Identification of novel anti-bacterial compounds from a chemically highly diverse pathways-based library using phenotypic screens in amoebae host models

Abstract

24Legionella pneumophila and tubercular Mycobacteria are the causative agents of potentially 25 fatal diseases due to their pathogenesis but also to the emergence of antibiotic resistance that limits 26 treatment strategies. The aim of our study is to explore the antimicrobial activity of a small ligand-based 27 chemical library of 1,255 structurally diverse compounds. These compounds were screened in a 28 combination of three assays, two monitoring the intracellular growth of the pathogenic bacteria, 29Mycobacterium marinum and L. pneumophila, and an additional anti-virulence "plaque" assay for 30 M. marinum. We set up these assays using two amoeba strains, the genetically tractable Dictyostelium 31 discoideum and the free-living amoeba Acanthamoeba castellanii. In summary, sixty-four compounds 32 showed anti-infective/anti-virulence activity in at least one of the 3 assays. The intracellular assays hit 33 rate varied between 1.7% (n=22) for M. marinum and 2.8% (n=35) for L pneumophila with 7 compounds 34 in common between both pathogens. In parallel, 1.2 % (n= 15) of the tested compounds were able to 35 restore D. discoideum growth in presence of M. marinum spiked in a lawn of Klebsiella pneumoniae. 36We also validated the generality of the hit compounds identified using the A. castellanii-M. marinum 37 anti-infective screen in the powerful D. discoideum-M. marinum host-pathogen model. The 38 characterization of anti-infective and antibacterial hits in the latter infection model revealed compounds 39 able to reduce intracellular growth more than 50% at 30 M. Our studies underline the relevance of 40 using a combination of low-cost and low-complexity assays with full 3R compliance associated with a 41 rationalized focused library of compounds to help identifying new chemical scaffolds and dissect some 42 of their properties prior to run further compounds development steps.43 44 45 48 the host animal model or human, which were successful during the 50-60's to identify the main antibiotic 49 classes used today, are now reaching their limit. Indeed, the dramatic lack of in vivo confirmation of 50 promising chemical scaffolds identified in vitro and/or against validated molecular targets, due to 51 pharmacokinetic or toxicity problems at later animal and clinical stages, have given a decisive impulse 52 to design new experimental strategies for the screening procedure 'per se', as well as for the design of53 the chemical library (Pethe et al. 2010). In addition, the development of new curative treatments against 54 pathogenic bacteria, coupled to rationalized political choices constitute a major challenge for the future 55 of public health (Carlet, Rambaud, and Pulcini 2014; Perez et al. 2015).56 Over the years, millions of compounds have been synthesized or extracted from natural sources 57 worldwide and are now available for biological screens (Diop et al. 2018; Farnsworth et al. 1985). The 58 general concept behind the re-screening or repurposing of compounds with new assay systems is that 59 small molecules hav...

Time-resolved RNA-seq profiling of the infection of Dictyostelium discoideum by Mycobacterium marinum reveals an integrated host response to damage and stress

“…Infections were performed as previously described (Kicka et al, 2018). In brief, GFP-expressing M. marinum grown overnight were centrifuged and re-suspended in Hl5c, and then spinoculated on adherent D. discoideum cells.…”

Identification of Anti-Mycobacterium and Anti-Legionella Compounds With Potential Distinctive Structural Scaffolds From an HD-PBL Using Phenotypic Screens in Amoebae Host Models

“…This manuscript has been released as a Pre-Print at BioRxiv (doi: 10.1101/497032) (Kicka et al, 2018).…”