Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure–activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.     

Targeting cysteine rich C1 domain of Scaffold protein Kinase Suppressor of Ras (KSR) with anthocyanidins and flavonoids – a binding affinity characterization study

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, ERK to provide spatial and temporal regulation of Ras-dependent ERK cascade signaling. Interruption of this mechanism can have a high influence in inhibiting the downstream signaling of the mutated tyrosine kinase receptor kinase upon ligand binding. Still none of the studies targeted to prevent the binding of Raf, MEK binding on kinase suppressor of RAS. In that perspective the cysteine rich C1 domain of scaffold proteins kinase suppressor of Ras-1 was targeted rather than its ATP binding site with small ligand molecules like flavones and anthocyanidins and analyzed through insilico docking studies. The binding energy evaluation shows the importance of hydroxyl groups at various positions on the flavone and anthocyanidin nucleus. Over all binding interaction shows these ligands occupied the potential sites of cysteine rich C1 domain of scaffold protein KSR.     

Structural basis for a high affinity inhibitor bound to protein kinase MK2

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.     

Structures of human N-Acetylglucosamine kinase in two complexes with N-Acetylglucosamine and with ADP/glucose: insights into substrate specificity and regulation

N-Acetylglucosamine (GlcNAc), a major component of complex carbohydrates, is synthesized de novo or salvaged from lysosomally degraded glycoconjugates and from nutritional sources. The salvage pathway requires that GlcNAc kinase converts GlcNAc to GlcNAc-6-phosphate, a component utilized in UDP-GlcNAc biosynthesis or energy metabolism. GlcNAc kinase belongs to the sugar kinase/Hsp70/actin superfamily that catalyze phosphoryl transfer from ATP to their respective substrates, and in most cases catalysis is associated with a large conformational change in which the N-terminal small and C-terminal large domains enclose the substrates. Here we report two crystal structures of homodimeric human GlcNAc kinase, one in complex with GlcNAc and the other in complex with ADP and glucose. The active site of GlcNAc kinase is located in a deep cleft between the two domains of the V-shaped monomer. The enzyme adopts a "closed" configuration in the GlcNAc-bound complex and GlcNAc interacts with residues of both domains. In addition, the N-acetyl methyl group contacts residues of the other monomer in the homodimer, a unique feature compared to other members of the sugar kinase/Hsp70/actin superfamily. This contrasts an "open" configuration in the ADP/glucose-bound structure, where glucose cannot form these interactions, explaining its low binding affinity for GlcNAc kinase. Our results support functional implications derived from apo crystal structures of GlcNAc kinases from Chromobacter violaceum and Porphyromonas gingivalis and show that Tyr205, which is phosphorylated in thrombin-activated platelets, lines the GlcNAc binding pocket. This suggests that phosphorylation of Tyr205 may modulate GlcNAc kinase activity and/or specificity.     

Study on the interaction of porphyrin with G-quadruplex DNAs

Interactions of 5,10,15,20-Tetrakis(N-propylpyridinium-4-yl)-21H,23H-porphyrin (TPrPyP4) with dimer hairpin (G(4)T(4)G(4))2 and parallel four-stranded (TG(4)T)4 G-quadruplex DNAs in Na(+)-containing buffer were studied. The results show that two TPrPyP4 molecules bind to both G-quadruplexes by a noncooperative and nonequivalent binding mode, and there are one high affinity site and one low affinity site, the respective binding constants are 8.06x10(8) and 1.13x10(6) M(-1) for (G(4)T(4)G(4))2-TPrPyP4, 8.04x10(7) and 9.08x10(5)M(-1) for (TG(4)T)4-TPrPyP4. TPrPyP4 presents two lifetimes of about 5.8 and 12.0 ns in the complexes of G-quadruplexes-TPrPyP4. The primary results suggest that two TPrPyP4 molecules bind to both G-quadruplexes by terminal stacking and outside binding mode.     

In-silico study of seaweed secondary metabolites as AXL kinase inhibitors

AXL kinase is an attractive cancer target for drug design and it is involved in different cancers. A set of molecule databases with 1072 secondary metabolites from seaweeds were screened against the AXL kinase active site and eight molecules were shortlisted for further studies. From the docking analysis of the complexes, four molecules GA011, BE005, BC010, and BC005 are showing prominent binging. From the 100 ns of molecular dynamics simulations and ligand-bound complex MM-PBSA free energy analysis, two molecules BC010 (ΔG = −135.38 kJ/mol) and BE005 (ΔG = −141.72 kJ/mol) are showing molecule stability in the active site also showing very strong binding free energies. It suggests these molecules could be the potent molecules for AXL kinase.     

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.     

Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.     

