Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2, 5-dibromophenyl)propenamide

In a systematic effort to design potent inhibitors of the anti-apoptotic tyrosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with apoptosis-promoting and chemosensitizing properties, we have constructed a three-dimensional homology model of the BTK kinase domain. Our modeling studies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu460, Tyr476, Arg525, and Asp539 residues occupying the corners of the rectangle. The dimensions of this rectangle are approximately 18 x 8 x 9 x 17 A, and the thickness of the pocket is approximately 7 A. Advanced docking procedures were employed for the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a Ki value of 1.4 microM, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 microM. Similarly, LFM-A13 inhibited recombinant BTK expressed in a baculovirus expression vector system with an IC50 value of 2.5 microM. The energetically favorable position of LFM-A13 in the binding pocket is such that its aromatic ring is close to Tyr476, and its substituent group is sandwiched between residues Arg525 and Asp539. In addition, LFM-A13 is capable of favorable hydrogen bonding interactions with BTK via Asp539 and Arg525 residues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was also discovered to be a highly specific inhibitor of BTK. Even at concentrations as high as 100 micrograms/ml (approximately 278 microM), this novel inhibitor did not affect the enzymatic activity of other protein tyrosine kinases, including JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin receptor kinase. In accordance with the anti-apoptotic function of BTK, treatment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivity to ceramide- or vincristine-induced apoptosis. To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leukemic agent targeting BTK.     

A remote substrate docking mechanism for the tec family tyrosine kinases

During T cell signaling, Itk selectively phosphorylates a tyrosine within its own SH3 domain and a tyrosine within PLCgamma1. We find that the remote SH2 domain in each of these substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. Additionally, we detect a stable interaction between the substrate SH2 domains and the kinase domain of Itk and find that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation. On the basis of these results, we show that the kinetic parameters of a generic peptide substrate of Itk are significantly improved via fusion of the peptide substrate to the SH2 domain of PLCgamma1. This work is the first characterization of a substrate docking mechanism for the Tec kinases and provides evidence of a novel, phosphotyrosine-independent regulatory role for the ubiquitous SH2 domain.     

The complete pathway for catalytic activation of the mitogen-activated protein kinase, ERK2

The mitogen-activated protein (MAP) kinase ERK2 is an essential signal transduction molecule that mediates extracellular signaling by all polypeptide growth factors. Full activation of ERK2 requires phosphorylation at both a threonine residue (Thr(183)) conserved in most protein kinases as well as a tyrosine residue (Tyr(185)) unique to members of the mitogen-activated protein kinase family. We have characterized the kinetic role of phosphorylation at each site with respect to the overall activation mechanism, providing a complete picture of the reaction steps involved. Phosphorylation at Tyr(185) serves to configure the ATP binding site, while phosphorylation at both residues is required to stabilize binding of the protein substrate, myelin basic protein. Similar control mechanisms are employed to stabilize ATP and myelin basic protein in the phosphoryl group transfer reaction, accounting for the enormous increase in turnover rate. The mechanism of ERK2 activation is kinetically similar to that of the cell cycle control protein, cdk2/cyclinA. Phosphorylation of Tyr(185) in ERK2 and association of cyclinA with cdk2 both serve to stabilize ATP binding. Subsequent phosphorylation of both enzymes on threonine serves to stabilize binding of the phosphoacceptor substrate.     

Kinetic mechanism for human Rho-Kinase II (ROCK-II)

Rho-Kinase is a serine/threonine kinase that is involved in the regulation of smooth muscle contraction and cytoskeletal reorganization of nonmuscle cells. While the signal transduction pathway in which Rho-Kinase participates has been and continues to be extensively studied, the kinetic mechanism of Rho-Kinase-catalyzed phosphorylation has not been investigated. We report here elucidation of the kinetic mechanism for Rho-Kinase by using steady-state kinetic studies. These studies used the kinase domain of human Rho-Kinase II (ROCK-II 1-534) with S6 peptide (biotin-AKRRRLSSLRA-NH(2)) as the phosphorylatable substrate. Double-reciprocal plots for two-substrate kinetic data yielded intersecting line patterns with either ATP or S6 peptide as the varied substrate, indicating that Rho-Kinase utilized a ternary complex (sequential) kinetic mechanism. Dead-end inhibition studies were used to investigate the order of binding for ATP and the peptide substrate. The ATP-competitive inhibitors AMP-PCP and Y-27632 were noncompetitive inhibitors versus S6 peptide, and the S6 peptide analogue S6-AA (acetyl-AKRRRLAALRA-NH(2)) was a competitive inhibitor versus S6 peptide and a noncompetitive inhibitor versus ATP. These results indicated a random order of binding for ATP and S6 peptide.     

