## 2 Results and Discussion
### Thermal Shift Assay on Selected Fragments vs. Rel\({}_{Seq}\) Protein Constructs  
With the aim of studying protein-ligand interactions between Rel\({}_{Seq}\) constructs and selected fragments, we evaluated the use of different techniques, such as tryptophan assay, microscale thermophoresis (MST), isothermal calorimetry (ITC) or thermal shift assay (TSA). The tryptophan assay is based on ligand-induced conformational changes in the local environment surrounding tryptophan residues in the target protein. Irradiation at 280 nm is followed by the detection of tryptophan fluorescence emission at different wavelengths. We used this technique to measure the Kd values of the natural substrates GDP and ATP (see below), but it is not applicable in the case of fragments due to their high absorbance at 280 nm. On the other hand, the currently available protein-labeling reagents for MST were not compatible with our protein constructs. We chose TSA over ITC due to its higher potential throughput and lower amount of protein required. Thermal shift is an experimental technique in which protein thermal denaturation is monitored following the increase in fluorescence reported by a protein-bound dye [49]. In particular, an environment-sensitive hydrophobic dye (e.g., SYPRO Orange) binds to hydrophobic regions that become progressively exposed during thermal denaturation, resulting in an increase in its fluorescence emission. Since the binding of small molecules (e.g., fragments) to the protein can cause conformational changes that affect its melting temperature, this technique allows the screening of several compounds in a range of concentrations without consuming sizeable amounts of protein. We preliminary determined by TSA the dissociation constants (\(K_{\text{d}}\)) of complexes engaged by Rel\({}_{Seq}\) with its natural substrates, ATP (0.49 \(\pm\) 0.09 mM) and GDP (0.26 \(\pm\) 0.06 mM), finding values comparable to those obtained by tryptophan assay (\(K_{\text{d}}\)\({}^{\text{ATP}}\) = 0.39 \(\pm\) 0.04 mM, \(K_{\text{d}}\)\({}^{\text{GDP}}\) = 0.15 \(\pm\) 0.01 mM) and confirming the robustness of this technique (Table 1, entries 1 and 2). Figure 4: Fragments selected for experimental validation (i.e., thermal shift assay) grouped by chemotype: benzimidazole (**B1–B4**), aminobenzic acids (**A1–A7**), indoles (**I1–I4**), and the three singletons (**BO1**, **BT1**, and **TP1**). We therefore evaluated the affinity of the 18 fragments selected from our _in silico_ screening for the bifunctional protein \(\mathrm{Rel}_{Seq}\) by titration of the protein in a thermal shift assay (Table 1). We initially used \(\mathrm{Rel}_{Seq}\) 1-385, a truncated construct lacking the abovementioned C-terminal regulatory domain with a catalytic activity intrinsically shifted towards (p)ppGpp synthesis (12-fold higher than the full-length protein) [50]. Interestingly, all but four of the tested fragments showed a dose-dependent interaction with the bifunctional protein with \(K_{\mathrm{d}}\) values in the low millimolar range. In particular, only one of the four tested benzimidazoles showed a measurable affinity for \(\mathrm{Rel}_{Seq}\) (1-385) (**B3**, Table 1, entry 5), whereas fragment **B2** yielded a biphasic curve that requires further investigation (Table 1, entry 4). All of the selected aminobenzoic acids showed good affinities for the protein, with the exception of **A2** (Table 1, entries 7-13), while among the four indoles tested, all interacted in a dose-dependent manner with \(\mathrm{Rel}_{Seq}\) (1-385) (Table 1, entries 14-17). Finally, two of the three singletons tested (**BO1** and **TP1**) showed a low mM affinity for the protein. We assessed the fragments' selectivity for \(\mathrm{Rel}_{Seq}\) synthetase domain by performing TSA experiments on two mono-functional truncated protein constructs: \(\mathrm{Rel}_{Seq}\) SYNTH (residues 79-385) and \(\mathrm{Rel}_{Seq}\) HYD (residues 1-224), presenting only the synthetase or hydrolase protein domain, respectively.