Mechanism of activation and the rewired network: New drug design concepts

Abstract

Precision oncology benefits from effective early phase drug discovery decisions. Recently, drugging inactive protein conformations has shown impressive successes, raising the cardinal questions of which targets can profit and what are the principles of the active/inactive protein pharmacology. Cancer driver mutations have been established to mimic the protein activation mechanism. We suggest that the decision whether to target an inactive (or active) conformation should largely rest on the protein mechanism of activation. We next discuss the recent identification of double (multiple) same-allele driver mutations and their impact on cell proliferation and suggest that like single driver mutations, double drivers also mimic the mechanism of activation. We further suggest that the structural perturbations of double (multiple) in cis mutations may reveal new surfaces/pockets for drug design. Finally, we underscore the preeminent role of the cellular network which is deregulated in cancer. Our structure-based review and outlook updates the traditional Mechanism of Action, informs decisions, and calls attention to the intrinsic activation mechanism of the target protein and the rewired tumor-specific network, ushering innovative considerations in precision medicine.

Keywords: K-Ras4B; KRAS; cancer network; driver mutations; drug discovery; inhibitor; kinases.

Figure 1

Bcr‐Abl kinase domain structure. Bcr‐Abl can be drugged with a combination of orthosteric and allosteric inhibitors to hinder the development of drug resistance. Crystal structure of Bcr‐Abl kinase domain (PDB: 3K5V) with the orthosteric inhibitor imatinib (green) and the allosteric inhibitor GNF‐2 (pink). Highlights of the ATP binding pocket with imatinib (upper right panel) and the myristate binding pocket with GNF‐2 (lower right panel) [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Structural insights into the driver mutations in kinases. (A) The L858R driver mutation in EGFR destabilizes the inactive structure (PDB: 1XKK) and stabilizes the active conformation (PDB: 6JX4). (B) The “gatekeeper” mutations in EGFR (PDB: 6JX4), Bcr‐Abl (PDB: 2GQG), Src (PDB: 1YI6), c‐Kit (PDB: 1PKG), and PDFGRα (PDB: 6JOI) stabilize the R‐spine for the active conformation. The mutated residues were modeled based on the crystal structures [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

B‐Raf mutations and inhibitors. B‐Raf kinase domain structure with highlighted ⁵⁹⁴DFG⁵⁹⁶ motif (left panel). Examples of B‐Raf inhibitors (right panels). Inhibitors can bind to active or inactive B‐Raf. GDC‐0879 is a Type 1 inhibitor that binds to the active form of B‐Raf with αC‐helix‐in and DFG‐in. Vemurafenib is a Type 1½ inhibitor that binds to an inactive form of B‐Raf with αC‐helix‐out and DFG‐in. Sorafenib is a Type 2 inhibitor that binds to an inactive form of B‐Raf with αC‐helix‐in and DFG‐out. The αC‐helix and the side chains of DFG motif are colored blue and black, respectively. In the cartoons, the crystal structures (PDB: 4MNE, 4MNF) were used to model the protein structures. The mechanism of activation for B‐Raf mutation classes (bottom panels). B‐Raf mutations are grouped into three classes based on activation mechanisms. B‐Raf kinase domain with Class I (pink), Class II (green), and Class III (gray) mutation sites highlighted. Class I mutations are Ras and dimer independent. Class II mutations are Ras independent but require homodimerization. Class III mutations require activation via mutated Ras and dimerization with wild‐type C‐Raf [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

PI3Kα structure and mutations. A modeled PI3Kα structure (left panel) based on the crystal structure (PDB: 4OVV). PI3Kα is an obligate heterodimer composed of the p110α catalytic and p85α regulatory subunits. Mutations in the p110α subunit of PI3Kα (right panel). The p110α subunit in PI3Kα contains the hotspot (E542K, E545K in the helical domain; H1047R in the kinase domain) and weak (R38H/C, R88Q, R93Q, R108H, and G118D in the ABD; N345R/K, C420R/K, and E453K/Q in the C2 domain; and E726K, M1043V/I in the kinase domain) driver mutations [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Co‐occurrence patterns of mutations on same genes and pathways. (A) Prevalence of the double mutant tumors on four tumor suppressor genes, TSGs (TSC1, APC, NF1, and PTEN) and four oncogenes, OGs (BRAF, KRAS, PIK3CA, and EGFR) among the tumors in brain, bowel, stomach, bladder, uterus, breast, and lung tissues. Source nodes are genes harboring significant double mutations, and target nodes are the tissues enriched with double mutant tumors. Green source nodes are tumor suppressors, red source nodes are oncogenes. Size of the arc proportional to the number of double mutant tumors, arc color is compatible with the target node color. (B) Heatmap shows fraction of different gene double mutant tumors where constituents of the double mutations belong to the pathways on the x‐axis and y‐axis. Fractions are calculated based on the ratio of the double mutant tumors from pathway 1 and pathway 2 to the number of double mutant tumors where one component from pathway 1 or pathway 2. (C) Fraction of different gene double mutant tumors in breast, brain, bowel, lung, and uterus tissues. More than 25% of double mutant tumors where one component from PI3K and the other from TP53 pathways are accumulated in breast tissue. The fraction of double mutant tumors with components from PI3K and RTK/Ras pathways is ~5% [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

BRCA cell lines and xenografts drug responses. (A) Violin plot showing drug response distributions of PIK3CA wild‐type, single (H1047), and double mutant (H1047/P539) cell lines. Drugs with z‐score < −0.5 in the double mutant cell line (BT‐20) are covered. 18 drugs (out of 39 common drugs) target the PI3K/mTOR signaling pathway. (B) Comparisons of tumor growth rates of wild type, single, and double mutant xenografts before and after treatments with several drugs. Double mutant xenograft shows better treatments with drugs, slowing down tumor growth rate [Color figure can be viewed at wileyonlinelibrary.com]