## 2 Results and Discussion  
### Fragment Libraries Virtual Screening in \(\mathrm{Rel}_{\mathrm{Sq}}\) Synthetase Site  
We chose as a protein model the X-ray crystal structure reported for \(\mathrm{Rel}_{\mathrm{Sq}}\) in a so-called synthetase-ON conformation, i.e., the chain A of the pdb structure 1 VJ7 (residues 1-385), which carries the GDP substrate in the synthetase catalytic site [25]. This is a truncated form of the protein, lacking the C-terminal regulatory domain.  
By analyzing the interactions of GDP within the catalytic site (Figure 1b), we could observe that it binds to the G-loop (Tyr299-Ser310), forming a \(\pi\)-\(\pi\) stacking interaction with the side chain of Tyr308 through its guanine ring. H-bonds with the side chains of Lys304 and Asn306 and with the backbone of Ala335 stabilize this core interaction. In addition, the GDP pyrophosphate moiety forms salt bridges with Lys304 and Lys297 side chains and H-bonds with Tyr299 and His312 side chains.  
Our analysis revealed that the \(\mathrm{Rel}_{\mathrm{Sq}}\) synthetase-ON catalytic site conformation could not be catalytically competent, as it lacks the space necessary to accommodate the pyrophosphate donor ATP, and the reported catalytic residues Asp264 and Glu323 are unfavorably oriented to promote the reaction (Figure 1b). On this basis, we constructed and reported a catalytically competent \(\mathrm{Rel}_{\mathrm{Sq}}\) chimera model based on the structure of the SAS RelP from _S. aureus_, where the resulting catalytic site is considerably more extended [36]. However, we performed an initial virtual screening on the \(\mathrm{Rel}_{\mathrm{Sq}}\) X-ray crystal structure in order to focus our binding site exploration on the region occupied by the enzyme substrate GDP.  
During protein preparation (1 VJ7, chain A, see Section 3), we took a closer look at the protonation state of His312, interacting with the \(\beta\)-phosphate group of GDP in the crystal. Although the neutral form should be more plausible, given the pH working conditions of the enzyme (activity usually tested at pH 7-9), we decided to generate a model with the residue in the protonated form as well (Hip312) in order to assess the validity of this assumption. We set up and validated a docking protocol within the synthetase binding site for both models by re-docking the co-crystallized GDP molecule using GLIDE v8.0 (Supplementary Figures S1 and S2). [37]  
Several chemical vendors currently make available virtual structure datasets of fragment libraries, often organized according to specific experimental properties (e.g., solubility). In order to maximize the chemical space explored in our screening [38; 39], we selected seven different libraries of commercially available fragments, _Maybridge_ Rule of 3, _Asinex_ Fragments, _Life Chemicals_ Fragment Library with Experimental Solubility Data I and II, _OTAVA_ Solubility fragment library, _Chembridge_ Fragment library, and _SPECS_ fragment library, amounting to a total of 58,321 2D entries (see Section 3 and Supplementary Table S1). We implemented the validated docking protocol for the virtual screening (VS) of the selected fragment libraries following the workflow shown in Figure 2. For each library, Ligprep [40] converted 2D entries into 3D structures considering stereoisomers, tautomers, and protonation states, leading to an increase in the total number of structures up to 114,966.  
Figure 2: Virtual screening workflow. Ligprep generated 3D structure from 2D fragments; Glide docked the 3D structures into both \(\mathrm{Rel}_{\mathrm{Sq}}\) models (His312 and Hip312). A state penalty filter excluded unfavorable states, and the resulting docking poses were ranked according to Gscore. Only fragments forming an aromatic interaction with Tyr308 were retained, and after removing PAINS, we evaluated the top 1% ranked poses identifying the most representative chemotypes.