Molecular docking and ligand specificity in fragment-based inhibitor discovery

Abstract

Fragment screens have successfully identified new scaffolds in drug discovery, often with relatively high hit rates (5%) using small screening libraries (1,000-10,000 compounds). This raises two questions: would other noteworthy chemotypes be found were one to screen all commercially available fragments (>300,000), and does the success rate imply low specificity of fragments? We used molecular docking to screen large libraries of fragments against CTX-M beta-lactamase. We identified ten millimolar-range inhibitors from the 69 compounds tested. The docking poses corresponded closely to the crystallographic structures subsequently determined. Notably, these initial low-affinity hits showed little specificity between CTX-M and an unrelated beta-lactamase, AmpC, which is unusual among beta-lactamase inhibitors. This is consistent with the idea that the high hit rates among fragments correlate to a low initial specificity. As the inhibitors were progressed, both specificity and affinity rose together, yielding to our knowledge the first micromolar-range noncovalent inhibitors against a class A beta-lactamase.

Figure 1

Crystal structures of fragment inhibitors and optimized derivatives against CTX-M. Five compounds have molecular weight below 250 Da, with two additional ones below 300. The surface of the protein active site is colored white (carbon), red (oxygen) and blue (nitrogen). The crystal pose of the ligand is colored yellow. The docking prediction is shown in cyan. The 2F_o − F_c electron density of the ligands is shown in white at 1σ. For compound 4, the active site is shared by the inhibitor and a phosphate molecule, both modeled at half occupancy. For compound 11, there is a partial-occupancy phosphate in the active site that is not shown here. Waters with half occupancy are also modeled in the structures of compounds 3 and 6.

Figure 2

Inhibition and binding of compound 12. (a) Dose-response curve for CTX-M. The error for any point is 2% activity or less. (b) CTX-M complex structure (stereoview). Potential hydrogen bonds are represented by black dash lines. A mediating water molecule is shown as a red sphere.