Pyrimidine-based inhibitors of CaMKIIdelta

Non-ATP competitive pyrimidine-based inhibitors of CaMKIIdelta were identified. Computational studies were enlisted to predict the probable mode of binding. The results of the computational studies led to the design of ATP competitive inhibitors with optimized hinge interactions. Inhibitors of this class possessed improved enzyme and cellular activity compared to early leads.     

Functional insensitivity of the cytochrome b6f complex to structure changes in the hinge region of the Rieske iron-sulfur protein

Structure analysis of the cytochrome bc1 complex in the presence and absence of Qp quinol analog inhibitors implied that a large amplitude motion of the Rieske iron-sulfur protein (ISP) is required to mediate electron transfer from ubiquinol to cytochrome c1. Studies of the functional consequences of mutagenesis of an 8-residue ISP "hinge" region in the bc1 complex showed it to be sensitive to structure perturbation, implying that optimum flexibility and length are required for the large amplitude motion. Mutagenesis-function analysis carried out on the ISP hinge region of the cytochrome b6 f complex using the cyanobacterium Synechococcus sp. PCC 7002 showed the following. (i) Of three petC genes, only that in the petCA operon codes for functional ISP. (ii) The function of the complex was insensitive to changes in the hinge region that increased flexibility, decreased flexibility by substitutions of 4-6 Pro residues, shortened the hinge by a 1-residue deletion, or elongated it by insertion of 4 residues. The latter change increased sensitivity to Qp inhibitors, whereas deletion of 2 residues resulted in a loss of inhibitor sensitivity and a decrease in activity, indicating a minimum hinge length of 7 residues required for optimum binding of ISP at the Qp site. Thus, in contrast to the bc1 complex, the function of the b6 f complex was insensitive to sequence changes in the ISP hinge that altered its length or flexibility. This implies that either the barriers to motion or the amplitude of ISP motion required for function is smaller than in the bc1 complex.     

Some insights into the binding mechanism of Aurora B kinase gained by molecular dynamics simulation

Aurora B kinase is essential in the process of mitosis, and its overexpression has been reported to be associated with a number of solid tumors. We therefore carried out molecular docking, molecular dynamics, and molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations on several structurally diverse inhibitors (pentacyclic, pyrimidine, quinazoline, and pyrrolopyridine derivatives) and Aurora B kinase to explore the structural and chemical features responsible for the binding recognition mechanism. Molecular simulations reveal that the binding site mainly consists of six binding regions (sites A-F). We have identified that sites B and C are required for optimum binding in Aurora B-inhibitor complexes, sites A and F are needed to improve pharmacokinetic properties, while sites D and E lead to enhanced stability. We verified that hydrogen bonding to the hinge region and hydrophobic contact with the conserved hydrophobic pocket are of critical importance in the systems studied. Specifically, the amino acids Glu171, Phe172, and Ala173 in the hinge region and Leu99, Val107, and Leu223 in the conserved hydrophobic pocket probably account for the high binding affinities of these systems, as shown by hydrogen-bonding analysis and energy decomposition analysis. Hydrophobic contact with Phe172 is also in agreement with experimental data. In addition, the MM-PBSA calculations reveal that the binding of these inhibitors to Aurora B kinase is mainly driven by van der Waals/nonpolar interactions. The findings of this study should help to elucidate the binding pattern of Aurora B inhibitors and aid in the design of novel active ligands.     

Structural and functional analysis of the flexible regions of the catalytic α-subunit of protein kinase CK2

CK2 is a Ser/Thr protein kinase essential for cell viability. Its activity is anomalously high in several solid (prostate, mammary gland, lung, kidney and head and neck) and haematological tumours (AML, CML and PML), creating conditions favouring the onset of cancer. Cancer cells become addicted to high levels of CK2 activity and therefore this kinase is a remarkable example of "non-oncogene addiction". CK2 is a validated target for cancer therapy with one inhibitor in phase I clinical trials. Several crystal structures of CK2 are available, many in complex with ATP-competitive inhibitors, showing the presence of regions with remarkable flexibility. We present the structural characterisation of these regions by means of seven new crystal structures, in the apo form and in complex with inhibitors. We confirm previous findings about the unique flexibility of the CK2α catalytic subunit in the hinge/αD region, the p-loop and the β4β5 loop, and show here that there is no clear-cut correlation between the conformations of these flexible zones. Our findings challenge some of the current interpretations on the functional role of these regions and dispute the hypothesis that small ligands stabilize an inactive state. The mobility of the hinge/αD region in the human enzyme is unique among protein kinases, and this can be exploited for the development of more selective ATP-competitive inhibitors. The identification of different ligand binding modes to a secondary site can provide hints for the design of non-ATP-competitive inhibitors targeting the interaction between the α catalytic and the β regulatory subunits.     

