ATP Binding Sites Of Protein Kinases Research Articles

Identification of selective dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) inhibitors and their effects on tau and microtubule

The overexpression of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), commonly observed in neurodegenerative diseases like Alzheimer's disease (AD) and Down syndrome (DS), can induce the formation of neurofibrillary tangles (NFTs) and amyloid plaques. Hence, designing a selective DYRK1A inhibitor would result in a promising small molecule for treating neurodegenerative diseases. Developing selective inhibitors for DYRK1A has been a difficult challenge due to the highly preserved ATP-binding site of protein kinases. In this study, we employed a structure-based virtual screening (SBVS) campaign targeting DYRK1A from a database containing 1.6 million compounds. Enzymatic assays were utilized to verify inhibitory properties, confirming that Y020-3945 and Y020-3957 showed inhibitory activity towards DYRK1A. In particular, the compounds exhibited high selectivity for DYRK1A over a panel of 120 kinases, reduced the phosphorylation of tau, and reversed the tubulin polymerization for microtubule stability. Additionally, treatment with the compounds significantly reduced the secretion of inflammatory cytokines IL-6 and TNF-α activated by DYRK1A-assisted NFTs and Aβ oligomers. These identified inhibitors possess promising therapeutic potential for conditions associated with DYRK1A in neurodegenerative diseases. The results showed that Y020-3945 and Y020-3957 demonstrated structural novelty compared to known DYRK1A inhibitors, making them a valuable addition to developing potential treatments for neurodegenerative diseases.

Mechanistic Insights into the Mechanism of Inhibitor Selectivity toward the Dark Kinase STK17B against Its High Homology STK17A.

As a member of the death-associated protein kinase (DAPK) family, STK17B plays an important role in the regulation of cellular apoptosis and has been considered as a promising drug target for hepatocellular carcinoma. However, the highly conserved ATP-binding site of protein kinases represents a challenge to design selective inhibitors for a specific DAPK isoform. In this study, molecular docking, multiple large-scale molecular dynamics (MD) simulations, and binding free energy calculations were performed to decipher the molecular mechanism of the binding selectivity of PKIS43 toward STK17B against its high homology STK17A. MD simulations revealed that STK17A underwent a significant conformational arrangement of the activation loop compared to STK17B. The binding free energy predictions suggested that the driving force to control the binding selectivity of PKIS43 was derived from the difference in the protein–ligand electrostatic interactions. Furthermore, the per-residue free energy decomposition unveiled that the energy contribution from Arg41 at the phosphate-binding loop of STK17B was the determinant factor responsible for the binding specificity of PKIS43. This study may provide useful information for the rational design of novel and potent selective inhibitors toward STK17B.

Chemoproteomic Analysis of Intertissue and Interspecies Isoform Diversity of AMP-activated Protein Kinase (AMPK)

AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that senses and governs changes in the cellular energy balance represented by concentrations of AMP, ADP, and ATP. Each of its three chains (α, β, and γ) exists as either two or three subtypes, theoretically allowing up to 12 different forms of the complete enzyme. Tissue specificity in the distribution of AMPK subtypes is believed to underpin a range of biological functions for AMPK, a central regulator of metabolic function and response. It is of particular interest for drug discovery purposes to compare AMPK isoforms that are most prevalent in human liver and muscle with isoforms present in key preclinical species. To complement immunocapture/immunodetection methods, which for AMPK are challenged by sequence similarities and difficulties of obtaining accurate relative quantitation, AMPK was captured from lysates of a range of cells and tissues using the ActivX ATP probe. This chemical probe covalently attaches desthiobiotin to one or more conserved lysyl residues in the ATP-binding sites of protein kinases, including AMPK, while also labeling a wide range of ATP-utilizing proteins. Affinity-based recovery of labeled proteins followed by gel-based fractionation of the captured sample was followed by proteomic characterization of AMPK polypeptides. In agreement with transcript-based analysis and previous indications from immunodetection, the results indicated that the predominant AMPK heterotrimer in human liver is α1β2γ1 but that dog and rat livers mainly contain the α1β1γ1 and α2β1γ1 forms, respectively. Differences were not detected between the AMPK profiles of normal and diabetic human liver tissues.

Conformation-Selective Inhibitors Reveal Differences in the Activation and Phosphate-Binding Loops of the Tyrosine Kinases Abl and Src

Over the past decade, an increasingly diverse array of potent and selective inhibitors that target the ATP-binding sites of protein kinases have been developed. Many of these inhibitors, like the clinically approved drug imatinib (Gleevec), stabilize a specific catalytically inactive ATP-binding site conformation of their kinases targets. Imatinib is notable in that it is highly selective for its kinase target, Abl, over other closely related tyrosine kinases, such as Src. In addition, imatinib is highly sensitive to the phosphorylation state of Abl's activation loop, which is believed to be a general characteristic of all inhibitors that stabilize a similar inactive ATP-binding site conformation. In this report, we perform a systematic analysis of a diverse series of ATP-competitive inhibitors that stabilize a similar inactive ATP-binding site conformation as imatinib with the tyrosine kinases Src and Abl. In contrast to imatinib, many of these inhibitors have very similar potencies against Src and Abl. Furthermore, only a subset of this class of inhibitors is sensitive to the phosphorylation state of the activation loop of these kinases. In attempting to explain this observation, we have uncovered an unexpected correlation between Abl's activation loop and another flexible active site feature, called the phosphate-binding loop (p-loop). These studies shed light on how imatinib is able to obtain its high target selectivity and reveal how the conformational preference of flexible active site regions can vary between closely related 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.

