A FRET-based microplate assay for human protein kinase CK2, a target in neoplastic disease

Besides cardiovascular diseases, cancer represents the major cause of death in developed countries. In many different human tumors, increased activity of serine/threonine protein kinase CK2 has been detected, and recent in vivo studies support a direct involvement of CK2 in tumor progression. Therefore, potent compounds to decrease CK2 activity to a non-pathogenic level would be a promising effort toward an antineoplastic therapy. In this study, an alternative to the established radiometric phosphorylation assay for quantification of CK2 activity was developed. For this purpose, the substrate peptide RRRDDDSDDD was coupled at the C-terminus to the fluorophore EDANS (5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid) and at the N-terminus to the quencher DABCYL (4-(4-dimethylaminophenylazo)benzoic acid). This resulted in quenched fluorescence of EDANS due to a FRET-based effect. After proteolytic cleavage of the peptide by elastase, the quenching effect was reduced and, as a consequence, fluorescence was increased. Because elastase is supposed to cleave at the S/D site of the peptide, phosphorylation of serine by CK2 hampered substrate binding of elastase and blocked the increase in fluorescence by proteolytic cleavage. This means that the new assay to quantify human CK2 activity is based on the differential accessibility of the proteolytic cleavage site, which is dependent on kinase phosphorylation. It could be used to measure inhibition of the human target in neoplastic diseases by the compounds TBB (4,5,6,7-tetrabromobenzotriazole) and Emodin.     

A Continuous Fluorescence Assay of Renin Activity

A sensitive fluorescence assay that employs a new fluorogenic peptide substrate has been developed to continuously measure the proteolytic activity of human renin. The substrate, DABCYL-gaba-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Thr-EDANS, has been designed to incorporate the renin cleavage site that occurs in the N-terminal peptide of human angiotensinogen. The assay relies upon resonance energy transfer-mediated, intramolecular fluorescence quenching that occurs in the intact peptide substrate. Efficient fluorescence quenching occurs as a result of favorable energetic overlap of the EDANS excited state and the DABCYL absorption, and the relatively long excited state lifetime of the EDANS fluorophore. Cleavage of the substrate by renin liberates the peptidyl-EDANS fragment from proximity with the DABCYL acceptor, restoring the higher, unattenuated fluorescence of the EDANS moiety. This leads to a time-dependent increase in fluorescence intensity, directly related to the extent of substrate consumed by renin cleavage. The kinetics of renin-catalyzed hydrolysis of this substrate have been shown to be consistent with a simple substrate inhibition model with a substrate K_m 1.5 μM at physiological pH; Cleavage of the substrate occurs specifically at the Leu-Val bond and corresponds to the renin cleavage site of angiotensinogen, as reported earlier. In this report, we describe in detail the synthesis of the fluorogenic renin substrate and its application in assays of renin activity. Assay sensitivity has been evaluated by a series of enzyme dilution experiments using the continuous assay format, showing that the assay can detect renin as low as 30 ng/ml after a incubation of only 3-5 min. It was estimated that with extended incubation time (2-3 h) the assay can detect renin at 0.5 ng/ml concentration level. An automated, high throughput fluorometric renin assay has been developed for a 96-well microtiter-plate fluorescence reader, which is useful for studies of enzyme inhibitors and enzyme stability.     

An improved solid-phase synthesis of resonance energy transfer fluorescent peptides and depsipeptides employing the EDANS/DABCYL donor-acceptor pair

Summary Fluorescent peptide substrates of the resonance energy transfer type, employing the donor-acceptor pair 5-[(2′-aminoethyl)amino]naphthalene sulphonic acid (EDANS) and 4-[[4′-(dimethylamino)phenyl]azo]benzoic acid (DABCYL), are widely used for continuous monitoring of the activity of various proteases. We describe here a flexible synthetic scheme which allows the synthesis of peptides having EDANS and DABCYL in any position of the sequence; the synthesis is entirely performed on solid phase with standard methods and makes use only of EDANS, DABCYL and commercially available amino acid derivatives. Moreover, we show that our scheme can be equally applied to the synthesis of EDANS/DABCYL-derivatised depsipeptides.     

