Lessons from Hot Spot Analysis for Fragment-Based Drug Discovery

Abstract

Analysis of binding energy hot spots at protein surfaces can provide crucial insights into the prospects for successful application of fragment-based drug discovery (FBDD), and whether a fragment hit can be advanced into a high-affinity, drug-like ligand. The key factor is the strength of the top ranking hot spot, and how well a given fragment complements it. We show that published data are sufficient to provide a sophisticated and quantitative understanding of how hot spots derive from a protein 3D structure, and how their strength, number, and spatial arrangement govern the potential for a surface site to bind to fragment-sized and larger ligands. This improved understanding provides important guidance for the effective application of FBDD in drug discovery.

Figure 1

Hot spots and selected ligands of HSP-90 and B-RAF. Top row: HSP-90 ligands. The HSP-90 hot spots shown, based on the mapping on an HSP-90 apo structure (PDB ID 1yes) are CC1 (cyan, 22 probe clusters), CC2 (magenta, 13 clusters), CC3 (salmon, 12 clusters), and CC5 (green, 9 clusters). Hot spots CC1, CC2, and CC5 overlap. (a) Fragment 2-methyl-4-diethylamide-phenol from PDB ID 2xdl (K_d = 790,000 nM) overlaps with hot spots CC1, CC2, and CC5. (b) Inhibitor from PDB ID 2xab (K_d = 0.54 nM) retains the binding mode of the fragment, but overlaps better with CC1, resulting in much improved affinity. (c) Inhibitor from PDB ID 2xjx (K_d = 0.71 nM) has been developed to have better pharmaceutical properties, but the added moiety did not interact with any hot spot, and the affinity did not improve. (d) Benzolactam inhibitor from PDB ID 4o0b (K_d = 5 nM). The inhibitor only partially overlaps with CC5, but protrudes into CC3. The reduced K_d value demonstrates that interactions with regions of the protein surface between the hot spots do not contribute much to the binding free energy. Bottom row: BRAF ligands. The BRAF hot spots shown, based on the mapping of a BRAF(V600E) structure co-crystallized with the ligand PLX4720 (PDB ID 3c4c) are CC1 (cyan, 16 probe clusters), CC2 (magenta, 15 clusters), CC3 (yellow, 15 clusters), and CC4 (salmon, 9 clusters). (e) Fragment N-phenyl-1H-pyrrolo[2,3-b]pyridin-3-amine, co-crystallized with Pim-1 kinase domain rather than BRAF, from PDB ID 3c4e (IC₅₀ = 100,000 nM) overlaps with CC1 and CC3. (f) Inhibitor PLX4720, co-crystallized with BRAF(V600E), from PDB ID 3c4c. PLX4720 retains the core 1H-Pyrrolo[2,3-b]pyridine fragment, but the fragment position is slightly shifted relative to its position in Pim-1. The overlap with CC3 is substantially improved, and PLX4720 reaches into both CC2 and CC4, resulting in much higher affinity (IC₅₀ = 13–160 nM). (g) PLX3203, co-crystallized with BRAF(V600E), from PDB ID 4fk3, retains the position of the core fragment in CC1 and CC3, and improves overlap with CC2 and CC4. (h) Vemurafenib (PLX4032) from PDB ID 3og7 also retains the position of the core, and further optimizes overlap with CC4 ((IC₅₀ =21–100 nM).

