Comprehensive screening of heterocyclic compound libraries to identify novel inhibitors for PfRIO-2 kinase through docking and substrate competition studies

Malaria is most prevalent in tropical climate and causes 1–3 million deaths annually. RIO-2 kinase, an atypical kinase regulates ribosome biogenesis and is necessary for cell cycle progression. Structural characterization of PFD0975w (PfRIO-2 kinase) indicates N-terminal DNA binding winged helix domain (1–84), a linker region (85–147), and C-terminal kinase domain (148–275). Heterocyclic compounds present in different databases represent an enormous reservoir to screen and develop the suitable inhibitor. An extensive screening of heterocyclic compounds present in zinc database, PubChem and ChemBank database was done to identify potent PfRIO-2 kinase specific inhibitors. Initial screening gave 41 compounds with high affinity toward PfRIO-2 kinase than natural substrate ATP. A substrate competition experiment and analysis of binding mode conformation within the ATP binding pocket identifies five compounds; Zc-49775260, Pc-44415375, Pc-44215930, Cb-2082655, and Cb-832054 as potential PfRIO-2 kinase inhibitors. Further analysis of top hits in ADMET parameters, Caco-2 cell permeability profile, human oral absorption, and drug-like properties indicates Pc-44215930 as the best suitable compound among the top hits. Searching top hits candidate heterocyclic compounds in the drug database picked up clindamycin, nelfinavir, methacycline, and other drugs in circulation. Most of these drugs are targeting ribosome maturation and highlights the possibility of PfRIO-2 kinase as a drug target. Hence, screening and substrate competition studies along with ADME analysis of top hit compounds allowed us to identify potential PfRIO-2 kinase inhibitors. We are hopeful that the current study will help to develop effective chemotherapy against malaria utilizing PfRIO-2 kinase as a target.     

Sequence and structural analysis of kinase ATP pocket residues

Protein kinases represent one of the largest known families of enzymes. Most kinases bind ATP and most synthetic kinase inhibitors are ATP-competitive, which makes selectivity a potential problem. However, despite the high sequence similarity in the ATP binding pocket, several groups including ours have been able to develop highly potent and selective ATP-competitive inhibitors. To systematically aid the design of specific inhibitors in our protein kinase projects, we aligned all known three-dimensional structures and all known sequences of human protein kinases. We identified a set of 38 residues that make up the ATP pocket and analyzed the variability among these residues. The most variable residues in the ATP pocket are targeted to design specificity into inhibitors in our various kinase projects.     

Polo-like kinases inhibitors

Polo-like kinases (PLKs) are a family of serine/threonine kinases that play crucial roles in multiple stages of mitosis. PLK1 is the most studied member of the family. It is overexpressed in a wide spectrum of cancer types and is a promising target in oncology. Most of PLK1 inhibitors are ATP-competitive. Despite the structural similarities among various kinases, several inhibitors are selective. Some areas of the PLK1 active site are important for selectivity against other kinases. These include a small pocket formed by Leu 132 in the hinge region, a bulky phenylalanine and a small cysteine at the bottom and in the roof of the ATP pocket, respectively, and an unusual concentration of positively charged residues in the solvent-exposed region. Many ATP-competitive inhibitors are heterocyclic systems able to interact with the unique features of the PLK1 binding site. Other inhibitors target regions outside the ATP pocket, such as the substrate binding domain or a hydrophobic pocket, formed when the kinase is in the inactive conformation. An alternative approach to obtain specificity and to overcome drug resistance often associated with kinase inhibitors is the inhibition of the polo-box domain (PBD) of PLK1. The PBD is unique for the family of PLKs and is essential for PLK functions; so it is a useful target for the development of selective and potent inhibitors for clinical uses. In this review some PLK inhibitors are reported, focusing on chemical structures, structure-activity-relationships (SAR) and biological activities. The great potential of these compounds could open promising perspectives. Moreover, a combination of polo-like kinases inhibitors with other anticancer drugs might offer new opportunities for cancer therapy.     