An improved solid-phase synthesis of resonance energy transfer fluorescent peptides and depsipeptides employing the EDANS/DABCYL donor-acceptor pair

Fluorescent peptide substrates of the resonance energy transfer type, employing thedonor–acceptor pair 5-[(2-aminoethyl)amino]naphthalene sulphonic acid(EDANS) and 4-[[4-(dimethylamino)phenyl]azo]benzoic acid (DABCYL), are widelyused for continuous monitoring of the activity of various proteases. We describe here aflexible synthetic scheme which allows the synthesis of peptides having EDANS andDABCYL in any position of the sequence; the synthesis is entirely performed on solid phasewith standard methods and makes use only of EDANS, DABCYL and commercially availableamino acid derivatives. Moreover, we show that our scheme can be equally applied to thesynthesis of EDANS/DABCYL-derivatised depsipeptides.     

Stage-specific profiling of Plasmodium falciparum proteases using an internally quenched multispecificity protease substrate

Novel internally quenched fluorescence peptide substrates containing sequence specific sites for cleavage by multiple proteases were designed and synthesized. The 28 and 29 residue peptides contain an N-terminal fluorescence acceptor group, 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL), and a C-terminal fluorescence donor group, 5-(2-aminoethylamino)naphthalene-1-sulfonic acid (EDANS). Efficient energy transfer between the donor and acceptor groups flanking the peptide sequence was achieved by incorporation of a central DPro-Gly segment, which serves as a conformation nucleating site, inducing hairpin formation. This multispecificity protease substrate was used to profile the proteolytic activities in the malarial parasite Plasmodium falciparum in a stage dependent manner using a combination of fluorescence and MALDI mass spectrometry. Cysteine protease activity was shown to be dominating at neutral pH, whereas aspartic protease activity contributed predominantly to the proteolytic repertoire at acidic pH. Maximum proteolysis was observed at the trophozoite stage followed by the schizonts and the rings.     

New intramolecularly quenched fluorogenic peptide substrates for the study of the kinetic specificity of papain.

A series of new substrates for determining the catalytic activity of cysteine proteinases is described. The rate of hydrolysis by papain was monitored by a fluorescence continuous assay based on internal resonance energy transfer using 5-[(2-aminoethyl)amino]naphtalene-1-sulfonic acid (EDANS) and 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) as fluorescent donor and quenching acceptor, respectively, in peptides with the general structure: DABCYL-Lys-Phe-Gly-Xxx-Ala-Ala-EDANS. The substrates were used to evaluate the effect of amino acid structure in the S1' position on the kinetic parameters for papain catalyzed hydrolysis.     

An internally quenched fluorescent substrate for collagenase

A synthetic collagenase substrate containing the internal peptide sequence--Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro--has been synthesized, with an N-terminus 4-((4-(dimethylamino)phenyl)azo)-benzoyl (DABCYL) group and C-terminus 5-[2-(acetamido)ethylamino] naphthalene-1-sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. K(m) and V(max) values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures.     

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.     

Inhibition of protein kinase CK2 by condensed polyphenolic derivatives. An in vitro and in vivo study

ATP site-directed inhibitors that can target individual kinases are powerful tools for use in signal transduction research, all the more so in the case of a pleiotropic, constitutively active protein kinase such as CK2, which is not turned on in response to specific stimuli. By screening a library of more than 200 derivatives of natural polyphenolic compounds, we have identified 16 molecules which inhibit CK2 with IC(50) values of 相似文献   

HTLV-I protease cleavage of P19/24 substrates is not dependent on NaCl concentration

Understanding the factors that affect the activity of Human T-cell Leukemia Virus type I (HTLV-I) protease is essential for the discovery of inhibitors to be used for the treatment of HTLV-I infection, but little has been reported on the protease to date. Here we report the production of HTLV-I protease in purified yields greater than 150 mg/L, determination of its extinction coefficient, and determination of the optimum conditions for cleavage of the p19/24 substrates (DABCYL)-(GABA)-PQVL-Nph-VMH-(EDANS), (DABSYL)-(GABA)-PQVL-Nph-VMH-(EDANS), and (DABSYL)-(GABA)-PQVLPVMH-(EDANS). The highest activity was found at pH 5.2-5.3 and 37 degrees C. There was no effect on activity upon change in sodium chloride concentration from 0 to 1500 mM. The values of K(m) and k(cat) for cleavage of these substrates by the protease with and without the histidine tag were determined.     