The structural basis for high affinity binding of α1-acid glycoprotein to the potent antitumor compound UCN-01

The α1-acid glycoprotein (AGP) is an abundant blood plasma protein with important immunomodulatory functions coupled to endogenous and exogenous ligand-binding properties. Its affinity for many drug-like structures, however, means AGP can have a significant effect on the pharmokinetics and pharmacodynamics of numerous small molecule therapeutics. Staurosporine, and its hydroxylated forms UCN-01 and UCN-02, are kinase inhibitors that have been investigated at length as antitumour compounds. Despite their potency, these compounds display poor pharmokinetics due to binding to both AGP variants, AGP1 and AGP2. The recent renewed interest in UCN-01 as a cytostatic protective agent prompted us to solve the structure of the AGP2–UCN-01 complex by X-ray crystallography, revealing for the first time the precise binding mode of UCN-01. The solution NMR suggests AGP2 undergoes a significant conformational change upon ligand binding, but also that it uses a common set of sidechains with which it captures key groups of UCN-01 and other small molecule ligands. We anticipate that this structure and the supporting NMR data will facilitate rational redesign of small molecules that could evade AGP and therefore improve tissue distribution.     

A molecular modeling study of the interaction of 2'-fluoro-substituted analogues of dUMP/FdUMP with thymidylate synthase

Molecular dynamics simulations and free energy calculations are presented, exploring previously described experimentally studied interactions of a series of 2'-fluoro-substituted dUMP/FdUMP analogues with thymidylate synthase (TS). The results show the inhibitory behaviors of 2'-F-ara-UMP, 2',2'-diF-dUMP and 2',5-diF-ara-UMP to be dependent upon the binding positions and orientations adopted by the molecules of these compounds in the active site of TS. The binding mode of 2',5-diF-ara-UMP suggests a novel role of the active site residue Trp 80, stabilizing through hydrophobic stacking the binding position of the pyrimidine ring in 2',5-diF-ara-UMP.     

Inhibition of epidermal growth factor receptor tyrosine kinase by chalcone derivatives.

In our previous study, butein, a chalcone derivative, was found to be an inhibitor of tyrosine kinases and the inhibition was ATP-competitive. In this work, chalcone and seven chalcone derivatives were used to analyse the relationship between the structure of these compounds and their inhibition of tyrosine kinase activity. Three of chalcone derivatives, including butein, marein and phloretin, were found to have an ability to inhibit the tyrosine kinase activity of epidermal growth factor receptor (EGFR) in vitro. IC(50) was 8 microM for butein, 19 microM for marein and 25 microM for phloretin. The structural characterisations of these inhibitors suggest that the hydroxylations at C4 and C4' of these molecules may be required for them to act as EGFR tyrosine kinase inhibitors. The inhibition of EGF-induced EGFR tyrosine phosphorylation by butein was also observed in human hepatocellular carcinoma HepG2 cells, while marein and phloretin were inactive at the doses tested. Molecular modelling suggests that butein, marein and phloretin can be docked into the ATP binding pocket of EGFR. Hydrogen bonds and hydrophobic interaction appear to be important in the binding of these inhibitors to EGFR.     

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.     

Two stable, conducting conformations of the selectivity filter in Shaker K+ channels

We have examined the voltage dependence of external TEA block of Shaker K(+) channels over a range of internal K(+) concentrations from 2 to 135 mM. We found that the concentration dependence of external TEA block in low internal K(+) solutions could not be described by a single TEA binding affinity. The deviation from a single TEA binding isotherm was increased at more depolarized membrane voltages. The data were well described by a two-component binding scheme representing two, relatively stable populations of conducting channels that differ in their affinity for external TEA. The relative proportion of these two populations was not much affected by membrane voltage but did depend on the internal K(+) concentration. Low internal K(+) promoted an increase in the fraction of channels with a low TEA affinity. The voltage dependence of the apparent high-affinity TEA binding constant depended on the internal K(+) concentration, becoming almost voltage independent in 5 mM. The K(+) sensitivity of these low- and high-affinity TEA states suggests that they may represent one- and two-ion occupancy states of the selectivity filter, consistent with recent crystallographic results from the bacterial KcsA K(+) channel. We therefore analyzed these data in terms of such a model and found a large (almost 14-fold) difference between the intrinsic TEA affinity of the one-ion and two-ion modes. According to this analysis, the single ion in the one-ion mode (at 0 mV) prefers the inner end of the selectivity filter twofold more than the outer end. This distribution does not change with internal K(+). The two ions in the two-ion mode prefer to occupy the inner end of the selectivity filter at low K(+), but high internal K(+) promotes increased occupancy of the outer sites. Our analysis further suggests that the four K(+) sites in the selectivity filter are spaced between 20 and 25% of the membrane electric field.     