Design of selective PI3Kδ inhibitors using an iterative scaffold-hopping workflow

PI3Kδ mediates key immune cell signaling pathways and is a target of interest for multiple indications in immunology and oncology. Here we report a structure-based scaffold-hopping strategy for the design of chemically diverse PI3Kδ inhibitors. Using this strategy, we identified several scaffolds that can be combined to generate new PI3Kδ inhibitors with high potency and isoform selectivity. In particular, an oxindole-based scaffold was found to impart exquisite selectivity when combined with several hinge binding motifs.     

Benzimidazole inhibitors of the protein kinase CHK2: Clarification of the binding mode by flexible side chain docking and protein–ligand crystallography

Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.     

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. Here, we demonstrate that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. We found that Doc expression was restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevented the lateral extension and deepening of these folds at the larval stage and delayed wing disc bending in the early pupal stage. Ectopic Doc expression was sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression expressed Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins were depleted. Overexpression of Doc along the blade/hinge folds caused precocious wing disc bending, which could be suppressed by co-expressing MMP2RNAi.     

Synthesis,structure–activity relationship and crystallographic studies of 3-substituted indolin-2-one RET inhibitors

The synthesis, structure–activity relationships (SAR) and structural data of a series of indolin-2-one inhibitors of RET tyrosine kinase are described. These compounds were designed to explore the available space around the indolinone scaffold within RET active site. Several substitutions at different positions were tested and biochemical data were used to draw a molecular model of steric and electrostatic interactions, which can be applied to design more potent and selective RET inhibitors. The crystal structures of RET kinase domain in complex with three inhibitors were solved. All three compounds bound in the ATP pocket and formed two hydrogen bonds with the kinase hinge region. Crystallographic analysis confirmed predictions from molecular modelling and helped refine SAR results. These data provide important information for the development of indolinone inhibitors for the treatment of RET-driven cancers.     

Hinge sequences as signaling agents?

We report an unexpected finding of common structural principles in two unrelated signaling systems: the FAS death domain transformation that initializes the extrinsic apoptotic pathway and signaling by calmodulin bending. The location and design of the hinge is postulated to be a general principle for creating potential signaling event. We suggest that already existing tool can predict the existence of such a hinge and formulate the hypothesis that the internal instabilities designed into the hinge sequences are necessary devices in effective signaling events.     

Non-enzymatic hinge region fragmentation of antibodies in solution

Liquid formulations of monoclonal antibodies (MAbs) typically undergo fragmentation near the papain cleavage site in the hinge region, resulting in Fab and Fab+Fc forms. The purpose of this study was to investigate whether this fragmentation is due to proteases. Four closely-related MAbs were exchanged into a pH 5.2 acetate buffer with NaCl and stored at -20 degrees C, 5 degrees C, 30 degrees C, or 40 degrees C for 1 month. Fragmentation generated size-exclusion chromatography (SEC) peak fractions that were analyzed by electrospray mass spectrometry to identify the cleavage sites. The effects of protein inhibitors or host cell proteins on fragmentation were also studied. The extent of fragmentation was equivalent for all four antibodies, occurring in the heavy chain hinge region Ser-Cys-Asp-Lys-Thr-His-Thr sequence. The fragment due to cleavage of the Asp-Lys bond showed two forms that differ by 18 Da. A synthetic peptide with the hinge region sequence terminating with Asp did not show fragmentation or the loss of 18 Da after incubation. Protease inhibitors did not affect rates of cleavage or modify sites of fragmentation. Degradation was not affected by host cell protein content. Fragmentation appears to be a kinetic process that is not caused by low levels of host cell proteases.     

The identification of novel p38α isoform selective kinase inhibitors having an unprecedented p38α binding mode

A novel series of p38 MAP kinase inhibitors with high selectivity for the p38α isoform over the other family members including the highly homologous p38β isoform has been identified. X-ray co-crystallographic studies have revealed an unprecedented kinase binding mode in p38α for representative analogs, 5c and 9d, in which a Leu108/Met109 peptide flip occurs within the p38α hinge region. Based on these findings, a general strategy for the rational design of additional promising p38α isoform selective inhibitors by targeting this novel binding mode is proposed.     

Structure-guided discovery of cyclin-dependent kinase inhibitors

CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.     

Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors.     

