Inhibition effects of 5-S-glutathionyl-N-beta-alanyl-L-dopa analogues against Src protein tyrosine kinase.

Twelve analogues of the antibacterial phenolic peptide 5-S-glutathionyl-N-beta-alanyl-L-dopa (5-S-GA-L-D, 1) were synthesized via orthoquinone using tyrosinase. Several synthesized compounds inhibited the v-Src autophosphorylation tyrosine kinase reaction with an IC50 value comparable to that of herbimycin A. The inhibition of c-Src substrate phosphorylation was much less active than v-Src autophosphorylation inhibition. 5-S-GA-L-D (1) and its analogous competed with peptide substrate and non-compared with ATP. The analogues showed no effects on substrate phosphorylation by epidermal growth factor receptor (EGFR), and this selectivity is the most characteristic feature of the 5-S-GA-L-D and its analogues (1-12).     

Recognition of multiple substrate motifs by the c-ABL protein tyrosine kinase

Using a combinatorial peptide library that is based on the one-bead one-peptide approach we identified 14 peptide substrates for the c-ABL protein tyrosine kinase, which define three distinct consensus sequence groups. This is distinct from many serine/threonine kinases, which often phosphorylate only one major consensus sequence. The three consensus sequences accurately predict phosphorylation sites in cellular ABL substrates proven to play a role in cell signaling. Our data suggest that protein tyrosine kinases have evolved to recognize multiple substrate motifs.     

Binding of a Diphosphorylated-ITAM peptide to spleen tyrosine kinase (Syk) induces distal conformational changes: A hydrogen exchange mass spectrometry study

Structural flexibility plays a crucial role in protein function. To assess whether specific structural changes are associated with the binding of an immunoreceptor tyrosine-based activation motif (ITAM) to the tandem Src homology-2 domains (tSH2) of the spleen tyrosine kinase [EC 2.7.7.112] (Syk), we used an approach based on protein hydrogen/deuterium exchange in the presence and absence of the diphosphorylated ITAM peptide. The protein deuterium uptake by the intact Syk protein was monitored in time by electrospray mass spectrometry, which revealed a dramatic relative decrease in deuterium uptake when the protein was bound to the ITAM peptide, suggesting an overall change in protein dynamics. Subsequently, the deuterium incorporation of individual segments of the protein was investigated using proteolysis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass-analysis, which revealed that several regions of Syk tSH2 are significantly more protected from exchange in the presence of the ITAM peptide. Four protected regions encompass the phosphotyrosine and hydrophobic binding sites on the SH2 domains, whereas two other protected regions are located in the inter-SH2 linker motif and do not make any direct contacts with the peptide. Interestingly, our data suggest that binding of the ITAM peptide to Syk tSH2 induces distal structural effects on the protein that stabilize the inter-SH2 linker region, possibly by raising the degree of helical structure upon binding.     

Auto-thiophosphorylation activity of Src tyrosine kinase

Background

Intermolecular autophosphorylation at Tyr416 is a conserved mechanism of activation among the members of the Src family of nonreceptor tyrosine kinases. Like several other tyrosine kinases, Src can catalyze the thiophosphorylation of peptide and protein substrates using ATPγS as a thiophosphodonor, although the efficiency of the reaction is low.

Results

Here, we have characterized the ability of Src to auto-thiophosphorylate. Auto-thiophosphorylation of Src at Tyr416 in the activation loop proceeds efficiently in the presence of Ni²⁺, resulting in kinase activation. Other tyrosine kinases (Ack1, Hck, and IGF1 receptor) also auto-thiophosphorylate in the presence of Ni²⁺. Tyr416-thiophosphorylated Src is resistant to dephosphorylation by PTP1B phosphatase.

Conclusions

Src and other tyrosine kinases catalyze auto-thiophosphorylation in the presence of Ni²⁺. Thiophosphorylation of Src occurs at Tyr416 in the activation loop, and results in enhanced kinase activity. Tyr416-thiophosphorylated Src could serve as a stable, persistently-activated mimic of Src.