Theoretical studies on the selective mechanisms of GSK3β and CDK2 by molecular dynamics simulations and free energy calculations

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase which is widely involved in cell signaling and controls a broad number of cellular functions. GSK3 contains α and β isoforms, and GSK3β has received more attention and becomes an attractive drug target for the treatment of several diseases. The binding pocket of cyclin‐dependent kinase 2 (CDK2) shares high sequence identity to that of GSK3β, and therefore, the design of highly selective inhibitors toward GSK3β remains a big challenge. In this study, a computational strategy, which combines molecular docking, molecular dynamics simulations, free energy calculations, and umbrella sampling simulations, was employed to explore the binding mechanisms of two selective inhibitors to GSK3β and CDK2. The simulation results highlighted the key residues critical for GSK3β selectivity. It was observed that although GSK3β and CDK2 share the conserved ATP‐binding pockets, some different residues have significant contributions to protein selectivity. This study provides valuable information for understanding the GSK3β‐selective binding mechanisms and the rational design of selective GSK3β inhibitors.     

Analysis of c‐Met Kinase Domain Complexes: A New Specific Catalytic Site Receptor Model for Defining Binding Modes of ATP‐Competitive Ligands

The receptor tyrosine kinase c‐Met have multiple roles during cancer development and is currently considered as an important target for molecularly targeted therapies. Structural knowledge of how compounds interact on c‐Met catalytic site could guide structure‐based drug design strategies towards more effective and selective anticancer drug candidates. However, although 17 crystal structures of c‐Met complexed with adenosine triphosphate (ATP)‐competitive kinase inhibitors are publicly available (August 2009), there are still open questions regarding the prediction of ligand binding modes. We have applied molecular modeling and molecular mechanics to analyze the distribution of ligands interaction energy on c‐Met residues, and deduced a new model of the active site allowing for an unambiguous identification of ligand binding modes. We demonstrate that the binding of known ligands on the c‐Met catalytic site involves seven identified structurally‐distinct areas. Five of these match the generic kinase ATP binding site model built by Novartis scientists in the 1990s, while the two others are distinct allosteric regions that can be exploited by second generation kinase inhibitors such as Gleevec. We show here that c‐Met can accept both such kinds of allosteric inhibitors, a very unusual feature in the kinase family that opens new grounds for highly specific drug design.     

X-ray crystal structure of bone marrow kinase in the x chromosome: a Tec family kinase

Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton’s tyrosine kinase and IL‐2‐inducible T‐cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X‐ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 Å resolution and PP2 at 1.9 Å resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well‐ordered protein conformation that includes an open/extended activation loop and a stabilized DFG‐motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG‐motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.     

Molecular modeling of G-protein coupled receptor kinase 2: Docking and biochemical evaluation of inhibitors

G-protein coupled receptor kinase 2 (GRK2) regulates the activity of many receptors. Because potent inhibitors of GRK2 are thus far limited to polyanionic compounds like heparin, we searched for new inhibitors with the aid of a molecular model of GRK2. We used the available crystal structure of cAMP dependent protein kinase (cAPK) as a template to construct a 3D homology model of GRK2. Known cAPK and GRK2 inhibitors were docked into the active sites of GRK2 and cAPK using DOCK v3.5. H8 docked into the hydrophobic pocket of the adenosine 5-triphosphate (ATP) binding site of cAPK, consistent with its known competitive cAPK inhibition relative to ATP. Similarly, 3 of 4 known GRK2 inhibitors docked into the ATP binding pocket of GRK2 with good scores. Screening the Fine Chemicals Directory (FCD, containing the 3D structures of 13,000 compounds) for docking into the active sites of GRK2 identified H8 and the known GRK2 inhibitor trifluoperazine as candidates. Whereas H8 indeed inhibited light-dependent phosphorylation of rhodopsin by GRK2, but with low potency, 3 additional FCD compounds with promising GRK2 scores failed to inhibit GRK2. This result demonstrates limitations of the GRK2 model in predicting activity among diverse chemical structures. Docking suramin, an inhibitor of protein kinase C (not present in FCD) yielded a good fit into the ATP binding site of GRK2 over cAPK. Suramin did inhibit GRK2 with IC50 32 μM (pA₂ 6.39 for competitive inhibition of ATP). Suramin congeners with fewer sulfonic acid residues (NF062, NF503 [IC50 14 μM]) or representing half of the suramin molecule (NF520) also inhibited GRK2 as predicted by docking. In conclusion, suramin and analogues are lead compounds in the development of more potent and selective inhibitors of GRK2.     

