Fig 1.
Kinase binding to type-II kinase inhibitors.
(A) The conformational state of protein kinases (e.g., KDR) including DFG-in (red) and DFG-out (blue) is determined by the DFG-motif. The DFG-pocket (cyan mesh) is unique to the DFG-out conformation. Sorafenib is shown in pink. Broken yellow lines indicate hydrogen bonds. (B) A scheme depicting the positive and negative effects of drug acting on genetic modifiers of medullary thyroid cancer in a Drosophila model. ptc-driven dRetM955T induces lethality during development. ‘Suppressors’ or ‘enhancers’ suppress or enhance, respectively, dRetM955T-induced disease phenotypes as revealed in genetic screening. A drug can suppress lethality by inhibiting the suppressors. It can also induce toxicity and/or worsen transformed phenotypes by inhibiting the enhancers, which results in enhanced lethality.
Fig 2.
Fly genetics and computational chemistry discovery platform.
Key steps include (A) determining suppressors and enhancers in a dominant modifier genetic screening and their in silico modelability, (B) generating DFG-out kinase models using DFGmodel, (C) virtual screenings of compound libraries against the modeled suppressors and enhancers, combining top-ranking screening results into consensus result, (D) testing top-ranking compounds for rescue of lethality (left panel) and migration of transformed cells in developing wing discs of ptc>dRetM955T flies (right panel), and (E) refining hits by combining structural elements of computationally derived hits and that of drugs and evaluating new targets.
Fig 3.
(A) Electrostatic potential (red, negative potential; blue, positive potential) on the surface of the DFG-pocket in various kinases, including the suppressors RET and SRC, the enhancer TTK, and ERK. (B) Accessible volume of the DFG-pocket (colored volume) for potential type-II kinase inhibitor. Hit molecule 1 is depicted in pink sticks. Broken yellow lines indicate hydrogen bonds.
Fig 4.
(A) Rescue of ptc>dRetM955T fly lethality by 1 and 1–1. Both showed improved efficacy (synergy) when co-administrated with 200 μM sorafenib (soraf). (-), vehicle DMSO control. Error bars represent standard error in triplicate experiments. *P < 0.05 in one-sided Student’s t-test as compared with vehicle control. (B) Docking pose of 1 and its analogs 1–1 and 1–2 (salmon sticks) with a DFG-out model of RET (broken yellow lines indicate hydrogen bonds), and their inhibition of migration of the dRetM955T-expressing cells. Right, suppression of cell migration by 1 and 1–1. Controls are shown in (C). (C) In vivo cell migration assay in ptc>dRetM955T flies. Left, a developing whole wing disc containing GFP-labeled, dRetM955T-expressing cells constituting a stripe in the midline. The disc margin is visualized with DAPI (red pseudocolor). There are wild-type cells in black areas. Center, overgrowth of dRetM955T-expressing cells resulting in the thickening of the stripe in the apical view (top). Virtual z-series view of confocal images derived from the plane indicated by yellow dotted lines (bottom) shows dRetM955T-expressing cells migrating away from the original domain (arrows). Right, sorafenib suppressed the migration. White scale bars, 50 μm.
Fig 5.
Rescue of ptc>dRetM955T flies by 2 and its analogs.
(A) ptc>dRetM955T viability assay. 2 showed increased efficacy when co-administrated with 200 μM sorafenib. (-), vehicle control. Error bars represent standard error in triplicate experiments. *P < 0.05 in one-sided Student’s t-test as compared with no-drug control. (B) Chemical structure of 2 and its analogs. (C) Docking pose of 2 and its analogs in a RET DFG-out model. These compounds are proposed to be putative type-II kinase inhibitors that bind in the DFG-pocket through their 1H-indole moiety and interact with the conserved αC-helix glutamate side chain and DFG-Aspartate backbone (broken yellow lines).
Table 1.
Kinase inhibition profile of compound 1 at 50 μM.
Table 2.
Kinase inhibition profile of compounds 2 and 2–3 at 50 μM.
Fig 6.
Hybrid compounds with improved efficacy.
(A) The kinase inhibitors imatinib, sorafenib, and 2–3 share the common N-phenylcarboxamide moiety (grey box), while the 1H-indole-2-carboxamide of 2–3 resembles the urea linker of sorafenib (blue box). (B) Hybridization of 2 and sorafenib and AD-80 yielded 3 and 4, respectively. Top, chemical structures of compounds. Bottom, docking poses of compounds in a RET DFG-out model. (C) 3 rescued ptc>dRetM955T flies more effectively than by either 2 or sorafenib alone. (-), vehicle control. Error bars represent standard error in triplicate experiments. *P < 0.05 in one-sided Student’s t-test as compared with no-drug control. (D) 3 suppresses migration of dRetM955T-expressing wing disc cells from the original domain (green) similarly to the positive control, sorafenib. Top and bottom, apical and z-series views at the yellow dotted lines in apical view, respectively. Arrows, migrating cells. White scale bars, 50 μm.
Table 3.
Kinase inhibition profile of compound 3 at 50 μM.