L

L. of pantothenate synthetase, an important target in tuberculosis therapy. Binding efficiencies were found to be distributed unevenly within a lead molecule derived using a fragment\based approach. Substitution of the less efficient parts of the molecule allowed systematic development of more potent compounds. This method of dissecting and analyzing different groups within a molecule offers a rational and general way of carrying out lead optimization, with potential broad application within drug discovery. pantothenate synthetase, an attractive target for developing new drugs against tuberculosis.10, 11, 12 Pantothenate synthetase catalyzes the ATP\dependent formation of an amide bond between pantoate and \alanine.11, 12 We have previously reported the identification of fragments 1 and 2 (see Schemes?1 and?2) from biophysical screens using thermal shift and NMR methods.13, 14 The stepwise growing of indole fragment 1 led to the generation of lead compound 5 (Scheme?1; see also, Figure?S1 in the Supporting Information).13 In a parallel study, linking of fragments 1 and 2 afforded compounds 6C9 (Scheme?2; Calcipotriol see also, Figure?S2 in the Supporting Information).[13,?15] Both fragment growing and linking approaches rapidly led to relatively potent inhibitors against pantothenate synthetase (5: pantothenate synthetase, generating lead compound 5. Open in a separate window Scheme 2 A fragment\linking approach applied against pantothenate synthetase generating lead compounds 6C9. (XCYCZ represents the approximate three\atom length of the linker.) Based solely on the values of the compounds were determined from titration experiments using ITC. The GE value is subsequently calculated by dividing the contribution from each group by the number of heavy atoms in the group. As shown in Figure?1, the majority of the binding energy resides in the original indole fragment (GE=0.75). A similar observation has been observed in other fragment elaboration strategies6 and is mainly due to the high intrinsic binding energies required for fragments to be detected (pantothenate synthetase was determined by isothermal titration calorimetry (ITC), and the structureCaffinity relationship (SAR) results are summarized in Table?1; ITC binding data for all compounds are presented in the Supporting Information). Replacing the methyl pyridine/benzofuran groups in 5 and 8 generated a series of sub\micromolar inhibitors (10C14).The substitution of the methyl pyridine ring (5) by a more electron\rich toluene group (10: values derived from ITC and the number of heavy atoms associated with the corresponding groups/compounds. [c]?cLog?values were derived from ChemDraw. Gratifyingly, the addition of a bulkier and more electronegative trifluromethyl group to the indole sulfonamide core gave rise to 11, the most potent compound of this series (pantothenate synthetase (PDB code: 4MQ6, 4MUE, 4MUF, Calcipotriol 4MUL, respectively). The ligands are shown as sticks with carbon atoms in light blue, nitrogen atoms in dark blue, oxygen atoms in red, and sulfur atoms in yellow. The cross\sectional area of the active pocket of pantothenate synthetase is shown in green. All figures were generated and rendered with PyMOL v.0.99.20 The X\ray crystal structures of 10C13 bound to pantothenate synthetase show binding at the active site, with a conserved binding mode for the indole sulfonamide fragment core. Less obviously, the substituted groups on all four compounds were seen to bind in the P1 pocket of the enzyme (see Figure?1?B). The P1 pocket DKFZp781B0869 binds the alkyl groups of the pantoate substrate and is primarily lipophilic, surrounded by the hydrophobic residues Pro?38, Met?40, Val?143, Leu?146 and Phe?157 (Figure?S5 in the Supporting Information). In contrast, the P2 site binds the phosphates of ATP and is relatively hydrophilic. As can be seen in Figure?2, the binding orientations of the added groups are Calcipotriol all similar, and no new hydrogen bonds are formed. The detailed binding interactions of the most potent compound (11) with the P1 pocket residues are shown in Figure?S5 in the Supporting Information. In addition to binding assays and X\ray crystallography studies, an inhibition study was carried out that demonstrated that compound 11 inhibits pantothenate synthetase with an IC50 value of 5.7?m (see the Supporting Information). The structural data on compounds 10C13 provided the impetus for further elaboration.