Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis

Abstract

FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).

FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics

“…Fluorescence cross-correlation spectroscopy (FCCS) is an extension of FCS [ 63 , 88 , 89 ] and relies on two-channel detection for the generation of cross-correlation curves indicative of complex formation. FCCS was used in high throughput screens to identify compounds that perturb FtsZ self-interactions [ 90 ]. In this assay, N- and C-terminal fragments of FtsZ were each labeled with fluorescent proteins emitting at different wavelengths, and hits were based on the ability to reduce the cross-correlation arising from GTP-induced interaction between the fragments [ 90 ].…”

State-of-the-Art Fluorescence Fluctuation-Based Spectroscopic Techniques for the Study of Protein Aggregation

“…Aβ and polyQ have been classically and traditionally analyzed using FCS [ 47 , 61 , 62 , 63 , 64 , 65 ]. Although these aggregation-prone proteins were initially regarded as one of the high molecular weight sample for FCS measurement, FCS gradually became used for the analysis of the aggregation process and determining the aggregation-suppressive effect of molecular chaperones and drugs [ 45 , 47 , 66 ]. Because of the high sensitivity of FCS, oligomers of Htt Q25, which contains a normal-length polyQ repeat and does not form IBs in the cell, have been successfully detected in a cell lysate [ 67 ].…”

Identification of Agents Targeting Ftsz Assembly

“…After an initial high-throughput virtual screen of approximately 210,000 compounds from the Open Innovation Centre for Drug Discovery (The University of Tokyo), the top 500 compounds were selected and evaluated by fluorescence cross-correlation spectroscopy, with the binding of the top six to S. aureus FtsZ being confirmed by surface plasmon resonance and their dissociation constants (Kd) determined. The compounds were weak inhibitors, with the most potent being the N-(dihydro-[1-2,4]triazolo[1,5-a] pyrimidinyl)sulfonamide 11 (Figure 4), which resulted in 89% inhibition (100 μM) and which had a Kd of 24.5 μM [92].…”