# FtsZ Interactions and Biomolecular Condensates as Potential Targets for New Antibiotics
## 3 Methods to Identify Drugs Targeting FtsZ Polymerization and Their Mechanisms
The identification of antibacterial candidates targeting heterotypic interactions within the FtsZ network will benefit from the many assays developed to detect and quantify these interactions in solution and in reconstructed systems (see Section 5). Probably the most widely explored interaction of FtsZ with the purpose of finding antibiotic leads is with ZipA (Figure 1b), a protein specific to enterobacteria, which anchors the Z-ring to the membrane [18]. Together with FtsZ and FtsA, in _E. coli_, ZipA forms the proto-ring, a protein complex that subsequently recruits other components of the divisome [20]. Since ZipA is a membrane protein whose N-terminal region is actually inserted in the bacterial inner membrane, assays in solution are usually conducted with mutants  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Method** & **Fundamentals** & **Information Obtained** & **Examples** \\  & **Refs. ** & **Refs. ** & **Refs. ** \\ \hline \multicolumn{4}{c}{**Techniques in solution**} \\ \hline
90\({}^{\circ}\) LS, Sedimentation, EM & [54] & Assessment of polymerization & [4; 66; 7; 74; 75; 76] \\ \hline \multirow{2}{*}{Fluorescence anisotropy} & [62; 63] & Quantification of drug binding & [65; 66; 67; 68; 69] \\  & & Assessment of polymerization & [91]\({}^{\star}\) \\  & & Interaction with Kil, ZipA, MinC, SlmA & [41; 92], [38; 39; 93]\({}^{\star}\) \\ \hline FCS, FCCS & [63; 88] & Assessment of polymerization & [41; 90] \\ \hline \multirow{2}{*}{ITC} & [70] & Quantification of drug binding & [71; 72] \\  & & Assessment of polymerization & [83]\({}^{\star}\) \\  & & Interaction with SulA & [40]\({}^{\star}\) \\ \hline DLS & [54; 78] & Assessment of polymerization & [80; 82] \\ \hline SV & [54] & Assessment of polymerization & [41] \\  & & Interaction with SImA & [93]\({}^{\star}\) \\ \hline FRET, intrinsic fluorescence & [63] & Assessment of polymerization & [30]\({}^{\star}\) \\  & & Interaction with FtsA, MinC & [94; 95]\({}^{\star}\) \\ \hline Biosensor & [56] & Interaction with SlmA, MinC & [12; 96]\({}^{\star}\) \\ \hline Fluorescence microscopy & [97] & Assessment of polymerization & [98] \\  & & Interaction with ZapA & [75; 99] \\ \hline \multicolumn{4}{c}{**Reconstruction systems**} \\ \hline Nanodiscs & [100] & Interaction with ZipA & [37]\({}^{\star}\) \\ \hline Microbeads & [56] & Interaction with ZipA & [101]\({}^{\star}\) \\  & & Interaction of FtsA, SlmA with membrane & [102; 103]\({}^{\star}\) \\ \hline SLBs & [55; 57] & Interaction with MinCDE, FtsA, ZipA & [104; 105; 106; 107; 108]\({}^{\star}\) \\ \hline Biosensor, plasmonic sensor & [56] & Interaction with ZipA & [109; 110]\({}^{\star}\) \\  & & Interaction of SlmA, MinDE with membrane & [103; 111]\({}^{\star}\) \\ \hline Microdroplets, liposomes & [55; 56; 57; 58; 112] & Interaction with FtsA, ZipA & [113; 114; 115; 116; 117; 118; 119]\({}^{\star}\) \\  & & Interaction of MinCDE with membrane & [108]\({}^{\star}\) \\  & & Arrangement \& distribution of FtsZ species & [116; 117; 118; 119; 120; 121; 122; 123; 124; 125; 126; 127; 128; 129; 130; 131; 132; 133; 134; 135; 136; 137; 138; 139; 140; 141; 142; 143; 144; 145; 146; 147; 148; 149; 150; 151; 152; 153; 154; 155; 156; 157;lacking this region but which keep the C-terminal globular domain recognized by FtsZ. The full-length ZipA protein has been also employed in experiments using cytomimetic membranes for its reconstruction and/or solubilization (see Section 5). Years ago, a fluorescence anisotropy assay was used to screen drug candidates that perturb the essential FtsZ-ZipA interaction in _E. coli_[92]. This assay consisted of a fluorescently labeled peptide derived from the C-terminal tail of FtsZ that interacts with ZipA, together with the cytoplasmic C-terminal domain of ZipA that participates in this interaction. NMR was also used for the screening of compounds that bind to this C-terminal region of ZipA [120].