Porphyrin binding mechanism is altered by protonation at the loops in G-quadruplex DNA formed near the transcriptional activation site of the human c-kit gene

Background

G-quadruplex DNA structures are hypothesized to be involved in the regulation of gene expression and telomere homeostasis. The development of small molecules that modulate the stability of G-quadruplex structures has a potential therapeutic interest in cancer treatment and prevention of aging.

Methods

Molecular absorption and circular dichroism spectra were used to monitor thermal denaturation, acid base titration and mole ratio experiments. The resulting data were analyzed by multivariate data analysis methods. Surface plasmon resonance was also used to probe the kinetics and affinity of the DNA–drug interactions.

Results

We investigated the interaction between a G-quadruplex-forming sequence in the human c-kit proto-oncogene and the water soluble porphyrin TMPyP4. The role of cytosine and adenine residues at the loops of G-quadruplex was studied by substitution of these residues by thymidines.

Conclusions

Here, we show the existence of two binding modes between TMPyP4 and the considered G-quadruplex. The stronger binding mode (formation constant around 107) involves end-stacking, while the weaker binding mode (formation constant around 106) is probably due to external loop binding. Evidence for the release of TMPyP4 upon protonation of bases at the loops has been observed.

General significance

The results may be used for the design of porphyrin-based anti-cancer molecules with a higher affinity to G-quadruplex structures which may have anticancer properties.     

Oxygen-evolving enhancer protein 2 is phosphorylated by glycine-rich protein 3/wall-associated kinase 1 in Arabidopsis

The Arabidopsis wall-associated receptor kinase, WAK1, is a member of WAK family that links the plasma membrane to the extracellular matrix. A glycine-rich secreted protein, AtGRP-3, was previously shown to regulate WAK1 functions through binding to the extracellular domain of WAK1. In this study, we sought to determine the downstream molecules of the AtGRP-3/WAK1 signaling pathway, by using two-dimensional gel electrophoresis combined with Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). We report here that a chloroplast protein, oxygen-evolving enhancer protein 2 (OEE2), specifically interacts with the cytoplasmic kinase domain of WAK1 and becomes phosphorylated in an AtGRP-3-dependent manner. The phosphorylation of OEE2 is also induced in Arabidopsis by treatment with avirulent Pseudomonas syringae. Taken together, these results suggest that OEE2 activity is regulated by AtGRP-3/WAK1.     

Differential and multiple binding of signal transducing molecules to the ITAMs of the TCR-ζ chain

A biotin-streptavidin-based technique was developed for high affinity, unidirectional, and specific immobilization of synthetic peptides to a solid phase. Biotinylated 23-mer carboxamide peptides corresponding to the three immunoreceptor tyrosine-based activation motifs (ITAMs) of the T cell antigen receptor associated ζ-chain (TCR-ζ) in their bis-, mono-, or unphosphorylated forms were used to study the binding of cellular proteins from human Jurkat T cells to these signal transduction motifs. The protein tyrosine kinase ZAP-70 bound specifically to all bisphosphorylated peptides but not to the mono- or unphosphorylated peptides. In contrast, Shc, phosphatidylinositol 3-kinase (P13K), Grb2, and Ras-GTPase activating protein (GAP) bound with different affinities to the bis- or monophosphorylated peptides, while the Src family protein tyrosine kinase (PTK) Fyn did not bind specifically to any of the tested peptides. The different preferences of the studied signaling molecules for distinct ITAMs, and in particular the binding of some of them preferentially to monophosphorylated peptides, suggests that the TCR-ζ may bind multiple signaling molecules with each ITAM binding a unique set of such molecules. In addition, partial phosphorylation of the ITAMs may result in recruitment of different proteins compared to double phosphorylation. This may be crucial for coupling of the TCR to various effector functions under different conditions of receptor triggering. © 1996 Wiley-Liss, Inc.     

Ligand binding affinity determined by temperature-dependent circular dichroism: cyclin-dependent kinase 2 inhibitors