Affinity-Based Probes Based on Type II Kinase Inhibitors

Protein kinases are key components of most mammalian signal transduction networks and are therapeutically relevant drug targets. Efforts to study protein kinase function would benefit from new technologies that are able to profile kinases in complex proteomes. Here, we describe active site-directed probes for profiling kinases in whole cell extracts and live cells. These probes contain general ligands that stabilize a specific inactive conformation of the ATP-binding sites of protein kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties that allow covalent modification and enrichment of kinases, respectively. A diverse group of serine/threonine and tyrosine kinases were identified as specific targets of these probes in whole cell extracts. In addition, a number of kinase targets were selectively labeled in live cells. Our chemical proteomics approach should be valuable for interrogating protein kinase active sites in physiologically relevant environments.

KinDOCK: a tool for comparative docking of protein kinase ligands

KinDOCK is a new web server for the analysis of ATP-binding sites of protein kinases. This characterization is based on the docking of ligands already co-crystallized with other protein kinases. A structural library of protein kinase–ligand complexes has been extracted from the Protein Data Bank (PDB). This library can provide both potential ligands and their putative binding orientation for a given protein kinase. After protein–protein structural superposition, the ligands are transferred from the template complexes to the target protein kinase. The resulting complexes are evaluated using the program SCORE to compute a theoretical affinity. They can be dynamically visualized to allow a rapid mapping of important steric clashes and potential substitutions relevant for specificity and affinity. These characteristics allow a quick characterization of protein kinase active sites including conformation changes potentially required to accommodate particular ligands. Additionally, promising pharmacophores can be identified in the focussed library. These features will help to rationalize or optimize virtual screening (VS) on larger chemical compound libraries. The server and its documentation are freely available at .

Classification and comparison of ligand-binding sites derived from grid-mapped knowledge-based potentials

We describe the application of knowledge-based potentials implemented in the MOE program to compare the ligand-binding sites of several proteins. The binding probabilities for a polar and a hydrophobic probe are calculated on a grid to allow easy comparison of binding sites of superimposed related proteins. The method is fast and simple enough to simultaneously use structural information of multiple proteins of a target family. The method can be used to rapidly cluster proteins into subfamilies according to the similarity of hydrophobic and polar fields of their ligand-binding sites. Regions of the binding site which are common within a protein family can be identified and analysed for the design of family-targeted libraries or those which differ for improvement of ligand selectivity. The field-based hierarchical clustering is demonstrated for three protein families: the ligand-binding domains of nuclear receptors, the ATP-binding sites of protein kinases and the substrate binding sites of proteases. More detailed comparisons are presented for serine proteases of the chymotrypsin family, for the peroxisome proliferator-activated receptor subfamily of nuclear receptors and for progesterone and androgen receptor. The results are in good accordance with structure-based analysis and highlight important differences of the binding sites, which have been also described in the literature.

Evaluation of kinase inhibitor selectivity by chemical proteomics.

Small-molecule inhibitors of protein kinases constitute a novel class of drugs for therapeutic intervention in a variety of human diseases. Most of these agents target the relatively conserved ATP-binding site of protein kinases and have only been tested against a rather small subset of all human protein kinases. Therefore, the selectivity of protein kinase inhibitors has remained a widely underestimated, but highly important issue in drug development programs. In this review, we focus on the recent advancement of chemical proteomic methods to evaluate drug selectivity in an unbiased, comprehensive way. Efficient affinity purification procedures using immobilized kinase inhibitors combined with the sensitivity of mass spectrometry detection permit the mapping of drug targets on a proteome-wide scale. Data from this type of assessment can be used to set up tailor-made selectivity panels, which guide compound development in the context of the most relevant off-targets during lead optimization. In cases in which identified alternative targets are of validated clinical relevance, chemical proteomics provides the opportunity to repeatedly exploit a once established kinase inhibitor principle for additional target kinases and can thereby dramatically shorten the time toward highly selective, preclinical candidates. Moreover, the identification of alternative targets for preclinical or clinical drugs can provide new insights into their cellular modes of action, which might help to define those disease settings in which the most beneficial therapeutic effect is likely to occur.

