DFGmodel: predicting protein kinase structures in inactive states for structure-based discovery of type-II inhibitors.

The protein kinase family is one of the largest protein families in human, comprising 518 different kinases that function as on/off switches in cellular signaling pathways and modulate almost all basic cellular activities.1,2 Malfunctions in protein kinases are associated with various diseases,3 such as cancer,4 and autoimmune disorders.5 Therefore, protein kinases are one of the most pursued targets for drug development.6,7 In fact, 25 kinase drugs have already been approved by the FDA, and many other potential kinase drugs are currently in clinical trials.8,9 All protein kinases share similar structural fold.10 This includes a catalytic domain comprising a smaller N-terminal subdomain (N-lobe) and a large C-terminal subdomain (C-lobe), in which the cleft between the two lobes forms the ATP-binding site. The N-lobe is composed of a β-sheet and a long α-helix (αC-helix), whereas the C-lobe is predominantly α-helical. The two lobes are connected by the hinge region, in which the N-lobe can adopt a wide range of positions relative to the C-lobe.11 Situated in the C-lobe, the Asp-Phe-Gly (DFG) motif is a highly conserved motif that forms part of the ATP-binding site and coordinates magnesium binding. Immediately following the DFG-motif is the activation loop (A-loop), a stretch of 20–30 residues, which serves as the regulator of kinase activities.12,13 Protein kinases are highly dynamic. The N-lobe, αC-helix, hinge region, and A-loop can undergo a wide range of movement and adopt multiple conformations, such as DFG-flip and rotation of αC-helix, that define catalytic activity.14−18 Particularly, the DFG-motif of protein kinases adopts two major conformations, including the DFG-in and DFG-out conformations, which are thought to determine active or inactive states, respectively, as well as various intermediate conformations. In the active state or DFG-in conformation, the DFG-Phe is packed into a hydrophobic pocket, the DFG-pocket, between the N- and C-lobes, and stabilizes this active conformation through interactions with hydrophobic residues in that region.19 In this conformation, the ATP-binding site is well-defined; the DFG-Asp faces outward to coordinate a magnesium ion for ATP binding, whereas the A-loop moves away from the ATP-binding site and forms a β-hairpin for substrate binding. Currently, there are 16 FDA-approved drugs that target the ATP-binding site in this conformation to competitively inhibit ATP binding (type-I kinase inhibitors, e.g., vandetanib).9 Conversely, in the inactive state or DFG-out conformation, the DFG-Asp flips, and the DFG-Phe moves out of the DFG-pocket (Figure ​(Figure1A),1A), which can adopt a range of conformations (Figure S1). This DFG-flip induces movement in the A-loop, thereby deforming and obstructing part of the ATP-binding site. The unoccupied DFG-pocket joins with the deformed ATP-binding site and becomes accessible for ligand binding. Drugs that target the DFG-out conformation (type-II kinase inhibitors), such as the cancer drug imitanib (Gleevec) and sorafenib (Nexavar), have been recently developed, and current effort has focused on the design of this class of inhibitors.9,20 Notably, structural data of kinases with the DFG-out conformation remains scarce, making it difficult to discover unique type-II inhibitor scaffolds for key drug targets such as mTOR and RET with structure-based drug design methods. Thus, several approaches have been developed to model kinase domains in the DFG-out conformation. For example, Kufareva and Abagyan deleted the DFG-motif in a DFG-in kinase structure and replaced it with an attractive potential density.21 Xu et al. modeled the DFG-out conformation by building the A-loop ab initio and rotating the N-lobe by a fixed amount of degree to account for the conformational changes.22 However, the extent of the N-lobe rotation can be highly variable and is still unknown for some kinases (e.g., mTOR). Furthermore, the modeling of the roughly 25-residue A-loop is time-exhaustive and subject to large errors.23 Figure 1 DFG-motif flips between two conformations. (A) Type-II inhibitor sorafenib (cyan sticks) is not compatible with the DFG-in conformation because it overlaps with the phenylalanine residue of the DFG-motif (red stick). (B) The DFG-motif and the residues ... Here, we introduce a method to generate structural models of the kinase domain in DFG-out conformations using homology modeling. We first perform a comprehensive analysis of all experimentally determined kinase structures in complex with small molecule ligands. We then describe the development of DFGmodel, a method that constructs DFG-out kinase models based on multiple structures of related kinases, and evaluate the method’s performance with a variety of measures (e.g., statistical potentials). Finally, we illustrate the utility of the approach by describing three case studies of relevant targets.