Transcription factor STAT5A is a substrate of Bruton's tyrosine kinase in B cells.

STAT5A is a molecular regulator of proliferation, differentiation, and apoptosis in lymphohematopoietic cells. Here we show that STAT5A can serve as a functional substrate of Bruton's tyrosine kinase (BTK). Purified recombinant BTK was capable of directly binding purified recombinant STAT5A with high affinity (K(d) = 44 nm), as determined by surface plasmon resonance using a BIAcore biosensor system. BTK was also capable of tyrosine-phosphorylating ectopically expressed recombinant STAT5A on Tyr(694) both in vitro and in vivo in a Janus kinase 3-independent fashion. BTK phosphorylated the Y665F, Y668F, and Y682F,Y683F mutants but not the Y694F mutant of STAT5A. STAT5A mutations in the Src homology 2 (SH2) and SH3 domains did not alter the BTK-mediated tyrosine phosphorylation. Recombinant BTK proteins with mutant pleckstrin homology, SH2, or SH3 domains were capable of phosphorylating STAT5A, whereas recombinant BTK proteins with SH1/kinase domain mutations were not. In pull-down experiments, only full-length BTK and its SH1/kinase domain (but not the pleckstrin homology, SH2, or SH3 domains) were capable of binding STAT5A. Ectopically expressed BTK kinase domain was capable of tyrosine-phosphorylating STAT5A both in vitro and in vivo. BTK-mediated tyrosine phosphorylation of ectopically expressed wild type (but not Tyr(694) mutant) STAT5A enhanced its DNA binding activity. In BTK-competent chicken B cells, anti-IgM-stimulated tyrosine phosphorylation of STAT5 protein was prevented by pretreatment with the BTK inhibitor LFM-A13 but not by pretreatment with the JAK3 inhibitor HI-P131. B cell antigen receptor ligation resulted in enhanced tyrosine phosphorylation of STAT5 in BTK-deficient chicken B cells reconstituted with wild type human BTK but not in BTK-deficient chicken B cells reconstituted with kinase-inactive mutant BTK. Similarly, anti-IgM stimulation resulted in enhanced tyrosine phosphorylation of STAT5A in BTK-competent B cells from wild type mice but not in BTK-deficient B cells from XID mice. In contrast to B cells from XID mice, B cells from JAK3 knockout mice showed a normal STAT5A phosphorylation response to anti-IgM stimulation. These findings provide unprecedented experimental evidence that BTK plays a nonredundant and pivotal role in B cell antigen receptor-mediated STAT5A activation in B cells.     

Kinetic parameters of the protein tyrosine kinase activity of EGF-receptor mutants with individually altered autophosphorylation sites.

