## 2 Methods and Materials
### Scaffold Hopping
#### 2.2.1 Scaffold 1 Hopping  
The Scaffold 1 growing points for hopping are shown in Figure 2B. Ligand-based and receptor-based scaffold hopping were performed on the parent molecule. To enhance the interactions exhibited in the protein-hit complexes, the minimum cut-off for the number of hydrogen bonds was set to eight for the receptor-based scaffold hopping. Heavy-atom steric clashes with the receptor were prevented by setting the maximum number of clashing ligand atoms to two and the clash criterion to <2.2 A. The Schrodinger core hopping library (named core_library_2014.1-86640.sqlite) was used for the scaffold searching. The Schrodinger scaffold library used in this study contains 86,640 cores that have been prepared from a compound database of ~6 million drug-like compounds. This core library is highly diverse and follows most of the fragment selection rules of drug discovery. We profiled the diversity of the library with molecular descriptors and included the analysis in the Supporting Information (Figure S1). For this, we used physicochemical properties that are commonly used to describe chemical libraries, such as the molecular weight, number of rotatable bonds, hydrogen bond donors, and hydrogen bond acceptors. The hits obtained from scaffold hopping are ordinarily grouped based on three measures: overlap score, side chain root-mean-square deviations (RMSDs), and synthesizability scores. A high overlap  
Figure 2: Definition of the scaffolds used in this study. (**A**) The most potent co-crystallized ligand (considered as the reference ligand) is characterized by two scaffolds (indicated in blue and violet), other than the sugar. The glycomimetic binding pocket in FmlH is shown in surface representation. (**B**) The two scaffold growing points (shown in blue) used to replace Scaffold 1 in this study. (**C**) The two scaffold growing points (shown in violet) used to replace Scaffold 2 in this study. score and a low RMSD score would indicate highly homologous compound structures. It is crucial to obtain significantly diverse chemical matter from the first step of the screening process. The more structurally diverse the compounds that are found, the higher the chance of finding novel hits. We, therefore, did not consider the overlap score or the RMSD score in the selection of compounds. It is possible that compounds with good synthetic accessibility could still have high overlap and low RMSD scores relative to the parent compound. However, if most short-listed compounds were highly homologous, that would indicate the tendency of FmlH to bind to a particular class of chemical matter. We have not observed that in this study. Our obtained hits were structurally diverse and good starting points to be considered for the subsequent steps in the screening process. So, to enable the design of chemical species with novel structures, the overlap scores and RMSD values were not considered in this study, but instead only the synthesizability scores were used for the compound ranking. For the ligand-based scaffold hopping, a synthesizability score of \(>\)0 was considered as a criterion to collect hits. The Schrodinger core hopping capability calculates the synthesizability score based on whether a particular substitution pattern has been observed in drug-like molecules, and on the number of attachment points that are substituted in the new core molecule--the higher the synthesizability score, the better [33]. A study by [PERSON] et al. used similar criteria for the synthesizability scores for selecting the hit compounds as our study [34]. To maximize our hit-finding chances, we needed to ensure that we had enough compounds from the first few steps of the screening funnel. Hence, we chose all the compounds with a positive synthesizability score. These hit compounds were subjected to molecular docking studies. The known most potent glycominetic exhibited a Glide score of \(-\)5.71 kcal\(\cdot\)mol\({}^{-1}\), as reported in our preceding study [14].