfonamide analogue 30 inhibited C. trachomatis infectivity in low micromolar concentrations. Further pharmacokinetic evaluation at an oral dose of 10 mg kg-1 showed an apparent bioavailability of 41%, compared to C8-cyclopropyl and -methoxy analogues which had negligible oral uptake. In vitro ADME (absorption, distribution, metabolism and excretion) testing of solubility and Caco-2 cell permeability revealed that both solubility and permeability is greatly improved with the C8-methyl sulfonamide 30, effectively moving it from BCS (Biopharmaceutical Classification System) class IV to II.

Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol.

Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a ~1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.

Towards small molecule inhibitors of mono-ADP-ribosyltransferases.

Protein ADP-ribosylation is a post-translational modification involved in DNA repair, protein degradation, transcription regulation, and epigenetic events. Intracellular ADP-ribosylation is catalyzed predominantly by ADP-ribosyltransferases with diphtheria toxin homology (ARTDs). The most prominent member of the ARTD family, poly(ADP-ribose) polymerase-1 (ARTD1/PARP1) has been a target for cancer drug development for decades. Current PARP inhibitors are generally non-selective, and inhibit the mono-ADP-ribosyltransferases with low potency. Here we describe the synthesis of acylated amino benzamides and screening against the mono-ADP-ribosyltransferases ARTD7/PARP15, ARTD8/PARP14, ARTD10/PARP10, and the poly-ADP-ribosyltransferase ARTD1/PARP1. The most potent compound inhibits ARTD10 with sub-micromolar IC50.

N-acylated derivatives of sulfamethoxazole and sulfafurazole inhibit intracellular growth of Chlamydia trachomatis.

Antibacterial compounds with novel modes of action are needed for management of bacterial infections. Here we describe a high-content screen of 9,800 compounds identifying acylated sulfonamides as novel growth inhibitors of the sexually transmitted pathogen Chlamydia trachomatis. The effect was bactericidal and distinct from that of sulfonamide antibiotics, as para-aminobenzoic acid did not reduce efficacy. Chemical inhibitors play an important role in Chlamydia research as probes of potential targets and as drug development starting points.

Statistical molecular design: a tool to follow up hits from small-molecule screening.

In high-throughput screening (HTS) a robust assay is used to interrogate a large collection of small organic molecules in order to find compounds, hits, with a desired biological activity. The hits are then further explored by an iterative process where new compounds are designed, purchased, or synthesized, followed by an evaluation in one or more assays. Statistical molecular design (SMD) is a useful method to select a balanced, varied, and information-rich compound collection based on hits from HTS in order to create a foundation for development of optimized compounds with improved properties. In this chapter, we describe the use of SMD to explore a hit obtained from small-molecule screening.

Chemical probes to study ADP-ribosylation: synthesis and biochemical evaluation of inhibitors of the human ADP-ribosyltransferase ARTD3/PARP3.

The racemic 3-(4-oxo-3,4-dihydroquinazolin-2-yl)-N-[1-(pyridin-2-yl)ethyl]propanamide, 1, has previously been identified as a potent but unselective inhibitor of diphtheria toxin-like ADP-ribosyltransferase 3 (ARTD3). Herein we describe synthesis and evaluation of 55 compounds in this class. It was found that the stereochemistry is of great importance for both selectivity and potency and that substituents on the phenyl ring resulted in poor solubility. Certain variations at the meso position were tolerated and caused a large shift in the binding pose. Changes to the ethylene linker that connects the quinazolinone to the amide were also investigated but proved detrimental to binding. By combination of synthetic organic chemistry and structure-based design, two selective inhibitors of ARTD3 were discovered.

Modulation of curli assembly and pellicle biofilm formation by chemical and protein chaperones.

Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by "chemical chaperones."

PARP inhibitor with selectivity toward ADP-ribosyltransferase ARTD3/PARP3.

Inhibiting ADP-ribosyl transferases with PARP-inhibitors is considered a promising strategy for the treatment of many cancers and ischemia, but most of the cellular targets are poorly characterized. Here, we describe an inhibitor of ADP-ribosyltransferase-3/poly(ADP-ribose) polymerase-3 (ARTD3), a regulator of DNA repair and mitotic progression. In vitro profiling against 12 members of the enzyme family suggests selectivity for ARTD3, and crystal structures illustrate the molecular basis for inhibitor selectivity. The compound is active in cells, where it elicits ARTD3-specific effects at submicromolar concentration. Our results show that by targeting the nicotinamide binding site, selective inhibition can be achieved among the closest relatives of the validated clinical target, ADP-ribosyltransferase-1/poly(ADP-ribose) polymerase-1.

Design, synthesis and evaluation of triazole functionalized ring-fused 2-pyridones as antibacterial agents.

Antibacterial resistance is today a worldwide problem and the demand for new classes of antibacterial agents with new mode of action is enormous. In the strive for new antibacterial agents that inhibit pilus assembly, an important virulence factor, routes to introduce triazoles in position 8 and 2 of ring-fused bicyclic 2-pyridones have been developed. This was made via Sonogashira couplings followed by Huisgen 1,3-dipolar cycloadditions. The method development made it possible to introduce a diverse series of substituted triazoles and their antibacterial properties were tested in a whole cell pili-dependent biofilm assay. Most of the twenty four candidates tested showed low to no activity but interestingly three compounds, one 8-substituted and two 2-substituted, showed promising activities with EC(50)'s between 9 and 50 µM.

Discovery of ligands for ADP-ribosyltransferases via docking-based virtual screening.

The diphtheria toxin-like ADP-ribosyltransferases (ARTDs) are an enzyme family that catalyzes the transfer of ADP-ribose units onto substrate proteins by using nicotinamide adenine dinucleotide (NAD(+)) as a cosubstrate. They have a documented role in chromatin remodelling and DNA repair, and inhibitors of ARTD1 and 2 (PARP1 and 2) are currently in clinical trials for the treatment of cancer. The detailed function of most other ARTDs is still unknown. By using virtual screening, we identified small ligands of ARTD7 (PARP15/BAL3) and ARTD8 (PARP14/BAL2). Thermal-shift assays confirmed that 16 compounds, belonging to eight structural classes, bound to ARTD7/ARTD8. Affinity measurements with isothermal titration calorimetry for two isomers of the most promising hit compound confirmed binding in the low micromolar range to ARTD8. Crystal structures showed anchoring of the hits in the nicotinamide pocket. These results form a starting point in the development of chemical tools for the study of the role and function of ARTD7 and ARTD8.