Discovery of novel small-molecule Src kinase inhibitors via a kinase-focused druglikeness rule and structure-based virtual screening

Overexpression of the non-receptor tyrosine kinase Src is implicated in the development and progression of various human cancers. Blocking signalling pathways mediated by Src is believed to be a promising anticancer strategy. We report herein the discovery of novel small-molecule Src inhibitors by crystal structure-based virtual screening. A kinase-focused druglikeness rule was proposed and used in the design of compound library. Combination of large-scale docking with DOCK and rescoring with GOLD resulted in 6 hits with moderate to potent inhibitory activity against Src. Among them, compound 1 with an IC₅₀ of 1.2 μM shows the most potent inhibitory activity. By using molecular docking, binding models of the top 3 hits (ranked by potency and ligand efficiency) with Src were constructed to provide a rational strategy that simultaneously exploits hydrogen bonding interaction in the hinge region and hydrophobic stacking in the back pocket. This approach is instructive and meaningful to further structure-based drug design of Src inhibitors.     

4-Phenyl-7-azaindoles as potent and selective IKK2 inhibitors

The synthesis and SAR of a novel series of IKK2 inhibitors are described. Modification around the hinge binding region of the 7-azaindole led to a series of potent and selective inhibitors with good cellular activity.     

Molecular inhibitory mechanism study on the potent inhibitor brigatinib against four crizotinib-resistant ALK mutations

As a potent and selective drug, brigatinib exhibits high efficacy against wild-type and mutant anaplastic lymphoma kinase (ALK) proteins to treat non–small cell lung cancer. In this work, the mechanisms of brigatinib binding to wild type and four mutant ALKs were investigated to gain insight into the dynamic energetic and structural information with respect to the design of novel inhibitors. Comparison between ALK-brigatinib and ALK-crizotinib suggests that the scaffold of brigatinib is well anchored to the residue Met1199 of hinge region by two hydrogen bonds, and the residue Lys1150 has the strong electrostatic interaction with the dimethylphosphine oxide moiety in brigatinib. These ALK mutations have significant influences on the flexibility of P-loop region and DFG sequences, but do not impair the hydrogen bonds between brigatinib and the residue Met1199 of hinge region. And mutations (L1196M, G1269A, F1174L, and R1275Q) induce diverse conformational changes of brigatinib and the obvious energy variation of residues Glu1167, Arg1209, Asp1270, and Asp1203. Together, the detailed explanation of mechanisms of those mutations with brigatinib further provide several guidelines for the development of more effective ALK inhibitors.     

Hit to lead optimization of pyrazolo[1,5-a]pyrimidines as B-Raf kinase inhibitors

Our continued effort towards optimization of the pyrazolo[1,5-a]pyrimidine scaffold as B-Raf kinase inhibitors is described. Structure guided design was utilized to introduce kinase hinge region interacting groups in the 2-position of the scaffold. This strategy led to the identification of lead compound 9 with enhanced enzyme and cellular potency, while maintaining good selectivity over a number of kinases.     

Synthesis and evaluation of heteroaryl substituted diazaspirocycles as scaffolds to probe the ATP-binding site of protein kinases

With the success of protein kinase inhibitors as drugs to target cancer, there is a continued need for new kinase inhibitor scaffolds. We have investigated the synthesis and kinase inhibition of new heteroaryl-substituted diazaspirocyclic compounds that mimic ATP. Versatile syntheses of substituted diazaspirocycles through ring-closing metathesis were demonstrated. Diazaspirocycles directly linked to heteroaromatic hinge binder groups provided ligand efficient inhibitors of multiple kinases, suitable as starting points for further optimization. The binding modes of representative diazaspirocyclic motifs were confirmed by protein crystallography. Selectivity profiles were influenced by the hinge binder group and the interactions of basic nitrogen atoms in the scaffold with acidic side-chains of residues in the ATP pocket. The introduction of more complex substitution to the diazaspirocycles increased potency and varied the selectivity profiles of these initial hits through engagement of the P-loop and changes to the spirocycle conformation, demonstrating the potential of these core scaffolds for future application to kinase inhibitor discovery.     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Mutations on the hinge region of leukocyte elastase inhibitor determine the loss of inhibitory function

Leukocyte elastase inhibitor (LEI) is a cytosolic component of lung macrophages and blood leukocytes that inhibits neutrophil elastase. LEI is a member of the serpin superfamily, these proteins, mostly protease inhibitors, are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the hinge region of the reactive centre loop into a beta-sheet of the inhibitor. In this work three mutations were produced in the hinge region of elastase inhibitor that abolish the inhibition activity of LEI and transform the protein in a substrate of the elastase. This result demonstrates that the inhibitory mechanism of serpin is common to LEI.