To support drug discovery efforts for cyclin-dependent kinase 2 (CDK2), a moderate-throughput binding assay that can rank order or estimate the affinity of lead inhibitors has been developed. The method referred to as temperature-dependent circular dichroism (TdCD) uses the classical temperature-dependent unfolding of proteins by circular dichroism (CD) to measure the degree of protein unfolding in the absence and presence of potential inhibitors. The midpoint of unfolding is the Tm value. Rank ordering the affinity and predictions of the dissociation constant of compounds is obtained by measuring the increase in Tm for different protein-inhibitor complexes. This is the first time an extensive characterization of the TdCD method has been described for characterizing lead inhibitors in a drug discovery mode. The method has several favorable properties. Using the new six-cell Peltier temperature controller for the Jasco 810 spectropolarimeter, one can determine the affinity of 12-18 compounds per day. The method also requires only 20-40 microg protein per sample and can be used to estimate the affinity of compounds with dissociation constants of picomolar to micromolar. An important property of the method for lead discovery is that dissociation constants of approximately 5 microM can be estimated from a single experiment using a low concentration of compound such as 20 microM, which is generally low enough for most small molecules to be soluble for testing. In addition, the method does not require labeling the compound or protein. Although other methods such as isothermal titration calorimetry (ITC) can provide a full thermodynamic characterization of binding, ITC requires 1-2 mg protein per sample, cannot readily determine binding constants below nanomolar values, is most versatile with soluble compounds, and has a throughput of two to three experiments per day. The ITC method is not usually used in a high-throughput drug discovery mode; however, using the thermodynamic information from several ITC experiments can make the TdCD method very robust in determining reliable binding constants. Using the kinase inhibitors BMS-250595, purvalanol B, AG-12275, flavopiridol, and several other compounds, it is demonstrated that one can obtain excellent comparisons between the Kd values of binding to CDK2 obtained by TdCD and ITC.     

Lanthanide ion luminescence as a probe of DNA structure. 2. Non- guanine-containing oligomers and nucleotides.

Oligo(dC)8, oligo(dA)8, and oligo(dT)8 as well as d-CMP, d-AMP, and d-TMP, when complexed to Eu(3+), possess two classes of Eu(3+) binding environment. The binding environments consist of two classes, tight sites which coordinate two H2O molecules, and weaker sites which coordinate six or seven, analogous to the previously studied guanine-containing molecules. It is inferred that the tight class of Eu(3+) ion site observed with these oligomers and nucleotides corresponds to dimeric or polymeric structures. Comparison of the results for the guanine and non-guanine containing oligomers suggests that Eu(3+) possibly coordinates base nitrogen atoms in the former and in an outer sphere mode (hydrogen bonding via the H2O molecules coordinated to Eu(3+)) in the species examined here.     

Advances in the structural biology, design and clinical development of VEGF-R kinase inhibitors for the treatment of angiogenesis

Initial studies with angiogenesis inhibitors showed little clinical benefit. However, recently reported clinical studies in colorectal cancer have shown that bevacizumab, a vascular endothelial growth factor (VEGF) monoclonal antibody, in combination with cytotoxic therapy has positive effects on patient survival. Furthermore, the VEGF receptor kinase (VEGF-R) tyrosine kinase inhibitor, vatalanib, has also shown encouraging results in colorectal cancer, with molecular resonance imaging providing evidence that the anti-tumor efficacy was indeed the result of anti-angiogenic activity. Both of these agents are progressing in phase III trials. This proof of concept has stimulated the desire for second-generation VEGF-R inhibitors having an improved profile. Structural biology insight regarding the binding mode of protein kinase inhibitors is valuable for the design of molecules possessing superior selectivity, efficacy and tolerability. Towards this goal, we have developed a new series of VEGF-R2 kinase inhibitors, based upon an anthranilic acid amide scaffold. An X-ray crystal structure of a representative compound, AAL993 (ZK260253), in complex with the catalytic domain of diphosphorylated VEGF-R2 has revealed that this molecule binds to an inactive conformation of the protein. This binding mode, similar to that observed for the anti-leukemia drug, imatinib in complex with c-Abl kinase, may be responsible for the high selectivity of AAL993 and provides valuable insight for the design of further compounds.     

Model for the interaction of amphiphilic helices with troponin C and calmodulin

Crystals of troponin C are stabilized by an intermolecular interaction that involves the packing of helix A from the N-terminal domain of one molecule onto the exposed hydrophobic cleft of the C-terminal domain of a symmetry related molecule. Analysis of this molecular recognition interaction in troponin C suggests a possible mode for the binding of amphiphilic helical molecules to troponin C and to calmodulin. From the template provided by this troponin C packing, it has been possible to build a model of the contact region of mastoporan as it might be bound to the two Ca2+ binding proteins. A possible binding mode of melittin to calmodulin is also proposed. Although some of the characteristics of binding are similar for the two amphiphilic peptides, the increased length of melittin requires a significant bend in the calmodulin central helix similar to that suggested recently for the myosin light chain kinase calmodulin binding peptide (Persechini and Kretsinger: Journal of Cardiovascular Pharmacology 12:501-512, 1988). Not only are the hydrophobic interactions important in this model, but there are several favorable electrostatic interactions that are predicted as a result of the molecular modeling. The regions of troponin-C and calmodulin to which amphiphilic helices bind are similar to the regions to which the neuroleptic drugs such as trifluoperazine have been predicted to bind (Strynadka and James: Proteins 3:1-17, 1988).