Epidermal growth factor (EGF)-receptor mutants in which individual autophosphorylation sites (Tyr1068, Tyr1148 or Tyr1173) have been replaced by phenylalanine residues were expressed in NIH-3T3 cells lacking endogenous EGF-receptors. Kinetic parameters of the kinase of wild-type and mutant receptors were compared. Both wild-type and mutant EGF-receptors had a Km(ATP) 1-3 microM for the autophosphorylation reaction, and a Km(ATP) of 3-7 microM for the phosphorylation of a peptide substrate. These are similar to the Km(ATP) values reported for EGF-receptor of A431 cells. A synthetic peptide representing the major in vitro autophosphorylation site Tyr1173 of the EGF-receptor (KGSTAENAEYLRV) was phosphorylated by wild-type receptor with a Km of 110-130 microM, and the peptide inhibited autophosphorylation with a Ki of 150 microM. Mutant EGF-receptors phosphorylated the peptide substrate with a Km of 70-100 microM. A similar decrease of Km (substrate) was obtained when the phosphorylation experiments were performed with the commonly applied substrates angiotensin II and a peptide derived from c-src. The Km of angiotensin II phosphorylation was reduced from 1100 microM for wild-type receptor to 890 microM for mutant receptor and for c-src peptide from 1010 microM to 770 microM respectively. The Vmax of the kinase was dependent on receptor concentration, but was not significantly affected by the mutation. Analogs of the Tyr1173 peptide in which the tyrosine residue was replaced by either a phenylalanine or an alanine residue also inhibited autophosphorylation with Ki of 650-750 microM. These analyses show that alterations of individual autophosphorylation sites do not have a major effect on kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

ATP-phosphopeptide conjugates as inhibitors of Src tyrosine kinases

A number of Src SH2 domain inhibitors enhance the kinase catalytic activity by switching the closed inactive to the open active conformation. ATP-phosphopeptide conjugates were designed and synthesized as Src tyrosine kinase inhibitors based on a tetrapeptide sequence pTyr-Glu-Glu-Ile (pYEEI) and ATP to block the SH2 domain signaling and substrate phosphorylation by ATP, respectively. In general, ATP-phosphopeptide conjugates with optimal linkers such as compounds 5 and 7 (K(i) = 1.7-2.6 microM) showed higher binding affinities to the ATP-binding site relative to the other ATP-phosphopeptide conjugates having short or long linkers, 1-4 and 6, (K(i) = 10.1-16.1 microM) and ATP (K(m) = 74 microM). These ATP-phosphopeptide conjugates may serve as novel templates for designing protein tyrosine kinase inhibitors to block SH2 mediated protein-protein interactions and to counter the activation of enzyme that resulted from the SH2 inhibition.     

Kinetic mechanism of fully activated S6K1 protein kinase

S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.     

Potent,non-covalent reversible BTK inhibitors with 8-amino-imidazo[1,5-a]pyrazine core featuring 3-position bicyclic ring substitutes

Bruton’s tyrosine kinase (BTK) is a Tec family kinase with a well-defined role in the B cell receptor (BCR) pathway. It has become an attractive kinase target for selective B cell inhibition, and for the treatment of B cell related diseases. Many BTK inhibitors have been discovered for the treatment of cancer and rheumatoid arthritis, including a series of BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine we recently reported. The X-ray crystal structures of BTK with inhibitors were also published, which provided great help for the SAR design. Here we report our SAR work introducing ring constraints for the 3-position piperidine amides on the BTK inhibitors based on 8-amino-imidazo[1,5-a]pyrazine. This modification improved the potency in BTK inhibitions, as well as the PK profile and the off-target selectivity. The dose-dependent efficacy of two BTK inhibitors was observed in the rat collagen induced arthritis (CIA) model.     

A rationale for the design of an inhibitor of tyrosyl kinase

Two gastrin analogs containing a D- and a L-tetrafluorinated tyrosyl residue (Arg-Arg-Leu-Glu-Glu-Glu-Glu-Glu-Ala-(F4)Tyr-Gly) were synthesized and tested as substrates and inhibitors of the insulin receptor kinase. No phosphorylation of these peptides was observed, but both gastrin analogs were effective inhibitors in the microM range. Although the D- and L-tetrafluorotyrosine-gastrin analogs differ in the sequence by only 1 amino acid residue, a different inhibitory pattern was obtained with the insulin receptor. The inhibition of all-L-isomer is competitive with respect to both the protein substrate, reduced, S-carboxymethylated, and maleylated lysozyme (RCMM-lysozyme), and ATP with a Ki value of 4 microM. This result corroborates a previous finding (Walker, D. H., Kuppuswamy, D., Visvanathan, A., and Pike, L. J. (1987) Biochemistry 26, 1428-1433) that the kinetic mechanism for insulin receptor is a random Bi Bi mechanism. Different from the L-isomer, the D-analog is competitive to RCMM-lysozyme and noncompetitive toward ATP and gives an apparent inhibition constant of 20 microM. A free tetrafluorotyrosine also shows a competitive inhibition to protein substrate, RCMM-lysozyme (Ki = 18 mM) whereas free tyrosine shows no effect on the activity of insulin receptor. These results show the importance of the charge state and nucleophilicity of the phenolic component in substrate recognition and catalysis and provide a rationale for the design of inhibitors of tyrosyl phosphorylation.     