Indeno[1,2-b]indole derivatives as a novel class of potent human protein kinase CK2 inhibitors

Herein we describe the synthesis and properties of indeno[1,2-b]indole derivatives as a novel class of potent inhibitors of the human protein kinase CK2. A set of 19 compounds was obtained using a convenient and straightforward synthesis protocol. The compounds were tested for inhibition of human protein kinase CK2, which was recombinantly expressed in Escherichia coli. New inhibitors with IC(50) in the micro- and sub-micromolar range were identified. Compound 4b (5-isopropyl-7,8-dihydroindeno[1,2-b]indole-9,10(5H,6H)-dione) inhibited human CK2 with an IC(50) of 0.11 μM and did not significantly inhibit 22 other human protein kinases, suggesting selectivity towards CK2. ATP-competitive inhibition by compound 4b was shown and a K(i) of 0.06 μM was determined. Our findings indicate that indeno[1,2-b]indoles are a promising starting point for further development and optimization of human protein kinase CK2 inhibitors.     

A comparison of human immunodeficiency virus type-1 protease inhibition activities by the aqueous and methanol extracts of Chinese medicinal herbs

The aqueous and methanol extracts of thirty-one herbs traditionally used as anti-fever remedies in China were screened for their in vitro inhibition on human immunodeficiency virus type-1 protease (HIV-1 PR). The activity of recombinant HIV-1 protease was determined by sequence-specific cleavage at the Tyr-Pro bond of the fluorogenic substrate (Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg) or by HPLC anaylsis of the cleavage products after incubation of the enzyme with a synthetic peptide substrate (Acetyl-Ser-Gln-Asn-Tyr-Pro-Val-Val-amide). Among the herbal extracts examined, the aqueous extracts of Prunella vulgaris and Scutellaria baicalensis and the methanol extracts of Woodwardia unigemmata, Paeonica suffruticosa and Spatholobus suberectus elicited significant inhibition (>90%) at a concentration of 200 microg/ml.     

Longer wavelength fluorescence resonance energy transfer depsipeptide substrates for hepatitis C virus NS3 protease

Maturation of the hepatitis C virus (HCV) polyprotein occurs by a series of proteolytic processes catalyzed by host cell proteases and the virally encoded proteases NS2 and NS3. Although several peptidomimetic inhibitors of NS3 protease have been published, only a few small molecule inhibitors have been reported. In an effort to improve screening efficiency by minimizing the spectral interference of various test compounds, a substrate that contains the longer wavelength fluorescence resonance energy transfer (FRET) pair, TAMRA/QSY-7, was devised. For the optimized substrate T-Abu-Q, with sequence Ac-Asp-Glu-Lys(TAMRA)-Glu-Glu-Abu-Psi(COO)Ala-Ser-Lys(QSY-7)-amide, the kinetic parameters with HCV NS3 protease are K(m)=30 microM, k(cat)=0.6s(-1), and k(cat)/K(m)=20,100s(-1)M(-1). We show that this substrate is suitable for inhibitor analysis and mechanistic studies so long as the substrate concentration is low enough (0.5 microM) to avoid complications from high inner filter effects. The substrate is especially useful with ultra-high-density screening formats, such as microarrayed compound screening technology, because there is less spectral interference from the compounds being tested than with more traditional (EDANS/DABCYL) FRET protease substrates. The merits of the new substrate, as well as potential applications of this FRET pair to other protease substrates, are discussed.     

