Structural Analysis of the Binding of Type I, I1/2, and II Inhibitors to Eph Tyrosine Kinases.
ABSTRACT: We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out). The three structures with type I1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases.
Project description:Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.
Project description:Understanding the governing factors of fast or slow inhibitor binding/unbinding assists in developing drugs with preferred kinetic properties. For inhibitors with the same binding affinity targeting different binding sites of the same protein, the kinetic behavior can profoundly differ. In this study, we investigated unbinding kinetics and mechanisms of fast (type-I) and slow (type-II/III) binders of p38? mitogen-activated protein kinase, where the crystal structures showed that type-I and type-II/III inhibitors bind to pockets with different conformations of the Asp-Phe-Gly (DFG) motif. The work used methods that combine conventional molecular dynamics (MD), accelerated molecular dynamics (AMD) simulations, and the newly developed pathway search guided by internal motions (PSIM) method to find dissociation pathways. The study focuses on revealing key interactions and molecular rearrangements that hinder ligand dissociation by using umbrella sampling and post-MD processing to examine changes in free energy during ligand unbinding. As anticipated, the initial dissociation steps all require breaking interactions that appeared in crystal structures of the bound complexes. Interestingly, for type-I inhibitors such as SB2, p38? keeps barrier-free conformational fluctuation in the ligand-bound complex and during ligand dissociation. In contrast, with a type-II/III inhibitor such as BIRB796, with the rearrangements of p38? in its bound state, ligand unbinding features energetically unfavorable protein-ligand concerted movement. Our results also show that the type-II/III inhibitors preferred dissociation pathways through the allosteric channel, which is consistent with an existing publication. The study suggests that the level of required protein rearrangement is one major determining factor of drug binding kinetics in p38? systems, providing useful information for development of inhibitors.
Project description:The success of the first approved kinase inhibitor imatinib has spurred great interest in the development of type II inhibitors targeting the inactive DFG-out conformation, wherein the Phe of the DFG motif at the start of the activation loop points into the ATP binding site. Nevertheless, kinase inhibitors launched so far are heavily biased toward type I inhibitors targeting the active DFG-in conformation, wherein the Phe of the DFG motif flips by approximately 180° relative to the inactive conformation, resulting in Phe and Asp swapping their positions. Data recently obtained with structurally validated type II inhibitors supported the conclusion that type II inhibitors are more selective than type I inhibitors. In our type II BRAF V600E inhibitor lead discovery effort, we identified phenylaminopyrimidine (PAP) and unsymmetrically disubstituted urea as two fragments that are frequently presented in FDA-approved protein kinase inhibitors. We therefore defined PAP and unsymmetrically disubstituted urea as privileged fragments for kinase drug discovery. A pharmacophore for type II inhibitors, 4-phenylaminopyrimidine urea (4-PAPU), was assembled based upon these privileged fragments. Lead compound SI-046 with BRAF V600E inhibitory activity comparable to the template compound sorafenib was in turn obtained through preliminary structure-activity relationship (SAR) study. Molecular docking suggested that SI-046 is a bona fide type II kinase inhibitor binding to the structurally validated "classical DFG-out" conformation of BRAF V600E. Our privileged fragments-based approach was shown to efficiently deliver a bona fide type II kinase inhibitor lead. In essence, the theme of this article is to showcase the strategy and rationale of our approach.
Project description:Protein kinases have been found to possess two characteristic conformations in their activation-loops: the active DFG-in conformation and the inactive DFG-out conformation. Recently, it has been very interesting to develop type-II inhibitors which target the DFG-out conformation and are more specific than the type-I inhibitors binding to the active DFG-in conformation. However, solving crystal structures of kinases with the DFG-out conformation remains a challenge, and this seriously hampers the application of the structure-based approaches in development of novel type-II inhibitors. To overcome this limitation, here we present a computational approach for predicting the DFG-out inactive conformation using the DFG-in active structures, and develop related conformational selection protocols for the uses of the predicted DFG-out models in the binding pose prediction and virtual screening of type-II ligands. With the DFG-out models, we predicted the binding poses for known type-II inhibitors, and the results were found in good agreement with the X-ray crystal structures. We also tested the abilities of the DFG-out models to recognize their specific type-II inhibitors by screening a database of small molecules. The AUC (area under curve) results indicated that the predicted DFG-out models were selective toward their specific type-II inhibitors. Therefore, the computational approach and protocols presented in this study are very promising for the structure-based design and screening of novel type-II kinase inhibitors.
Project description:Structural coverage of the human kinome has been steadily increasing over time. The structures provide valuable insights into the molecular basis of kinase function and also provide a foundation for understanding the mechanisms of kinase inhibitors. There are a large number of kinase structures in the PDB for which the Asp and Phe of the DFG motif on the activation loop swap positions, resulting in the formation of a new allosteric pocket. We refer to these structures as "classical DFG-out" conformations in order to distinguish them from conformations that have also been referred to as DFG-out in the literature but that do not have a fully formed allosteric pocket. We have completed a structural analysis of almost 200 small molecule inhibitors bound to classical DFG-out conformations; we find that they are recognized by both type I and type II inhibitors. In contrast, we find that nonclassical DFG-out conformations strongly select against type II inhibitors because these structures have not formed a large enough allosteric pocket to accommodate this type of binding mode. In the course of this study we discovered that the number of structurally validated type II inhibitors that can be found in the PDB and that are also represented in publicly available biochemical profiling studies of kinase inhibitors is very small. We have obtained new profiling results for several additional structurally validated type II inhibitors identified through our conformational analysis. Although the available profiling data for type II inhibitors is still much smaller than for type I inhibitors, a comparison of the two data sets supports the conclusion that type II inhibitors are more selective than type I. We comment on the possible contribution of the DFG-in to DFG-out conformational reorganization to the selectivity.