A structure-function perspective of Jak2 mutations and implications for alternate drug design strategies: the road not taken

Jak2 is a non-receptor tyrosine kinase that is involved in the control of cellular growth and proliferation. Due to its significant role in hematopoiesis, Jak2 is a frequent target for mutations in cancer, especially myeloid leukemia, lymphoid leukemia and the myeloproliferative neoplasms (MPN). These mutations are common amongst different populations all over the world and there is a great deal of effort to develop therapeutic drugs for the affected patients. Jak2 mutations, whether they are point, deletion, or gene fusion, most commonly result in constitutive kinase activation. Here, we explore the structure-function relation of various Jak2 mutations identified in cancer and understand how they disrupt Jak2 regulation. Current Jak2 inhibitors target the highly conserved active site in the kinase domain and therefore, these inhibitors may lack specificity. Based on our knowledge regarding structure-function correlations as they pertain to regulation of Jak2 kinase activity, an alternative approach for specific Jak2 targeting could be via allosteric inhibitor design. Successful reports of allosteric inhibitors developed against other kinases provide precedent for the development of Jak2 allosteric inhibitors. Here, we suggest plausible target sites in the Jak2 structure for allosteric inhibition. Such targets include the type II inhibitor pocket and substrate binding site in the kinase domain, the kinase-pseudokinase domain interface, SH2-JH2 linker region and the FERM domain. Thus, future Jak2 inhibitors that target these sites via allosteric mechanisms may provide alternative therapeutic strategies to existing ATP competitive inhibitors.     

Discovery of an inhibitor of insulin-like growth factor 1 receptor activation: Implications for cellular potency and selectivity over insulin receptor

Insulin-like growth factor 1 receptor (IGF-1R) is an attractive target for anti-cancer therapy due to its anti-apoptotic effect on tumor cells, but inhibition of insulin receptor (IR) may have undesired metabolic consequences. The primary sequences of the ATP substrate-binding sites of these receptors are identical and the crystal structures of the activated kinase domains are correspondingly similar. Thus, most small-molecule inhibitors described to date are equally potent against the activated kinase domains of IGF-1R and IR. In contrast, the non-phosphorylated kinase domains of these receptors have several structural features that may accommodate differences in binding affinity for kinase inhibitors. We used a cell-based assay measuring IGF-1R autophosphorylation as an inhibitor screen, and identified a potent purine derivative that is selective compared to IR. Surprisingly, the compound is a weak inhibitor of the activated IGF-1R tyrosine kinase domain. Biochemical and structural studies are presented that indicate the compound preferentially binds to the ATP site of non-phosphorylated IGF-1R compared to phosphorylated IGF-1R. The potential selectivity and potency advantages of this binding mode are discussed.     

