Achieving selectivity between highly homologous tyrosine kinases: a novel selective erbB2 inhibitor

The discovery of small molecule kinase inhibitors for use as drugs is a promising approach for the treatment of cancer and other diseases, but the discovery of highly specific agents is challenging because over 850 kinases are expressed in mammalian cells. Systematic modification of the 4-anilino functionality of a selective quinazoline inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase can invert selectivity to favor inhibition of the highly homologous erbB2 tyrosine kinase. The selectivity pattern was demonstrated in assays of recombinant kinases and recapitulated in measures of kinase activity in intact cells. The most potent and selective erbB2 inhibitor of the analog series has anti-proliferative activity against an erbB2-overexpressing cell line that was lacking in the original EGFR-selective compound. Subtle changes to the molecular structure of ATP-competitive small molecule inhibitors of tyrosine kinases can yield dramatic changes in potency and selectivity. These results suggest that the discovery of highly selective small molecule inhibitors of very homologous kinases is achievable.     

Rational design of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones as a novel class of inhibitors of epidermal growth factor receptor (EGF-R) and Her2(p185(erbB)) tyrosine kinases

A novel class of 4,5-disubstituted-5,7-dihydro-pyrrolo[2,3-d]pyrimidin-6-ones has been discovered as potent and selective inhibitors of the EGF-R tyrosine kinase family. These compounds selectively inhibit EGF-R kinase activity at low nanomolar concentration and tyrosine autophosphorylation in cells expressing EGF-R or Her2 (p185(erbB)). Structure-activity relationships (SARs) for this class of compounds are presented.     

Extracellular domains drive homo- but not hetero-dimerization of erbB receptors

Many different growth factor ligands, including epidermal growth factor (EGF) and the neuregulins (NRGs), regulate members of the erbB/HER family of receptor tyrosine kinases. These growth factors induce erbB receptor oligomerization, and their biological specificity is thought to be defined by the combination of homo- and hetero-oligomers that they stabilize upon binding. One model proposed for ligand-induced erbB receptor hetero-oligomerization involves simple heterodimerization; another suggests that higher order hetero-oligomers are 'nucleated' by ligand-induced homodimers. To distinguish between these possibilities, we compared the abilities of EGF and NRG1-beta1 to induce homo- and hetero-oligomerization of purified erbB receptor extracellular domains. EGF and NRG1-beta1 induced efficient homo-oligomerization of the erbB1 and erbB4 extracellular domains, respectively. In contrast, ligand-induced erbB receptor extracellular domain hetero-oligomers did not form (except for s-erbB2-s-erbB4 hetero-oligomers). Our findings argue that erbB receptor extracellular domains do not recapitulate most heteromeric interactions of the erbB receptors, yet reproduce their ligand-induced homo-oligomerization properties very well. This suggests that mechanisms for homo- and hetero-oligomerization of erbB receptors are different, and contradicts the simple heterodimerization hypothesis prevailing in the literature.     

S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors

Increased activity of the src family of oncogenic tyrosine kinases is seen in many human tumors and pharmacologic inhibitors of these kinases are investigated as potential anti-tumor agents. A family of pyrido [2, 3-d] pyrimidine compounds (PD) has been characterized as selective inhibitors of Src kinases. We studied the effects of this class of compounds on cancer cell lines and found that they were highly specific inhibitors of cell cycle progression. These compounds inhibit cells either in the mitotic phase or in mid S-phase; these two activities are mutually exclusive: no compound exerts both activities. We undertook experiments to determine the mechanistic basis for these differences and found additional biochemical activities associated with the S-phase inhibitors. Treatment of cells with the S-phase blocker PD179483 causes abnormal and persistent hyperactivation of Cdk2 and Cdc2 due to Tyr-15 dephosphorylation. These effects were associated with hyperphosphorylation of the upstream regulatory kinase Myt1 and Wee1. They were not observed with the anti-mitotic compounds. Furthermore, the S-phase inhibitors PD179483 and PD166326, but not the anti-mitotic compounds, inhibit Wee1 in vitro at concentrations that cause S-phase block in vivo. These data identify a novel subset of pyridopyrimidine compounds which are inhibitors of src and Wee1 kinases and which inhibit tumor cell growth through cell cycle arrest in mid S-phase.     

