# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 2 Detection and Quantification of Direct FtsZ-Drug Binding in Solution  
Identification of novel antimicrobials directed towards FtsZ requires suitable methods to evaluate direct drug-protein interactions (Figure 3, Table 1). Moreover, binding affinity measurements of the identified candidates are a necessary starting point for the determination of minimum inhibitory concentrations (MICs). One of the methods that have been employed for this purpose is fluorescence anisotropy. Anisotropy is very sensitive to size changes, such as those that occur when a small fluorescent species binds to a large macromolecule [62; 63]. Among the most widely employed techniques for drug screening [64], fluorescence anisotropy can precisely measure high affinity interactions, and several low volume samples can be simultaneously and rapidly analyzed by usinga plate reader equipped with polarizers. Anisotropy has been used to determine the binding affinity between a boron-dipyrromethene (BODIYP)-labeled oxazole-benzamide inhibitor and FtsZs from a variety of Gram-negative and Gram-positive bacteria [65]. In addition, this technique has been used to determine interactions between FtsZ and 4\({}^{\prime}\),6-Diamidino-2-phenylindole (DAPI), a DNA intercalating dye that is also a modulator of FtsZ assembly [66], and the guanidinomethyl biaryl compound 13, a broad spectrum bactericidal agent [67]. An anisotropy-based competition assay has been devised to identify compounds targeting the GTP binding site of FtsZ by using the fluorescent analog 2\({}^{\prime}\)/3\({}^{\prime}\)-O-(N-methylanthraniloyl) (_mant_)-GTP. This assay has been used to analyze the binding of compounds such as C8-substituted GTP analogs to FtsZ from the archaeon _Methanococcus jannaschi_, or the interaction of a variety of synthetic inhibitors of bacterial cell division with _Bacillus subtilis_ FtsZ [68; 69]. Insight into the mechanism of action of the above mentioned compound 13 has also been obtained through anisotropy measurements of BODIPY-labeled GTP\(\gamma\)S, showing competition with the drug for binding to the GTP site of FtsZ [67].  
Figure 3: Illustration of techniques and reconstruction approaches useful for the detection and characterization of FtsZ interactions. Experimental approaches to study FtsZ homo- and hetero-association are represented at the outer circumference. Minimal membrane systems and cytomimetic platforms are shown at the center.  
A reference technique for the thermodynamic characterization of binding reactions is isothermal titration calorimetry (ITC), based on the measurement of the heat change produced upon interaction [70]. This technique is therefore ideal for detection of inhibitory compounds and selection of candidates showing optimal affinities for their targets. Binding isotherms of the inhibitory MciZ peptide to FtsZs from _B. subtilis_ or _Staphylococcus aureus_ demonstrated that the peptide, produced by the former during sporulation to arrest cytokinesis by disrupting FtsZ polymerization, binds FtsZ from different bacteria by establishing entropically favorable interactions [71]. This method also allowed determination of the binding constants of MciZ mutants with FtsZ. Likewise, ITC was employed to characterize the binding of _E. coli_ FtsZ to cinnamaldehyde, a plant-based small molecule that inhibits FtsZ polymerization and GTPase activity [72].  
As for other targets, the identification and characterization of hit compounds acting on FtsZ has benefited from the use of bioinformatics tools and structural approaches like nuclear magnetic resonance spectroscopy (NMR) and X-ray crystallography [48]. With these methods, the precise binding sites of the candidates can be ascertained and chemical modifications improving affinity or specificity can be foreseen by docking onto the FtsZ structures available. The use of methods such as NMR and X-ray crystallography to assess direct binding of inhibitors to FtsZ has been thoroughly analyzed elsewhere [4].