Identification of Allosteric PIF‐Pocket Ligands for PDK1 using NMR‐Based Fragment Screening and 1H‐15N TROSY Experiments

Aberrant activation of the phosphoinositide 3‐kinase pathway because of genetic mutations of essential signalling proteins has been associated with human diseases including cancer and diabetes. The pivotal role of 3‐phosphoinositide‐dependent kinase‐1 in the PI3K signalling cascade has made it an attractive target for therapeutic intervention. The N‐terminal lobe of the 3‐phosphoinositide‐dependent kinase‐1 catalytic domain contains a docking site which recognizes the non‐catalytic C‐terminal hydrophobic motifs of certain substrate kinases. The binding of substrate in this so‐called PDK1 Interacting Fragment pocket allows interaction with 3‐phosphoinositide‐dependent kinase‐1 and enhanced phosphorylation of downstream kinases. NMR spectroscopy was used to a screen 3‐phosphoinositide‐dependent kinase‐1 domain construct against a library of chemically diverse fragments in order to identify small, ligand‐efficient fragments that might interact at either the ATP site or the allosteric PDK1 Interacting Fragment pocket. While majority of the fragment hits were determined to be ATP‐site binders, several fragments appeared to interact with the PDK1 Interacting Fragment pocket. Ligand‐induced changes in ¹H‐¹⁵N TROSY spectra acquired using uniformly ¹⁵N‐enriched PDK1 provided evidence to distinguish ATP‐site from PDK1 Interacting Fragment‐site binding. Caliper assay data and ¹⁹F NMR assay data on the PDK1 Interacting Fragment pocket fragments and structurally related compounds identified them as potential allosteric activators of PDK1 function.     

Identification of Type‐II Inhibitors Using Kinase Structures

Spleen tyrosine kinase is a non‐receptor tyrosine kinase, overactivation of which is thought to contribute to autoimmune diseases as well as allergy and asthma. Protein kinases have a highly conserved ATP binding site, thus making challenging the design of selective small molecule inhibitors. It has been well documented that some protein kinases can be stabilized in their inactive conformations (Type‐II inhibitors). Herein, we describe a protein structure/ligand‐based approach to successfully identify ligands that bind to novel conformations of spleen tyrosine kinase. By utilizing kinase protein crystal structures both in the public domain (RCSB) and within Pfizer’s protein crystal database, we report the discovery of the first spleen tyrosine kinase Type‐II ligands. Compounds 1 and 3 were found to bind to the DFG‐out conformation of spleen tyrosine kinase, while compound 2 binds to a DFG‐in, C‐Helix‐out conformation. In this instance, the C‐helix moved significantly to create a large hydrophobic pocket rarely seen in kinase protein crystal structures.     

A novel approach for the development of selective Cdk4 inhibitors: library design based on locations of Cdk4 specific amino acid residues.

Identification of a selective inhibitor for a particular protein kinase without inhibition of other kinases is critical for use as a biological tool or drug. However, this is very difficult because there are hundreds of homologous kinases and their kinase domains including the ATP binding pocket have a common folding pattern. To address this issue, we applied the following structure-based approach for designing selective Cdk4 inhibitors: (1) identification of specifically altered amino acid residues around the ATP binding pocket in Cdk4 by comparison of 390 representative kinases, (2) prediction of appropriate positions to introduce substituents in lead compounds based on the locations of the altered amino acid residues and the binding modes of lead compounds, and (3) library design to interact with the altered amino acid residues supported by de novo design programs. Accordingly, Asp99, Thr102, and Gln98 of Cdk4, which are located in the p16 binding region, were selected as first target residues for specific interactions with Cdk4. Subsequently, the 5-position of the pyrazole ring in the pyrazol-3-ylurea class of lead compound (2a) was predicted to be a suitable position to introduce substituents. We then designed a chemical library of pyrazol-3-ylurea substituted with alkylaminomethyl groups based on the output structures of de novo design programs. Thus we identified a highly selective and potent Cdk4 inhibitor, 15b, substituted with a 5-chloroindan-2-ylaminomethyl group. Compound 15b showed higher selectivity on Cdk4 over those on not only Cdk1/2 (780-fold/190-fold) but also many other kinases (>430-fold) that have been tested thus far. The structural basis for Cdk4 selective inhibition by 15b was analyzed by combining molecular modeling and the X-ray analysis of the Cdk4 mimic Cdk2-inhibitor complex. The results suggest that the hydrogen bond with the carboxyl group of Asp99 and hydrophobic van der Waals contact with the side chains of Thr102 and Gln98 are important. Compound 15b was found to cause cell cycle arrest of the Rb(+) cancer cell line in the G(1) phase, indicating that it is a good biological tool.     

