# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 3 Methods to Identify Drugs Targeting FtsZ Polymerization and Their Mechanisms
Another well-studied case of hetero-association is that of FtsZ with the bacterial protease Clp (Figure 1d), also considered a novel target for antibiotic action because of its implication in vital cellular functions [121]. Clp is found in most bacterial species and involved in regulatory proteolysis. In _E. coli_, the proteolytic complex is formed by ClpP that includes the proteolytic core, and the unfoldase ClpX that controls access of the protein substrate to that core [21]. Antibiotic acyldepisopeptides (ADEPs) have been described to induce rapid degradation of FtsZ in vivo and in vitro [1,122] by deregulating the function of Clp [123]. Degradation studies with _B. subtilis_ FtsZ have identified the disordered N-terminus and, at high concentrations of ADEP2, also the C-terminus of FtsZ as the targets for the ADEP-ClpP complex, and suggested it leads to depletion of the FtsZ cytoplasmic pool under certain conditions [121]. Along these lines, treadmilling within early Z-rings seems to be more affected by FtsZ depletion, as determined by time-lapse fluorescence and super-resolution microscopy experiments [98]. A number of these ADEP compounds proved to have significant antimicrobial activity, mostly in Gram-positive bacteria, and have resistance-breaking potential, thus highlighting the inhibition of cell division as a powerful strategy for the development of new antibiotics [1]. An attractive interaction for the identification of broad-spectrum antibacterials is that of FtsZ with FtsA (Figure 1b), the other membrane anchor of the division ring, more widely conserved than ZipA. FtsA is an amphitropic protein that interacts with the membrane through its C-terminal amphipathic helix and with FtsZ through its core conserved domain [124]. FtsA interacts also with numerous divisionsome proteins, being directly or indirectly involved in septal wall and peptidoglycan synthesis [2]. The interaction between FtsA and FtsZ from _E. coli_ decreased FtsZ's GTP hydrolysis [125] although conflicting results have been obtained in this regard with FtsAs from different microorganisms, possibly related to species-specific regulatory mechanisms [2]. The binding affinity of FtsZ-FtsA from _Vibrio cholerae_ has been measured by following the variation of the intrinsic tryptophan fluorescence of FtsA upon interaction with FtsZ, which lacks tryptophan residues [94]. As FtsA is an amphitropic protein, minimal membrane systems have been also used to reconstruct its interactions (see Section 5). The formation of the Z-ring at the correct time and place largely depends on the spatial and temporal regulation of FtsZ assembly/disassembly dynamics by agonists and antagonists, mostly exerted through the direct binding of protein factors to FtsZ (Figure 1). Chemicals altering these interactions would therefore constitute potential antimicrobial leads. Broad-spectrum antibiotics could be obtained by targeting the interactions of FtsZ with ZapA (Figure 1e), a widely conserved protein that positively regulates Z-ring formation, by linking FtsZ with the chromosomal replication terminus region (Ter macrodomain) in the case of _E. coli_[22]. Fluorescent chimeras have been employed to show delocalization of this ZapA from midcell upon treatment of _B. subtilis_ cells with the benzimidazole derivative BT-benzo-29 [99] or colocalization with FtsZ foci dispersed all over the cytoplasm, induced by compound 8j [75]. Among the negative regulators of FtsZ assembly, the nucleoid occlusion factor SImA from _E. coli_ and the Min system have been widely studied in order to unravel their mechanisms of action. SImA binds to specific DNA sequences (SBS) found along the bacterial chromosome except in the Ter macrodomain, blocking inappropriate Z-ring assembly over un-partitioned chromosomes through direct interaction with FtsZ ([126], Figure 1a). The MinCDE complex blocks FtsZ polymerization at the cell poles through pole-to-pole oscillatory waves along the cytoplasmic membrane (Figure 1f), where MinC directly interacts with FtsZ [9].