     

Protein tyrosine kinase Csk-catalyzed phosphorylation of Src containing unnatural tyrosine analogues

Using expressed protein ligation, five unnatural tyrosine analogues (amino-phenylalanine, homotyrosine, 2-methyl-tyrosine, (alphaS,betaR)-beta-methyl-tyrosine, and 2,6-difluoro-tyrosine) were incorporated into Src in place of the natural tail tyrosine residue. These semisynthetic substrates were evaluated as Csk substrates or allosteric activators. It appears that the tyrosine phenol hydroxyl is unlikely to be contributing significantly to Src's ground-state binding affinity for Csk. It has been observed that stabilizing tyrosine conformers can further optimize Src's already high substrate efficiency. These latter findings contrast similar studies with synthetic peptide substrates and highlight the value of investigation of protein kinase substrate selectivity with protein substrates.     

Light-regulated sampling of protein tyrosine kinase activity

Protein tyrosine kinases serve as key mediators of signaling pathways, biochemical highways that control various aspects of cell behavior. Although fluorescent reporters of tyrosine kinases have been described, these species can suffer immediate phosphorylation upon exposure to the cellular milieu, thereby hindering a detailed analysis of kinase activity as a function of the cell cycle or exposure to environmental stimuli. The first example of a light-regulated tyrosine kinase reporter is described herein, which allows the investigator to control when kinase activity is sampled. In addition, the set of sensors created in this study contain different fluorophores, each with its own unique photophysical properties, thereby furnishing an array of choices that can be used in combination with other intracellular probes.     

Cytotoxicity,tyrosine kinase inhibition of novel pyran,pyridine, thiophene,and imidazole derivatives

In this work, we are interested to use multicomponent reactions of cyclohexan-1,3-dione with different reagents for synthesizing new derivatives of pyran, pyridine, thiophene, and imidazole with antitumor activities. Twenty-two newly synthesized derivatives were selected and tested for their anticancer potency. Several of these compounds exhibited quite interesting potencies toward three human tumor cell lines, namely NCI-H460 (non-small cell lung cancer), SF-268 (CNS cancer), and MCF-7 (breast adenocarcinoma), especially when compared to that of reference drugs, doxorubicin and 5-Fu.Compounds 5b , 5c , 7b , 9b , 14a , 16c , 18a , 19c , 20b , and 22b , were found to be the most cytotoxic compounds toward the selected cell lines. On the other hand, 7b , 14a , 16c , 19c , and 22b revealed high inhibitions toward the tyrosine kinases. Active compounds against VEGFR-2, 14a , 16c , and 19c, were docked inside VEGFR-2enzyme to show the interaction between the tested compounds and the amino acids of the active site.     

Novel sesquiterpenoids as tyrosine kinase inhibitors produced by Stachybotrys chortarum

In the course of screening for small-molecule inhibitors to Tyrosine kinase receptor seven novel K-76 derivatives (1-7) have been isolated from the fungal culture of Stachybotrys chortarum. The structures were elucidated by extensive mono- and bi- dimensional spectroscopy and mass spectrometry.     

Inhibition of polo-like kinase 1 by blocking polo-box domain-dependent protein-protein interactions

The serine/threonine kinase Polo-like kinase 1 (Plk1) is overexpressed in many types of human cancers, and has been implicated as an adverse prognostic marker for cancer patients. Plk1 localizes to its intracellular anchoring sites via its polo-box domain (PBD). Here we show that Plk1 can be inhibited by small molecules which interfere with its intracellular localization by inhibiting the function of the PBD. We report the natural product thymoquinone and, especially, the synthetic thymoquinone derivative Poloxin as inhibitors of the Plk1 PBD. Both compounds inhibit the function of the Plk1 PBD in vitro, and cause Plk1 mislocalization, chromosome congression defects, mitotic arrest, and apoptosis in HeLa cells. Our data validate the Plk1 PBD as an anticancer target and provide a rationale for developing thymoquinone derivatives as anticancer drugs.     

