Impact of linker strain and flexibility in the design of a fragment-based inhibitor

Abstract

The linking together of molecular fragments that bind to adjacent sites on an enzyme can lead to high-affinity inhibitors. Ideally, this strategy would use linkers that do not perturb the optimal binding geometries of the fragments and do not have excessive conformational flexibility that would increase the entropic penalty of binding. In reality, these aims are seldom realized owing to limitations in linker chemistry. Here we systematically explore the energetic and structural effects of rigid and flexible linkers on the binding of a fragment-based inhibitor of human uracil DNA glycosylase. Analysis of the free energies of binding in combination with cocrystal structures shows that the flexibility and strain of a given linker can have a substantial impact on binding affinity even when the binding fragments are optimally positioned. Such effects are not apparent from inspection of structures and underscore the importance of linker optimization in fragment-based drug discovery efforts.

Figure 1

Substrate fragment tethering strategy and application to human uracil DNA glycosylase (hUNG2). (a) The method involves linking a substrate- derived aldehyde fragment to a library of aldehydes using bivalent oxyamine linkers (n = 2 – 6). The tethering reactions are performed in high-throughput and high-yield (>90%) using 96-well plates ^-. Without the need for purification, the libraries are directly screened against a desired enzyme target to rapidly identify inhibitors. (b) Substrate fragment tethering using 6-formyl uracil (11) as the substrate fragment yielded the first small molecule inhibitor of the DNA repair enzyme hUNG2 (13, K_D = 6 μM). The interactions of the uracil and library fragments of dioxime 13 with hUNG2 are shown (PDB ID 2HXM). The tether does not directly interact with the enzyme and has an unusual kinked conformation (see text).

Figure 2

Diversification of rigid bivalent oxime linkers into flexible monoamine and diamine linkers. (a) The rigid and planar sp² centers of the dioxime (DO) linkers can be systematically converted into more flexible sp³ linkages using amine chemistry. Thus, the original uracil fragment libraries can be transformed into three different amine libraries. The monoamine (MA) libraries have a flexible sp³ amine center at the uracil end of the tether (MA1), or the diversified end of the tether (MA2). The diamine (DA) library has flexible amine centers at both ends of the tether. (b) The oxime and amine linkers can present the uracil and 4-carboxybenzaldehyde (29) binding fragments in the observed productive binding mode shown in Figure 1b. MMF3 molecular mechanics computations were used to superimpose the corresponding MA1, MA2 and DA linker versions with the crystallographically determined bound conformation of 13. In this computation, the uracil and carboxylate atoms of each compound were superimposed and frozen while the linkers were allowed to equilibrate.

Figure 3

Structures and inhibition profiles of library aldehyde fragments containing DO, MA1, MA2 and DA linkers. (a) Structure of library aldehydes used in the synthesis of DO, MA1, MA2 and DA libraries. (b) Concentration dependence of hUNG2 inhibition by the amine and oxime linker compounds generated from library aldehyde 30. The IC₅₀ values were DO (14) = 40 μM; MA1 (6) = 1.3 μM; MA2 (22) = 100 μM; DA (27) = 315 μM (c) IC₅₀ values for library aldehyde fragments 29 – 34 in the context of DO, MA1, MA2 and DA linkages. For aldehyde fragments 32 – 34, only DO and MA1 linkers were tested. Experiments were repeated in triplicate and errors are standard deviations of the data from the fitted curve. (d) Conformations and interactions of the bound DO (14) and MA1 (6) inhibitors derived from library aldehyde 30 (see Supplemental Figure 1 for electron density map of the complex with 6). The structures of MA2 (22) and DA (27) revealed that, for these compounds, fragment 30 did not interact with its binding site (Supplemental Figure 2 online).

Figure 4

Free energy changes (ΔΔG) arising from switching between flexible amine and rigid oxime linkages that connect the uracil and benzoic acid (30) binding fragments. Difference free energies are in kcal/mol relative to the DA (27) compound. The individual NH linkages that are changed when switching from DA (27) to MA1 (6), DO (14), or MA2 (22) are numbered as indicated (see text for further details).

Scheme 1

Synthesis of rigid and flexible oxime and amine substrate fragment libraries. (a) Monoamine 1 (MA1) library. Compounds 5 - 10 were constructed using the six aldehydes 29 - 34 shown in Figure 3a. (b) Dioxime (DO) library. Compounds 13 - 18 were constructed using the six aldehydes 29 - 34 shown in Figure 3a. (c) Monoamine 2 (MA2) library. Compounds 21 - 23 were constructed using the three aldehydes 29 - 31 shown in Figure 3a. (c) Diamine (DA) library. Compounds 26 – 28 were constructed using the three aldehydes 29 - 31 shown in Figure 3a. DMSO, dimethylsulfoxide; TEA, triethylamine; DMF, dimethylformamide; AcCl, acetyl chloride.