An enzyme-linked immunosorbent assay to measure insulin receptor dephosphorylation by PTP1B

Considerable effort exists within drug discovery to develop novel compounds to improve the underlying metabolic defects in type 2 diabetes. One approach is focused on inhibition of the tyrosine phosphatase, PTP1B, an important negative regulator of both insulin and leptin signaling. Historically, tyrosine phosphatase assays have used either small organic phosphates or, alternatively, phosphorylated peptides from the target proteins themselves. In characterizing inhibitors of PTP1B, measuring turnover of small organic phosphates is limited to evaluation of compounds that bind the active site itself. Peptide substrates allow identification of additional subsets of inhibitors (e.g., those that bind the second aryl-phosphate site), but assays of peptide turnover often involve detection steps that then limit full kinetic evaluation of inhibitors. Here we use a polyclonal antibody specific for the phosphorylated insulin receptor to allow much more sensitive detection of peptide phosphorylation. This kinetically robust enzyme-linked immunosorbent assay (ELISA) gives k(cat) and K(m) values for a phosphorylated insulin receptor peptide consistent with values determined by a continuous fluorescence-based assay. Furthermore, IC50 values determined for well-behaved active site inhibitors agree well with values determined for p-nitrophenyl phosphate cleavage. This assay permits full characterization of a larger subset of inhibitors as drug candidates for this promising target.     

Identification of small molecule inhibitors of the mitotic kinase haspin by high-throughput screening using a homogeneous time-resolved fluorescence resonance energy transfer assay

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at K(m) concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC( 50) determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC( 50) values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics.     

Enzyme assay for protein kinase using micellar electrokinetic chromatography with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection has been developed for a protein kinase assay. This protein kinase assay could readily determine the phosphorylation activity of substrate peptide kemptide using cAMP-dependent protein kinase (PKA) as a model enzyme. Kemptide and phosphorylated kemptide could be reacted with 7-fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-F) as a fluorescence derivatization reagent for LIF detection by directly adding NBD-F into the PKA enzymatic reaction mixture. These derivatives of substrate and product were separated and detected within the analysis time of 5 min by micellar electrokinetic mode using a mixture of sodium dodecylsulfate and methanol as a running buffer. Good linearity of the peak response of the phosphorylated kemptide was obtained over the range of 1-20 mU/tube of PKA in the assay. The relative standard deviation of the peak areas of the phosphorylated kemptide using 2, 5 and 10 mU/tube of PKA were calculated to <10.4%, indicating that the assay was reproducible. Also, IC(50) values of six PKA inhibitors, the K(i) value and the inhibition pattern of one inhibitor, which were calculated to estimate by the variation of the peak area of the phosphorylated kemptide using 5 mU/tube of PKA, were consistent with the published data. The sensitivity of the assay was higher than that of enzyme-linked immunosorbent assay (ELISA) for PKA phosphorylation activity, as IC(50) values, K(i) value, and the inhibition mechanism of inhibitors could be estimated using one-tenth amounts of PKA, compared with that of ELISA. The MEKC-LIF is expected to be very useful for protein kinase assay and its application to the estimation of inhibitors because this method does not entail experimentally troublesome procedures such as the preparation of antibody or fluorescence-labeled substrate.     

Development and characterization of potent and specific peptide inhibitors of p60c-src protein tyrosine kinase using pseudosubstrate-based inhibitor design approach.

The cytoplasmic protein p60c-src, an ubiquitous non-receptor protein tyrosine kinase (PTK) is a potential anticancer target as it is over-expressed and/or constitutively active in several cancer types. In addition, the phenotype of c-src knock-out mice is consistent with osteopetrosis, which suggests that inhibitors against this enzyme may also be therapeutic for osteoporosis. Using a known peptide substrate for c-src, MIYKYYF, as a template, we have developed a series of pseudosubstrate-based peptide inhibitors. Structure-activity relationship studies have been performed on one of these inhibitors, CIYKYYF. In a kinase assay using YIYGSFK as the substrate, CIYKYY has been demonstrated to inhibit p60c-src, with an IC50 of 0.6 microm. Further truncation has led to the determination that even the smaller peptide, CIYK, is a moderately potent inhibitor with IC50 of 15 microm. Some improvement in inhibitory potency (IC50 = 11.8 microm) has been observed with the replacement of Tyr3 in CIYK with beta-phenylalanine (beta-Phe). The tetrapeptide CI(beta-Phe)K will be used as a lead compound for future development of peptidomimetics and small molecule inhibitors that have the capacity to penetrate the plasma membrane of intact cells.     

Development and validation of a competitive AKT serine/threonine kinase fluorescence polarization assay using a product-specific anti-phospho-serine antibody.