Application of nonparametric statistics to the estimation of the accuracy of Monte Carlo confidence intervals in regression analysis

Confidence intervals and their uncertainties for nonlinear regression parameters are obtained using nonparametric statistical methods. The confidence intervals are calculated by means of a Monte Carlo procedure. Their uncertainties depend on the confidence level desired and on the number of Monte Carlo simulations of the data set. They are obtained by calculating the uncertainties in the boundaries of the confidence intervals using a generalization of the nonparametric method used to calculate confidence intervals for medians. The method described here provides reliable confidence intervals at relatively low computational expense. It seems especially suited to the statistical analysis of nonlinear regression problems that are difficult to deal with using conventional methods.     

The first total synthesis of hibarimicinone,a potent v-Src tyrosine kinase inhibitor

The first total synthesis of hibarimicinone has been achieved. The polyhydroxydecalin moieties (AB and GH rings) have been synthesized from sulfonylenone 4 derived from d-arabinose. The chiral biaryl 20 was coupled with two polyhydroxydecalins 11 by Michael–Dieckmann type condensation to give the eight rings system. Aromatization and oxidation with Ag⁺ gave quinone 24, and the subsequential transannular etheration gave the hibarimicinone skeleton. Deprotection and tautomerization were performed in one pot to give hibarimicinone (1).     

Inhibition of peroxynitrite- and peroxidase-mediated protein tyrosine nitration by imidazole-based thiourea and selenourea derivatives

In the present study, the synthesis and characterization of a series of N-methylimidazole-based thiourea and selenourea derivatives are described. The new compounds were also studied for their ability to inhibit peroxynitrite (PN)- and peroxidase-mediated nitration of protein tyrosine residues. It has been observed that the selenourea derivatives are more efficient than the thiourea-based compounds in the inhibition of protein nitration. The higher activity of selenoureas as compared to that of the corresponding thioureas can be ascribed to the zwitterionic nature of the selenourea moiety. Single crystal X-ray diffraction studies on some of the thiourea and selenourea derivatives reveal that the C=S bonds in thioureas possess more of double bond character than the C=Se bonds in the corresponding selenoureas. Therefore, the selenium compounds can react with PN or hydrogen peroxide much faster than their sulfur analogues. The reactions of thiourea and selenourea derivatives with PN or hydrogen peroxide produce the corresponding sulfinic or seleninic acid derivatives, which upon elimination of sulfurous/selenous acids produce the corresponding N-methylimdazole derivatives.     

Regulation of c-Src nonreceptor tyrosine kinase activity by bengamide A through inhibition of methionine aminopeptidases

Methionine aminopeptidases (MetAPs) remove the N-terminal initiator methionine during protein synthesis, a prerequisite step for N-terminal myristoylation. N-myristoylation of proto-oncogene c-Src is essential for its membrane association and proper signal transduction. We used bengamides, a family of general MetAP inhibitors, to understand the downstream physiological functions of MetAPs. c-Src from bengamide A-treated cells retained its N-terminal methionine and suffered a decrease in N-terminal myristoylation, which was accompanied by a shift of its subcellular distribution from the plasma membrane to the cytosol. Furthermore, bengamide A decreased the tyrosine kinase activities of c-Src both in vitro and in vivo and eventually delayed cell-cycle progression through G(2)/M. Thus, c-Src is a physiologically relevant substrate for MetAPs whose dysfunction is likely to account for the cell-cycle effects of MetAP inhibitors including bengamide A.     

Inhibition of protein tyrosine phosphatase 1beta by hispidin derivatives isolated from the fruiting body of Phellinus linteus

