# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 3 Methods to Identify Drugs Targeting FtsZ Polymerization and Their Mechanisms  
Given the crucial role of GTP-induced polymerization of FtsZ in cell division, alteration of this process by chemicals that either block assembly or prevent disassembly has been so far the most pursued strategy to perturb the function of this protein. Methods to measure FtsZ polymerization are of great utility in the identification of such compounds and in the determination of their precise mechanisms of action and active concentrations (Figure 3, Table 1). 90\({}^{\circ}\) Light scattering (LS), which can detect polymer formation, monitor time-dependent polymer disassembly, and determine the critical concentration for polymerization [73], is probably the most widely employed method to this end. This technique has been used to investigate how DAPI inhibits FtsZ polymer dynamics, how compound 13 [67] stimulates FtsZ polymerization in a concentration-dependent manner, or how the aminobenzamide derivative compound 28 [74] inhibits assembly of FtsZ from _S. aureus_[4, 66, 77, 74]. GTP-induced FtsZ polymers can also be detected by centrifugation, followed by evaluation of the fraction of protein in the pellet and supernatant, for example by polyacrylamide gel electrophoresis. This approach has been employed to study the impact of the polyhydroxy aromatic compound UCM05 [69] and the benzamide antibiotic compound 8 [75] on the polymerization of FtsZ from _B. subtilis_[69, 75] and of bacteriophage \(\lambda\) Kil peptide (Figure 1c) on the assembly of _E. coli_ FtsZ filament bundles [76]. Procedures for the determination of the GTPase activity of the protein (reviewed in [48]) are frequently employed in studies of its druggability (see for instance [77]). Another method for the characterization of FtsZ polymers is dynamic light scattering (DLS). The autocorrelation functions obtained by DLS result from the random diffusive motion of macromolecules in solution that correlate with their mass and shape, which translates into fluctuations in scattered light. Analysis of these curves yields translational diffusion coefficients [78] that can reflect changes in mass, for example from dissociation of protein-protein interactions upon drug binding, as long as there is a mass difference of at least four-fold. Information about the polymerization/depolymerization kinetics can be obtained by following the intensity of scattered light with time. Commercial plate readers are available that make DLS measurements amenable for high throughput screening of compounds [79]. DLS was used to determine the absence of any stabilizing effect of the benzamide derivative PC190723 in FtsZ filaments of _Caubobacter crescentus_[80]. This is consistent with the idea of this type of small molecule binding only weakly to FtsZ from Gram-negative bacteria [5], as previously suggested by inhibition of cell division in _S. aureus_ and _B. subtilis_ but not _E. coli_[81]. In a different approach, DLS was used to test the activity of Temporin L, an antimicrobial peptide active against Gram-positiveand Gram-negative bacteria, on _E. coli_ cells [82]. Specifically, DLS data revealed the formation of elongated structures by the inability of the cells to divide, which arose from the interaction of the peptide with FtsZ as determined by parallel fluorescence and GTPase activity measurements. A complementary technique, ITC, was used in an unconventional application to discern the different assembly mechanisms governing FtsZ oligomerization and polymerization [83]. It would be possible in principle to characterize the effect of compounds that interfere with these assemblies, although this would require the application of complex mechanistic models to analyze the data. Tunable resistive pulse sensing (TRPS) is an emerging sizing method applicable to a wide variety of particles ranging from single molecules to whole cells within 40 nm-10 \(\upmu\)m in size [84].