A competitive fluorescence polarization (FP) assay has been developed for the serine/threonine kinase, AKT. The FP assay has been formatted in a 384-well microtiter plate and automated using a pipeting workstation with performance suitable for high-throughput screening. The assay design utilizes a fluorescent phosphorylated peptide complexed to a product-specific anti-phospho-serine antibody. When unlabeled substrate is phosphorylated, by the kinase, the product competes with the fluorescent phosphorylated peptide for the antibody. The fluorescent phosphorylated peptide is then released from the antibody into solution resulting in a loss in polarization signal. Seven fluorescent phosphorylated peptides and 19 antibodies were evaluated for this assay. RARTSpSFAEPGK-Fl peptide and anti-phospho-GSK-3alpha Ser21 antibody gave the best affinity and change in polarization signal. The apparent kinetic constants were calculated for the FP assay and were consistent with reported values. The FP assay was validated with known inhibitors and the results compared to a radioactive Flashplate transfer assay, utilizing [(33)P]ATP and a biotinylated substrate, also developed in our laboratory. The IC(50) values generated were comparable between the two methods suggesting the competitive FP assay and Flashplate assay have similar sensitivities and abilities to identify inhibitors during screening.     

Fluorescent substrates for soluble epoxide hydrolase and application to inhibition studies

Inhibition of the mammalian soluble epoxide hydrolase (sEH) is a promising new therapy in the treatment of disorders resulting from hypertension and vascular inflammation. A spectrophotometric assay (4-nitrophenyl-trans-2,3-epoxy-3-phenylpropyl carbonate, NEPC) is currently used to screen libraries of chemicals; however this assay lacks the required sensitivity to differentiate the most potent inhibitors. A series of fluorescent alpha-cyanoester and alpha-cyanocarbonate epoxides that produce a strong fluorescent signal on epoxide hydrolysis by both human and murine sEH were designed as potential substrates for an in vitro inhibition assay. The murine enzyme showed a broad range of specificities, whereas the human enzyme showed the highest specificity for cyano(6-methoxy-naphthalen-2-yl)methyl trans-[(3-phenyloxiran-2-yl)methyl] carbonate. An in vitro inhibition assay was developed using this substrate and recombinant enzyme. The utility of the fluorescent assay was confirmed by determining the IC(50) values for a series of known inhibitors. The new IC(50) values were compared with those determined by spectrophotometric NEPC and radioactive tDPPO assays. The fluorescent assay ranked these inhibitors on the basis of IC(50) values, whereas the NEPC assay did not. The ranking of inhibitor potency generally agreed with that determined using the tDPPO assay. These results show that the fluorescence-based assay is a valuable tool in the development of sEH inhibitors by revealing structure-activity relationships that previously were seen only by using the costly and labor-intensive radioactive tDPPO assay.     

Inhibition of rat liver cyclic AMP-dependent protein kinase by flavonoids.

Rat liver cyclic AMP-dependent protein kinase catalytic subunit (cAK), assayed using the synthetic peptide substrate, LRRASLG, is inhibited by a range of plant-derived flavonoids. In general, maximal inhibitory effectiveness (IC50 values 1 to 2 microM) requires 2,3-unsaturation and polyhydroxylation involving at least two of the three flavonoid rings. 3-Hydroxyflavone (IC50 value 4 microM), 3,5,7,2',4'-pentahydroxyflavone (IC50 = 10 microM) and 5,7,4'-trihydroxyflavone (IC50 = 7 microM) represent somewhat less active variations from this pattern. Flavonoid O-methylation or O-glycosylation greatly decreases inhibitory effectiveness, as does 2,3-saturation. Various flavonoid-related compounds, notably gossypol (IC50 = 10 microM), also inhibit cAK. Flavonoids and related compounds are in general much better inhibitors of cAK than of avian Ca(2+)-calmodulin-dependent myosin light chain kinase or of plant Ca(2+)-dependent protein kinase. Tricetin (IC50 = 1 microM) inhibits cAK in a fashion that is non-competitive with respect to both peptide substrate and ATP (Ki value 0.7 microM). When histone III-S is used as a substrate, inhibition of cAK requires much higher flavonoid concentrations.