Protein tyrosine phosphatase 1beta (PTP1beta) acts as a negative regulator of insulin signaling. Selective inhibition of PTP1beta has served as a potential drug target for the treatment of type 2 diabetes mellitus. We evaluated the inhibitory effect of Phellinus linteus against PTP1beta as part of our ongoing search for natural therapeutic and preventive agents for diabetes mellitus. Fractions of the P. linteus extract were found to exhibit significant inhibitory activities against PTP1beta. In an attempt to identify bioactive components, we isolated, from the most active ethyl acetate fraction, five hispidin derivatives (phelligridimer A, davallialactone, hypholomine B, interfungins A, and inoscavin A) and four phenolic compounds (protocatechuic acid, protocatechualdehyde, caffeic acid, and ellagic acid). The chemical structures of these compounds were elucidated from spectroscopic evidence and by comparison with published data. All the compounds strongly inhibited PTP1beta activity in an in vitro assay; their IC50 values ranged from 9.0 +/- 0.01 to 58.2 +/- 0.3 microM. Our results indicated that the hispidin skeleton may be an important moiety for inhibitory activity of the above compounds against PTP1beta. Thus, hispidin derivatives could be a potent new class of natural PTP1beta inhibitors.     

A short, rigid, structurally pure carbon nanotube by stepwise chemical synthesis

The inaccessibility of uniform-diameter, single-chirality carbon nanotubes (CNTs) in pure form continues to thwart efforts by scientists to use these ultrathin materials in innovative applications that could revolutionize nanoscale electronics. Stimulated by the challenge to address this long-standing problem, we and other organic chemists have envisioned a new production strategy involving the controlled elongation of small hydrocarbon templates, such as hemispherical nanotube end-caps, prepared by bottom-up chemical synthesis; the diameter and rim structure encoded in the template would dictate the diameter and chirality of the resulting CNT. Toward that objective, a short [5,5] CNT has now been synthesized by stepwise chemical methods. This C(50)H(10) geodesic polyarene has been isolated, purified, crystallized, and fully characterized by NMR spectroscopy, UV-vis absorption spectroscopy, high resolution mass spectrometry, and X-ray crystallography.     

Conversion of a tyrosine kinase protein substrate to a high affinity ligand by ATP linkage

Protein kinases often show low affinity for their protein substrates, which makes it difficult to study kinase-substrate interactions. Here, we show using expressed protein ligation with the signaling protein Src that it is feasible to install a covalently linked ATP moiety into the tail of Src, generating a semisynthetic protein with a high affinity for its cognate tyrosine kinase, Csk. It is also established that this Src-ATP conjugate can be used to selectively pull down Csk from a complex protein mixture. This work outlines a general strategy for identifying an unknown kinase that is responsible for the phosphorylation of a protein substrate on a site of interest.     

A concise synthesis of a novel antiangiogenic tyrosine kinase inhibitor

An efficient synthesis of the potent KDR inhibitor 3-[5-[[4-(methylsulfonyl)-1-piperazinyl]methyl]-1H-indole-2-yl]quinolin-2(1H)-one (1) is described. The process features a noncryogenic indole boronation and a dicyclohexylamine-mediated Suzuki coupling.     

Mechanism of covalent binding of ibrutinib to Bruton's tyrosine kinase revealed by QM/MM calculations

Ibrutinib is the first covalent inhibitor of Bruton''s tyrosine kinase (BTK) to be used in the treatment of B-cell cancers. Understanding the mechanism of covalent inhibition will aid in the design of safer and more selective covalent inhibitors that target BTK. The mechanism of covalent inhibition in BTK has been uncertain because there is no appropriate residue nearby that can act as a base to deprotonate the cysteine thiol prior to covalent bond formation. We investigate several mechanisms of covalent modification of C481 in BTK by ibrutinib using combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics reaction simulations. The lowest energy pathway involves direct proton transfer from C481 to the acrylamide warhead in ibrutinib, followed by covalent bond formation to form an enol intermediate. There is a subsequent rate-limiting keto–enol tautomerisation step (ΔG^‡ = 10.5 kcal mol⁻¹) to reach the inactivated BTK/ibrutinib complex. Our results represent the first mechanistic study of BTK inactivation by ibrutinib to consider multiple mechanistic pathways. These findings should aid in the design of covalent drugs that target BTK and other similar targets.

QM/MM simulations show that covalent modification of BTK by ibrutinib proceeds via an intramolecular proton transfer from C481 to the acrylamide warhead of ibrutinib, followed by covalent bond formation and subsequent keto–enol tautomerisation.