Figure 2

Hot spots and selected ligands of protein kinase B/A (PKB/PKA) and urokinase-type plasminogen activator (uPA). Top row: PKB/PKA ligands. The PKB/PKA hot spots, based on the mapping a PKA-PKB chimera co-crystallized with (2R)-2-(4-chlorophenyl)-2-phenyl-ethanamine (PDB ID 2uw8) are CC1 (cyan, 21 probe clusters), CC3 (yellow, 14 clusters), CC7 (orange, 5 clusters), and CC8 (green, 3 clusters). (a) 3-methyl-4-phenyl-1h-pyrazole fragment (binding to the PKA-PKB chimera) from PDB ID 2uw3 (IC₅₀ = 80,000 nM) overlaps very well with CC1, but does not reach any other hot spot. The high level of overlaps predicts the conservation of fragment binding mode. (b) Extension of the fragment into CC3 yields the potent inhibitor from PDB ID 2uw5 (IC₅₀ = 34 nM). Again, the X-ray structure was determined for a PKA-PKB chimera. (c) Different chemotype but fairly similar binding mode in the potent inhibitor binding to PKA from PDB ID 1sve (IC₅₀ = 5 nM). The inhibitor interacts well with CC1, and reaches all other hot spots. (d) Potent inhibitor of PKA from PDB ID 2f7e (IC₅₀ = 2.1 nM) retains the binding mode in CC1 and has very good overlap with CC3. It appears that this is sufficient for strong binding, in spite of no interactions with CC7 and CC8. Bottom row: uPA ligands. The uPA hot spots, based on the mapping of a uPA apo structure (PDB ID 2o8t) are CC1 (cyan, 29 probe clusters), CC2 (magenta, 26 clusters), CC3 (yellow, 13 clusters), and CC6 (blue, 3 clusters). (e) Fragment from PDB ID 2vin (IC₅₀ = 1,000,000 nM) overlaps only with CC1, but the high level of overlap predicts conservation of the binding mode. (f) Fragment based inhibitor from PDB ID 2viw (IC₅₀ = 72 nM) extends into CC2 and somewhat into CC3. (g) Inhibitor with different chemotype from PDB ID 1gj9 (K_i = 11 – 140 nM), resulting in better overlaps with CC1 and CC2. (h) An orally bioavailable, non-amidine Inhibitor with a third chemotype and binding mode from PDB ID 3ig6 (IC₅₀ = 25 nM) has good overlaps with CC1 and CC2, and reaches into CC3.

Figure 3

Hot spots and selected ligands of β-secretase (BACE-1) and cyclin dependent kinase 2 (CDK2). Top row: BACE-1 ligands. The BACE-1 hot spots, based on the mapping of a BACE-1 apo structure (PDB ID 1w50) are CC1 (cyan, 19 probe clusters), CC3 (yellow, 16 clusters), CC5 (white, 7 clusters), CC7 (orange, 5 clusters) and CC8 (green, 5 clusters). The hot spot structure is very diffuse, and some hot spots partially overlap. (a) 4-butoxy-3-chlorobenzyl imidothiocarbamate fragment from PDB ID 3kmx (K_d = 15,000 nM) only partially overlaps with any hot spot. As discussed in the paper, the overlap with CC1 is not sufficient for the conservation of the binding mode, and the fragment was used only to capture some of the important interactions. (b) Inhibitor from PDB ID 3l5f (IC₅₀ = 605 nM) overlaps better with CC1, CC3, and CC7, and reaches CC8, resulting in higher affinity. (c) The potent inhibitor LY2811376 from PDB ID 4ybi retains the binding mode of the inhibitor in 3b, and also reaches into CC8. (d) The inhibitor by Amgen from PDB ID 4xkx also retains the binding mode. Bottom row: CDK2 ligands. The CDK2 hot spots, from the mapping of a CDK2 apo structure (PDB ID 4ek3) are CC1 (cyan, 24 probe clusters), CC3 (yellow, 8 clusters), and CC5 (white, 6 clusters). (e) Fragment from PDB ID 2vtm (IC₅₀ = 1,000,000 nM) overlaps only with CC1, and the level of overlap seems to be too low to yield conservation of the binding mode. (f) Inhibitor containing parts of the fragment from PDB ID 2vtn (IC₅₀ = 850 nM) extends into CC3. (g) Inhibitor from PDB ID 2vtp (IC₅₀ = 3 nM) was developed from the previous compound, but it also extends into CC5, resulting in much improved binding affinity. (h) An imidazole piperazine inhibitor with another binding mode from PDB ID 2vv9 (IC₅₀ = 17 nM). The inhibitor overlaps well with CC1 and CC3, and slightly extends toward CC5.