Molecular modeling studies of the Akt PH domain and its interaction with phosphoinositides

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.     

A Back-to-Front Fragment-Based Drug Design Search Strategy Targeting the DFG-Out Pocket of Protein Tyrosine Kinases

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Design and characterization of a potent and selective dual ATP- and substrate-competitive subnanomolar bidentate c-Jun N-terminal kinase (JNK) inhibitor

c-Jun N-terminal kinases (JNKs) represent valuable targets in the development of new therapies. Present on the surface of JNK is a binding pocket for substrates and the scaffolding protein JIP1 in close proximity to the ATP binding pocket. We propose that bidentate compounds linking the binding energies of weakly interacting ATP and substrate mimetics could result in potent and selective JNK inhibitors. We describe here a bidentate molecule, 19, designed against JNK. 19 inhibits JNK kinase activity (IC(50) = 18 nM; K(i) = 1.5 nM) and JNK/substrate association in a displacement assay (IC(50) = 46 nM; K(i) = 2 nM). Our data demonstrate that 19 targets for the ATP and substrate-binding sites on JNK concurrently. Finally, compound 19 successfully inhibits JNK in a variety of cell-based experiments, as well as in vivo where it is shown to protect against Jo-2 induced liver damage and improve glucose tolerance in diabetic mice.     

Clarifying binding difference of ATP and ADP to extracellular signal‐regulated kinase 2 by using molecular dynamics simulations

Extracellular signal‐regulated kinase 2 is a promising target for designs and development of anticancer drugs. Molecular dynamics simulations and molecular mechanics Poisson–Boltzmann method were applied to study binding difference of ADP and ATP to extracellular signal‐regulated kinase 2. The results prove that the binding ability of ATP to extracellular signal‐regulated kinase 2 is stronger than that of ADP. Principal component analysis performed by using molecular dynamics trajectories suggests that binding of ADP and ATP to extracellular signal‐regulated kinase 2 change motion directions of two helices α1 and α2. Residue‐based free energy decomposition method was adopted to calculate contributions of separate residues to associations of ADP and ATP with extracellular signal‐regulated kinase 2. The results show that ADP and ATP produce strong CH–π interactions with five residues Ile29, Val37, Ala50, Leu105, and Leu154. In addition, five hydrogen bonding interactions of ADP and ATP with residues Lys52, Gln103, Asp104, and Met106 also stabilize bindings of ADP and ATP to extracellular signal‐regulated kinase 2. Overall, the CH–π interactions of ATP with five residues Ile29, Val37, Ala50, Leu105, and Leu154 are stronger than ADP. This study is expected to contribute a significant theoretical hint for designs of anticancer drugs targeting extracellular signal‐regulated kinase 2.     

Design and crystal structures of protein kinase B-selective inhibitors in complex with protein kinase A and mutants

Protein kinase B (PKB)-selective inhibitors were designed, synthesized, and cocrystallized using the AGC kinase family protein kinase A (PKA, often called cAMP-dependent protein kinase); PKA has been used as a surrogate for other members of this family and indeed for protein kinases in general. The high homology between PKA and PKB includes very similar ATP binding sites and hence similar binding pockets for inhibitors, with only few amino acids that differ between the two kinases. A series of these sites were mutated in PKA in order to improve the surrogate model for a design of PKB-selective inhibitors. Namely, the PKA to PKB exchanges F187L and Q84E enable the design of the selective inhibitors described herein which mimic ATP but extend further into a site not occupied by ATP. In this pocket, selectivity over PKA can be achieved by the introduction of bulkier substituents. Analysis of the cocrystal structures and binding studies were performed to rationalize the selectivity and improve the design.