Considerations of Protein Subpockets in Fragment-Based Drug Design

Abstract

While the fragment-based drug design approach continues to gain importance, gaps in the tools and methods available in the identification and accurate utilization of protein subpockets have limited the scope. The importance of these features of small molecule-protein recognition is highlighted with several examples. A generalized solution for the identification of subpockets and corresponding chemical fragments remains elusive, but there are numerous advancements in methods that can be used in combination to address subpockets. Finally, additional examples of approaches that consider the relative importance of small-molecule co-dependence of protein conformations are highlighted to emphasize an increased significance of subpockets, especially at protein interfaces.

Keywords: computational screening; fragment; fragment-based drug design; subpocket.

Figure 1

The conformation of the adenine portion of ACP is conserved between two structurally and sequentially diverse proteins, demonstrating a shared subpocket at the binding site (HSP90 N-terminal domain, yellow, PDB: 3t10; chemotaxis protein CheA, blue, PDB: 1i5a).

Figure 2

Conserved subpockets in protein kinases contribute to their inherent promiscuity. (A) Staurosporine (shown as partially transparent) is known to have inhibitory activity against a plethora of protein kinases from different families (107). Conserved subpockets between the TAO2 MAP3-level kinase (green loop) and serine-threonine kinase 16 (STK16) (blue loop) allow staurosporine to bind with comparable affinities to both proteins (TAO2, PDB ID: 2GCD; STK16, PDB ID: 2BUJ). (B) The promiscuous RET tyrosine kinase (orange surface) contains a binding site subpocket that allows for the binding of overall structurally distinct inhibitors. The red highlighted portion of each inhibitor overlaps in the subpocket, shown with the red circle on the right (green, PDB ID: 2IVU; blue, PDB ID: 2IVV). (C) Within the binding pocket of JNK3 MAP kinase, residues Glu147, Met149, and Val196 form a microenvironment that binds comparable chemical features between a natural ligand, adenosine monophosphate (AMP) (PDB ID: 4KKE), and inhibitors such as the dihydroanthrapyrazole-based antagonist shown on the right (PDB ID: 1PMV).

Figure 3

Protein interface hotspots contain inducible microenvironments that bind conserved fragments between molecule types. (A) Three residues of p53 (F19, W23 and L25; green sticks) become buried in the surface of MDM2 (blue surface), inducing the formation of hydrophobic subcavities. Residue W23, in particular, likely acts as an anchoring residue, substantially contributing to the binding affinity (PDB ID: 1YCR). (B) Four key residue positions (green sticks) in the highly conserved PIP box sequence are essential for the binding of PIP box-containing proteins (p21 shown as green loop) to PCNA (orange surface). The fifth, seventh, and eighth residues in the sequence bind at a surface pocket on PCNA made up of several hydrophobic microenvironments (PDB ID: 1AXC). (C) Within the hydrophobic PIP box-binding site on PCNA (orange surface), the subpocket defined by the relative orientation of I128, Y133, Y250, P234, and V236 to one another has affinity for an aromatic ring moiety, with a tyrosine residue (green sticks) of p21’s PIP box sequence (green loop) anchored in the same location as the tyrosine-analogous fragment of the small-molecule inhibitor T3 (127) (PDB ID: 3VKX, p21 peptide from 1AXC). (D) Some receptors exist that themselves accept a variety of structurally diverse substrates, the best example being the large family of GPCR olfactory receptor proteins. OR1G1, a member of this family, becomes activated upon exposure to numerous diversified odorants (130).