Project description:Aurora kinase B (AKB), a Ser/Thr kinase that plays a crucial role in mitosis, is overexpressed in several cancers. Clinical inhibitors targeting AKB bind to the active DFG "in" conformation of the kinase. It would be beneficial, however, to understand if AKB is susceptible to type II kinase inhibitors that bind to the inactive, DFG "out" conformation, since type II inhibitors achieve higher kinome selectivity and higher potency in vivo. The DFG "out" conformation of AKB is not yet experimentally determined which makes the design of type II inhibitors exceedingly difficult. An alternate approach is to simulate the DFG "out" conformation from the experimentally determined DFG "in" conformation using atomistic molecular dynamics (MD) simulation. In this work, we employed metadynamics (MTD) approach to simulate the DFG "out" conformation of AKB by choosing the appropriate collective variables. We examined structural changes during the DFG-flip and determined the interactions crucial to stabilize the kinase in active and inactive states. Interestingly, the MTD approach also identified a unique transition state (DFG "up"), which can be targeted by small molecule inhibitors. Structural insights about these conformations is essential for structure-guided design of next-generation AKB inhibitors. This work also emphasizes the usefulness of MTD simulations in predicting macromolecular conformational changes at reduced computational costs.
Project description:Type-II kinase inhibitors represent a class of chemicals that trap their target kinases in an inactive, so-called DFG-out state, occupying a hydrophobic pocket adjacent to the ATP binding site. These compounds are often more specific than those that target active DFG-in kinase conformations. Unfortunately, the discovery of novel type-II scaffolds presents a considerable challenge, partially because the lack of compatible kinase structures makes structure-based methods inapplicable. We present a computational protocol for converting multiple available DFG-in structures of various kinases (approximately 70% of mammalian structural kinome) into accurate and specific models of their type-II bound state. The models, described as deletion-of-loop Asp-Phe-Gly-in (DOLPHIN) kinase models, demonstrate exceptional performance in various inhibitor discovery applications, including compound pose prediction, screening, and in silico activity profiling. Given the abundance of the DFG-in structures, the presented approach opens possibilities for kinome-wide discovery of specific molecules targeting inactive kinase states.
Project description:Tyrosine kinase domains dynamically fluctuate between two main structural forms that are referred to as type I (DFG-in) or type II (DFG-out) conformations. Comprehensive data comparing type I and type II inhibitors are currently lacking for NTRK fusion-driven cancers. Here we used a type II NTRK inhibitor, altiratinib, as a model compound to investigate its inhibitory potential for larotrectinib (type I inhibitor)-resistant mutations in NTRK. Our study shows that a subset of larotrectinib-resistant NTRK1 mutations (V573M, F589L and G667C) retains sensitivity to altiratinib, while the NTRK1<sup>V573M</sup> and xDFG motif NTRK1<sup>G667C</sup> mutations are highly sensitive to type II inhibitors, including altiratinib, cabozantinib and foretinib. Moreover, molecular modeling suggests that the introduction of a sulfur moiety in the binding pocket, via methionine or cysteine substitutions, specifically renders the mutant kinase hypersensitive to type II inhibitors. Future precision treatment strategies may benefit from selective targeting of these kinase mutants based on our findings.
Project description:To increase kinase selectivity in an aminopyrazole-based PAK1 inhibitor series, analogues were designed to interact with the PAK1 deep-front pocket pre-DFG residue Thr-406, a residue that is hydrophobic in most kinases. This goal was achieved by installing lactam head groups to the aminopyrazole hinge binding moiety. The corresponding analogues represent the most kinase selective ATP-competitive Group I PAK inhibitors described to date. Hydrogen bonding with the Thr-406 side chain was demonstrated by X-ray crystallography, and inhibitory activities, particularly against kinases with hydrophobic pre-DFG residues, were mitigated. Leveraging hydrogen bonding side chain interactions with polar pre-DFG residues is unprecedented, and similar strategies should be applicable to other appropriate kinases.
Project description:Targeted inhibition of anaplastic lymphoma kinase (ALK) dramatically improved therapeutic outcomes in the treatment of ALK-positive cancers, but unfortunately patients invariably progressed due to acquired resistance mutations in ALK. Currently available drugs are all type-I inhibitors bound to the ATP-binding pocket and are most likely to be resistant in patients harboring genetic mutations surrounding the ATP pocket. To overcome drug resistance, we rationally designed a novel kind of "bridge" inhibitor, which specially bind into an extended hydrophobic back pocket adjacent to the ATP-binding site of ALK. The novel type-I1/2 inhibitors display excellent antiproliferation activity against ALK-positive cancer cells and appear superior to two clinically used drugs, crizotinib and ceritinib. Structural and molecular modeling analyses indicate that the inhibitor induces dramatic conformational transition and stabilizes unique DFG-shifted loop conformation, enabling persistent sensitivity to different genetic mutations in ALK. These data highlight a rationale for further development of next-generation ALK inhibitors to combat drug resistance.