Reporter-Based Assays for High-Throughput Drug Screening against Mycobacterium abscessus

Abstract

Mycobacterium abscessus is a non-tuberculous mycobacterium that causes pulmonary and non-pulmonary infections. M. abscessus is resistant to many chemotherapeutic agents and the current treatment options show poor clinical outcomes. Thus, there is a dire need to find new antimicrobials effective at killing M. abscessus. Screening drug libraries to identify potential antimicrobials has been impeded by the lack of validated HTS assays for M. abscessus. In this study, we developed two 384-well high-throughput screening assays using fluorescent and bioluminescent reporter strains of M. abscessus for drug discovery. Optimization of inoculum size, incubation time and the volume-per-well based on Z-factor and signal intensity yielded two complementary, robust tools for M. abscessus drug discovery with Z-factor > 0.8. The MIC of known drugs, amikacin and clarithromycin, as determined by bioluminescence was in agreement with the published MIC values. A proof-of-concept screen of 2,093 natural product-inspired compounds was conducted using the 384-well bioluminescent assay to identify novel scaffolds active against M. abscessus. Five active “hit” compounds identified in this pilot screen were confirmed and characterized by a CFU assay and MIC determination. Overall, we developed and validated a 384-well screen that offers simple, sensitive and fast screening of compounds for activity against this emerging pathogen. To our knowledge, this is the first reporter–based high-throughput screening study aimed at M. abscessus drug discovery.

Novel Screen to Assess Bactericidal Activity of Compounds Against Non-replicating Mycobacterium abscessus

“…These factors make it difficult for drugs not only to enter the cell but also to remain effective if they get past the cell wall. In the few small-scale screens for compounds active against M. abscessus , screens suffered from low hit rates compared to other mycobacterial species ( Chopra et al, 2011 ; Gupta et al, 2017 ; Low et al, 2017 ).…”

“…Together with the similarity in disease progression to TB disease, we believe it is highly likely M. abscessus can survive in patients in a dormant state. Previous screens against M. abscessus specifically addressed drug activity against actively growing, metabolically active bacteria ( Chopra et al, 2011 ; Gupta et al, 2017 ; Low et al, 2017 ). Such efforts will miss compounds which are active against non-replicating or metabolically inactive cells.…”

Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates Targeting Mycobacterium tuberculosis and Mycobacterium abscessus

“…MIC assay. The MICs of 5 active compounds (compounds 8, 10, 11, 14, and 15) identified from the initial screening were determined using bioluminescent strains of M. abscessus and M. tuberculosis (74,76) in solid white 384-well microtiter plates (Corning). Compounds 8, 10, and 11 are gold(III) macrocycles, while compounds 14 and 15 are simple gold(III) chelates.…”

Creating and screening natural product libraries

“…The category of cell-based HTS includes a wide variety of targets and detection technologies which have been the subjects of a number of recent reviews, [47][48][49][50][51][52][53][54][55][56] including those detailing use for natural product discovery. 47,52,57 While there are many examples of non-mammalian cell-based assays that have been utilized for identication and characterization of anti-infective agents in natural product extracts, including anti-fungal, 54,58 anti-parasitic, [59][60][61][62] anti-bacterial [63][64][65] as well as in model organism-based cellular assay platforms including yeast, 66,67 Xenopus oocytes, 68 zebra sh 69 and C. elegans, 70 this section will focus on screens involving human cells. Mammalian and/or human cell-based natural products HTS has been employed in a wide range of disease areas and cellular phenomena.…”