Pig liver phosphomevalonate kinase: kinetic mechanism

Phosphomevalonate kinase catalyzes the phosphorylation of phosphomevalonate to diphosphomevalonate by ATP, one of the initial steps in the biosynthesis of steroids and isoprenoids. In previous studies, the enzyme from pig liver was purified and characterized, and preliminary work showed that the enzyme follows hyperbolic kinetics and a sequential mechanism. The present work is a more detailed analysis of its kinetic mechanism, using initial velocity and isotope exchange at equilibrium measurements. The results are compatible with a Bi Bi sequential ordered mechanism with phosphomevalonate as the first substrate and ADP the last product. The Km values estimated are 43+/-7 microM for Mg-ATP and 12+/-3 microM for phosphomevalonate, with a Vmax of 51+/-2 micromol min-1 mg of protein-1.     

The cyclin-dependent kinases cdk2 and cdk5 act by a random, anticooperative kinetic mechanism

cdk2.cyclin E and cdk5.p25 are two members of the cyclin-dependent kinase family that are potential therapeutic targets for oncology and Alzheimer's disease, respectively. In this study we have investigated the mechanism for these enzymes. Kinases catalyze the transfer of phosphate from ATP to a protein acceptor, thus utilizing two substrates, ATP and the target protein. For a two-substrate reaction, possible kinetic mechanisms include: ping-pong, sequential random, or sequential ordered. To determine the kinetic mechanism of cdk2.GST-cyclin E and cdk5.GST-p25, kinase activity was measured in experiments in which concentrations of peptide and ATP substrates were varied in the presence of dead-end inhibitors. A peptide identical to the peptide substrate, but with a substitution of valine for the phosphoacceptor threonine, competed with substrate with a K(i) value of 0.6 mm. An aminopyrimidine, PNU 112455A, was identified in a screen for inhibitors of cdk2. Nonlinear least squares and Lineweaver-Burk analyses demonstrated that the inhibitor PNU 112455A was competitive with ATP with a K(i) value of 2 microm. In addition, a co-crystal of PNU 112455A with cdk2 showed that the inhibitor binds in the ATP binding pocket of the enzyme. Analysis of the inhibitor data demonstrated that both kinases use a sequential random mechanism, in which either ATP or peptide may bind first to the enzyme active site. For both kinases, the binding of the second substrate was shown to be anticooperative, in that the binding of the first substrate decreases the affinity of the second substrate. For cdk2.GST-cyclin E the kinetic parameters were determined to be K(m, ATP) = 3.6 +/- 1.0 microm, K(m, peptide) = 4.6 +/- 1.4 microm, and the anticooperativity factor, alpha = 130 +/- 44. For cdk5.GST-p25, the K(m, ATP) = 3.2 +/- 0.7 microm, K(m, peptide) = 1.6 +/- 0.3 microm, and alpha = 7.2 +/- 1.8.     

Kinetic mechanism of AKT/PKB enzyme family

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.     

Identification and characterization of a constitutively T-loop phosphorylated and active recombinant S6K1: expression, purification, and enzymatic studies in a high capacity non-radioactive TR-FRET Lance assay

