Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity

Abstract

The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper’s natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10,000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family’s presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections.

Differential Susceptibility of Mycoplasma and Ureaplasma Species to Compound-Enhanced Copper Toxicity

“…Little work has been carried out with respect to antimicrobial effects of transition metals on human mollicutes. Previously published work with S. aureus and M. tuberculosis has demonstrated that compounds that chelate copper can exhibit antibacterial effects (Dalecki et al, 2016; Shah et al, 2016). Previous work in another laboratory during the 1980s examined the effect of Neo on Cu stress with M. gallisepticum (Smit et al, 1982; Gaisser et al, 1987b).…”

“…Screening compounds for copper-dependent killing activity has resulted in the identification of new compounds with copper-dependent antimicrobial activity against Mycobacterium tuberculosis and Staphylococcus aureus (Dalecki et al, 2016; Shah et al, 2016). Previous work has been described examining the effect of antimicrobial compounds on copper-induced stress in Mycoplasma gallisepticum (Smit et al, 1982; Gaisser et al, 1987a,b; de Zwart et al, 1991), but to date studies in this area have not been conducted with human mycoplasmas or ureaplasmas.…”

High-throughput screening and Bayesian machine learning for copper-dependent inhibitors of Staphylococcus aureus

“…aureus strain Newman was grown overnight in Mueller Hinton broth, used to reinoculate fresh broth, and grown to an optical density at 600nm (OD600) of ~1. Cells were washed twice in Roswell Park Memorial Institute medium (RPMI; Corning CellGrow #17105CV) and then resuspended in a mixture of RPMI plus trace elements (TE) as previously described 28 . Cells were adjusted to an OD600 of 0.004 and alamarBlue (AB; Life Technologies DAL1001) was added as a viability readout.…”

“…Previously, we reported a pilot screen for CDIs against S. aureus, as well as a lead series resulting from that screen 28 . Here, we describe the continuation of that study, with two primary goals: adaptation to and deployment on an industrial high-throughput screening platform, and the identification of new and unrecognized chemical motifs associated with copper-dependent antibacterial activity.…”

Development of a web-based tool for automated processing and cataloging of a unique combinatorial drug screen

“…Recently, we described a novel approach to drug screening in which we specifically identify compounds acting in concert with copper ions, more closely mimicking in vivo conditions, as well as offering an ability to more deeply screen existing compound libraries, defraying the cost of screening studies when compared to the alternative option of synthesizing additional compounds (7-10). However, this screen, which directly compares the activities of compounds in a copper-activating medium (Cu+) against activity in a copper-deficient medium (Cu−), requires two assay plates run in parallel, and time consuming analysis to identify promising hit compounds (Figure 1).…”