# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 3 Methods to Identify Drugs Targeting FtsZ Polymerization and Their Mechanisms
It measures current fluctuations produced by the species passing through a dynamically resizable pore, allowing simultaneous determination of size and concentration of the species present in polydisperse samples [85]. TRPS is a highly accurate and precise method potentially useful to detect and quantitate the effect of drugs on FtsZ polymerization. FtsZ polymerization has also been studied by fluorescence correlation spectroscopy (FCS), a single molecule technique that obtains autocorrelation curves from fluctuations in fluorescence intensities, which can then be used to derive translational diffusion parameters of the fluorescent molecules [63; 86]. FCS methods are suitable for high throughput screening [87]. An orthogonal combination of FCS, DLS, and sedimentation velocity (SV) approaches has been used to study FtsZ polymers [54]. Sedimentation velocity experiments yield sedimentation coefficients that correlate with the sizes and shapes of the molecular species, and parallel determination of translational diffusion coefficients by DLS or FCS allows calculation of masses using the Svedberg equation [54]. FCS and SV have been jointly applied to characterize the inhibition of FtsZ polymerization by Kil peptide when it is expressed from infecting \(\lambda\) phage (Figure 1c), with potential application in the development of therapies against bacterial infections [41]. That study also confirmed direct interaction between Kil and FtsZ by fluorescence anisotropy. 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]. Procedures based on fluorescence methods such as Forster resonance energy transfer (FRET) or anisotropy for the measurement of the critical concentration of polymerization of FtsZ are also potentially applicable to the screening of drugs that interfere with FtsZ assembly [30; 91]. The biophysical and biochemical methods described above are usually complemented with imaging approaches that provide structural information, the most popular being electron microscopy. This technique has been used to detect FtsZ polymer bundling induced by drugs such as compound 13 or DAPI, or morphological changes in FtsZ polymers induced, for example, by compound 8j [66; 67; 75]. The use of conventional fluorescence imaging methods for this purpose is more limited, as their lower resolution precludes characterization of the small polymers FtsZ forms in dilute solution conditions. They may be used, however, for studies of the modification of FtsZ bundles observed in crowding conditions in vitro [31] by antibacterial compounds and they are one of the preferred tools for the in vivo investigation of drug mechanisms of action. Exploiting the Interactions of FtsZ with Binding Partners in Solution to Discover New Antimicrobials  
Relatively unexplored targets to arrest bacterial cell division with therapeutics are the multiplicity of interactions that FtsZ establishes with a variety of proteins, some of them conserved across multiple bacterial species (Figure 1). There is agreement, however, that hetero-associations of FtsZ would constitute excellent targets for the identification of anti-infectives, probably with lower propensity to develop resistance [1; 50]. In addition to the opportunities for the identification of broad-spectrum antibacterials acting on FtsZ, targeting its hetero-associations may help in the quest for antibiotics directed towards certain bacterial species where those partners specifically operate.