Mutants of Protein Kinase A that Mimic the ATP-binding Site of Protein Kinase B (AKT)

The mutation of well behaved enzymes in order to simulate less manageable cognates is the obvious approach to study specific features of the recalcitrant target. Accordingly, the prototypical protein kinase PKA serves as a model for many kinases, including the closely related PKB, an AGC family protein kinase now implicated as oncogenic in several cancers. Two residues that differ between the α isoforms of PKA and PKB at the adenine-binding site generate differing shapes of the binding surface and are likely to play a role in ligand selectivity. As the corresponding mutations in PKA, V123A would enlarge the adenine pocket, while L173M would alter both the shape and its electronic character of the adenine-binding surface. We have determined the structures of the corresponding double mutant (PKAB2: PKAα V123A, L173M) in apo and MgATP-bound states, and observed structural alterations of a residue not previously involved in ATP-binding interactions: the side-chain of Q181, which in native PKA points away from the ATP-binding site, adopts in apo double mutant protein a new rotamer conformation, which places the polar groups at the hinge region in the ATP pocket. MgATP binding forces Q181 back to the position seen in native PKA. The crystal structure shows that ATP binding geometry is identical with that in native PKA but in this case was determined under conditions with only a single Mg ion ligand. Surface plasmon resonance spectroscopy studies show that significant energy is required for this ligand-induced transition. An additional PKA/PKB mutation, Q181K, corrects the defect, as shown both by the crystal structure of triple mutant PKAB3 (PKAα V123A, L173M, Q181K) and by surface plasmon resonance spectroscopy binding studies with ATP and three isoquinoline inhibitors. Thus, the triple mutant serves well as an easily crystallizable model for PKB inhibitor interactions. Further, the phenomenon of Q181 shows how crystallographic analysis should accompany mutant studies to monitor possible spurious structural effects.

Molecular scaffold-based design and comparison of combinatorial libraries focused on the ATP-binding site of protein kinases

Compound libraries were designed to target specifically the ATP cofactor-binding site in protein kinases by combining knowledge- and diversity-based design elements. A key aspect of the approach is the identification of molecular building blocks or scaffolds that are compatible with the binding site and therefore capture some aspects of target specificity. Scaffolds were selected on the basis of docking calculations and analysis of known inhibitors. We have generated 75 molecular scaffolds and applied different strategies to compute diverse compounds from scaffolds or, alternatively, to screen compound databases for molecules containing these scaffolds. The resulting libraries had a similar degree of molecular diversity, with at most 12% of the compounds being identical. However, their scaffold distributions differed significantly and a small number of scaffolds dominated the majority of compounds in each library.

Crystal Structures of Catalytic Subunit of cAMP-dependent Protein Kinase in Complex with Isoquinolinesulfonyl Protein Kinase Inhibitors H7, H8, and H89: STRUCTURAL IMPLICATIONS FOR SELECTIVITY

The discovery of several hundred different protein kinases involved in highly diverse cellular signaling pathways is in stark contrast to the much smaller number of known modulators of cell signaling. Of these, the H series protein kinase inhibitors (1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8) N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89)) are frequently used to block signaling pathways in studies of cellular regulation. To elucidate inhibition mechanisms at atomic resolution and to enable structure-based drug design of potential therapeutic modulators of signaling pathways, we determined the crystal structures of corresponding complexes with the cAPK catalytic subunit. Complexes with H7 and H8 (2.2 A) and with H89 (2.3 A) define the binding mode of the isoquinoline-sulfonamide derivatives in the ATP-binding site while demonstrating effects of ligand-induced structural change. Specific interactions between the enzyme and the inhibitors include the isoquinoline ring nitrogen ligating to backbone amide of Val-123 and an inhibitor side chain amide bonding to the backbone carbonyl of Glu-170. The conservation of the ATP-binding site of protein kinases allows evaluation of factors governing general selectivity of these inhibitors among kinases. These results should assist efforts in the design of protein kinase inhibitors with specific properties.

A point mutation at the putative ATP-binding site of protein kinase C alpha abolishes the kinase activity and renders it down-regulation-insensitive. A molecular link between autophosphorylation and down-regulation.

A protein kinase C alpha (PKC alpha) cDNA confers increased phorbol ester binding activity to intact cells when transiently expressed in COS cells or expressed stably in transfected rat 3Y1 fibroblasts. A point mutant (PKC alpha K----R) of PKC alpha, where Lys368 at the putative ATP-binding site is replaced with Arg, confers enhanced phorbol ester binding activity to both transiently and stably expressed COS and 3Y1 cells, respectively. Like endogenous and exogenously expressed wild type PKC alpha, the mutant PKC alpha K----R is translocated from the cytosol to the particulate fraction when cells are treated with a phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). On the other hand, the mutant PKC alpha K----R is not degraded when cells are treated with TPA, making a clear contrast to wild type PKC alpha; i.e. the mutant is resistant to TPA-mediated down-regulation. The mutant lacks kinase activity as expected, as judged by autophosphorylation and by a kinase assay using a peptide substrate, although the phorbol ester binding activity remains intact. These results suggest a link between the kinase activity of PKC alpha and the sensitivity to TPA-mediated proteolytic degradation. We propose that autophosphorylation of PKC alpha is a prerequisite for proteolytic cleavage associated with the down-regulation of PKC alpha.