High-throughput screening system for antimycobacterial compounds usinginfected cells: construction and application to compounds in the Ōmura Natural Compound Library.
Source: PubMed, NCBI / U.S. National Library of Medicine
The global increase in multidrug-resistant tuberculosis strains poses a major threat; consequently, there is an urgent need to identify and develop novel drugs. We have previously presented a simple fibroblast assay (SFA) screening method that enables the simultaneous assessment of antibacterial activity and cytotoxicity toward host cells. This method was developed based on our finding that live (but not dead)bacilli exhibit cytotoxic activity against human lung fibroblasts, and that this cytotoxicity, which is proportional to the number of viable bacterial cells, is inhibited by known antimycobacterial drugs. While conventional methods to measuregrowth require 2-4 weeks to detect antibacterial activity; the SFA method enables detection within 2-3 days and can detect compounds that are active within host cells. Here, we applied high-throughput screening, based on assessment of cytotoxicity, to identify candidate antimycobacterial agents. In parallel, by comparing the direct antibacterial activity of compounds in liquid medium (using the broth dilution test) with that observed via SFA, we developed a new SFA-based screening system to select compounds with low cytotoxicity that are active outside or inside the host cells. Using this dual system, we screened 742 compounds from the Ōmura Natural Compound Library (Kitasato University, Japan), with 62 hits. Of these, 19 were excluded owing to their toxicity, and six exhibited antibacterial activity only via SFA screening. Fou
Abstract
The global increase in multidrug-resistant tuberculosis strains poses a major threat; consequently, there is an urgent need to identify and develop novel drugs. We have previously presented a simple fibroblast assay (SFA) screening method that enables the simultaneous assessment of antibacterial activity and cytotoxicity toward host cells. This method was developed based on our finding that live (but not dead)bacilli exhibit cytotoxic activity against human lung fibroblasts, and that this cytotoxicity, which is proportional to the number of viable bacterial cells, is inhibited by known antimycobacterial drugs. While conventional methods to measuregrowth require 2-4 weeks to detect antibacterial activity; the SFA method enables detection within 2-3 days and can detect compounds that are active within host cells. Here, we applied high-throughput screening, based on assessment of cytotoxicity, to identify candidate antimycobacterial agents. In parallel, by comparing the direct antibacterial activity of compounds in liquid medium (using the broth dilution test) with that observed via SFA, we developed a new SFA-based screening system to select compounds with low cytotoxicity that are active outside or inside the host cells. Using this dual system, we screened 742 compounds from the Ōmura Natural Compound Library (Kitasato University, Japan), with 62 hits. Of these, 19 were excluded owing to their toxicity, and six exhibited antibacterial activity only via SFA screening. Four promising antimycobacterial candidates with no cytotoxicity at the 10 μg/mL threshold-aridicin A, lankamycin, streptovaricin, and lariatin A-were identified. This novel system supports the rapid identification of low-cytotoxicity antimycobacterial compounds that are active inside or outside the host cells. This screening system, comprising the simple fibroblast assay (SFA) and broth dilution test (BDT), effectively identifies low-cytotoxicity compounds that exhibit antimycobacterial activity inside or outside the host cells.