The p70 S6 ribosomal protein kinase 1 (S6K) is a substrate and effector of the mammalian target of rapamycin (mTOR). The mTOR/S6K pathway is implicated in cancer and metabolic disorders. To study the molecular regulation of S6K and identify specific inhibitors, availability of active recombinant S6K and robust enzyme assays are critically needed. To date, however, expression of active recombinant S6K has not been feasible as S6K activation requires a cascade of phosphorylation events. We have compared several engineered S6K enzymes. Expression of the Flag-S6KDeltaCT(T389E) in HEK293 cells resulted in a highly active S6K that was constitutively phosphorylated on T229 in the activation-loop (T-loop). The active enzyme was readily purified in large scale by anti-Flag affinity chromatography achieving a high purity. We developed a high capacity homogeneous time-resolved fluorescence resonance energy transfer. Lance assay for measurement of substrate phosphorylation and analysis of kinetic parameters. The Michaelis constant (Km) values of S6K for ATP and the Biotin-S6 substrate peptide were determined to be 21.4+/-0.29 and 0.9+/-0.48 microM, respectively. The Lance assay was further validated with a diverse panel of literature inhibitors, in which the PKC inhibitors staurosporine, Ro-318220, and the PKA inhibitor Balanol potently inhibited S6K. Dose-response and inhibition mechanism by these inhibitors were also studied. Our data provide a new simplified strategy to achieve rapid production of active S6K and demonstrate utility of the Lance assay for S6K enzyme screen in searching for specific inhibitors.     

Examination of the kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2

The kinetic mechanism of mitogen-activated protein kinase activated protein kinase-2 (MAPKAPK2) was investigated using a peptide (LKRSLSEM) based on the phosphorylation site found in serum response factor (SRF). Initial velocity studies yielded a family of double-reciprocal lines that appear parallel and indicative of a ping-pong mechanism. The use of dead-end inhibition studies did not provide a definitive assignment of a reaction mechanism. However, product inhibition studies suggested that MAPKAPK2 follows an ordered bi-bi kinetic mechanism, where ATP must bind to the enzyme prior to the SRF-peptide and the phosphorylated product is released first, followed by ADP. In agreement with these latter results, surface plasmon resonance measurements demonstrate that the binding of the inhibitor peptide to MAPKAPK2 requires the presence of ATP. Furthermore, competitive inhibitors of ATP, adenosine 5'-(beta,gamma-imino)triphosphate (AMPPNP) and a staurosporine analog (K252a), can inhibit this ATP-dependent binding providing further evidence that the peptide substrate binds preferably to the E:ATP complex.     

High yield expression of non-phosphorylated protein tyrosine kinases in insect cells

The key role of kinases in signal transduction and cell growth regulation has been a long standing interest among academics and the pharmaceutical industry. Recombinant enzymes have been used to understand the mechanism of action as well as to screen for chemical inhibitors. The baculo-insect system has been the primary method used to obtain soluble and active kinases, usually producing a mixture of the kinase in various phosphorylation states in different conformations. To obtain a homogenous preparation of non-phosphorylated kinases is critical for biochemical, biophysical and kinetic studies aimed at understanding the mechanism of kinase activation. Taking advantage of the eukaryotic expression property of insect cells, we were able to obtain high yield expression of non-phosphorylated protein tyrosine kinases BTK, JAK3 and Eph2A through coexpression with the tyrosine phosphatase YopH, which suggests that this method can be applied to protein tyrosine kinases in general. We have demonstrated that the fully non-phosphorylated BTK obtained with this method is suitable for various biochemical and kinetic studies.     

Bruton tyrosine kinase-like protein, BtkSD, is present in the marine sponge Suberites domuncula

Sponges, the simplest and most ancient phylum of Metazoa, encode in their genome complex and highly sophisticated proteins that evolved together with multicellularity and are found only in metazoan animals. We report here the finding of a Bruton tyrosine kinase (BTK)-like protein in the marine sponge Suberites domuncula (Demospongiae). The nucleotide sequence of one sponge cDNA predicts a 700-aa-long protein, which contains all of the characteristic domains for the Tec family of protein tyrosine kinases (PTKs). The highest homology (38% identity, 55% overall similarity) was found with human BTK and TEC PTKs. Sponge PTK was therefore named BtkSD. Human BTK is involved in the maturation of B cells and mutations in the BTK gene cause X-linked agammaglobulinemia. Kinases from the Tec family are not present in Caenorhabditis elegans and, until now, they were found only in insects and higher animal taxa. Our finding implies that the BTK/TEC genes are of a very ancient origin.