Disabling receptor ensembles with rationally designed interface peptidomimetics

Members of the erbB family receptor tyrosine kinases (erbB1, erbB2, erbB3, and erbB4) are overexpressed in a variety of human cancers and represent important targets for the structure-based drug design. Homo- and heterodimerization (oligomerization) of the erbB receptors are known to be critical events for receptor signaling. To block receptor self-associations, we have designed a series of peptides derived from potential dimerization surfaces in the extracellular subdomain IV of the erbB receptors (erbB peptides). In surface plasmon resonance (BIAcore) studies, the designed peptides have been shown to selectively bind to the erbB receptor ectodomains and isolated subdomain IV of erbB2 with submicromolar affinities and to inhibit heregulin-induced interactions of erbB3 with different erbB receptors. A dose-dependent inhibition of native erbB receptor dimerization by the erbB peptides has been observed in 32D cell lines transfected with different combinations of erbB receptors. The peptides effectively inhibited growth of two types of transformed cells overexpressing different erbB receptors, T6-17 and 32D, in standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cell viability assays. The study identifies distinct loops within the membrane-proximal part of the subdomain IV as potential receptor-receptor interaction sites for the erbB receptors and demonstrates the possibility of disabling receptor activity by structure-based targeting of the dimerization interfaces. Molecular models for possible arrangement of the erbB1.EGF complex, consistent with the involvement of subdomain IV in inter-receptor interactions, are proposed. Small dimerization inhibitors described herein can be useful as probes to elucidate different erbB signaling pathways and may be developed as therapeutic agents.     

Pyrazolo[3,4-b]quinoxalines. A new class of cyclin-dependent kinases inhibitors

Protein kinases are involved in most physiological processes and in numerous diseases. Therefore, inhibitors of protein kinases have therefore a wide therapeutic potential. While screening for inhibitors of cyclin-dependent kinases (CDK's) and glycogen synthase kinase-3 (GSK-3), we identified pyrazolo[3,4-b]quinoxalines as sub-micromolar inhibitors of CDK1/cyclin B. A preliminary structure-activity relationship study suggests that this family of compounds can be optimized to inhibit CDK's and GSK-3. Compounds were tested for their anti-proliferative activity and the results show that several of them displayed a significant inhibitory effect on CDK1/cyclin B. The most active compound (1) was also tested against the brain kinases CDK5/p25 and GSK-3, and proved to be a good inhibitor of both of them. On the contrary, none of the compounds showed any activity in the CDC25 phosphatase assay. As an additional approach, affinity chromatography on immobilized pyrazolo[3,4-b]quinoxalines will be used to identify the intracellular targets of this family of compounds.     

Targeting erbB receptors

Our work is concerned with the origins and therapy of human cancers. Members of the epidermal growth factor receptor (EGFR) family of tyrosine kinases, also known as erbB or HER receptors, are over expressed and/or activated in many types of human tumors and represent important therapeutic targets in cancer therapy. Studies from our laboratory identified targeted therapy as a way to treat cancer. Rational therapeutics targeting and disabling erbB receptors have been developed to reverse the malignant properties of tumors. Reversal of the malignant phenotype, best seen with disabling the HER2 receptors using monoclonal antibodies is a distinct process from that seen with blocking of ligand binding to cognate receptors as has been done for EGFr receptors. Here we review the mechanisms of action deduced from a number of approaches developed in our laboratory and elsewhere, including monoclonal antibodies, peptide mimetics, recombinant proteins and small molecules. The biochemical and biological principles which have been uncovered during these studies of disabling HER2 homomeric or HER2-EGFr heteromeric receptors will help the development of novel and more efficient therapeutics targeting erbB family receptors.     

Small tyrosine kinase inhibitors interrupt EGFR signaling by interacting with erbB3 and erbB4 in glioblastoma cell lines

Signaling through the epidermal growth factor receptor (EGFR) is relevant in glioblastoma. We have determined the effects of the EGFR inhibitor AG1478 in glioblastoma cell lines and found that U87 and LN-229 cells were very sensitive to this drug, since their proliferation diminished and underwent a marked G₁ arrest. T98 cells were a little more refractory to growth inhibition and A172 cells did not undergo a G₁ arrest. This G₁ arrest was associated with up-regulation of p27^kip1, whose protein turnover was stabilized. EGFR autophosphorylation was blocked with AG1478 to the same extent in all the cell lines. Other small-molecule EGFR tyrosine kinase inhibitors employed in the clinic, such as gefitinib, erlotinib and lapatinib, were able to abrogate proliferation of glioblastoma cell lines, which underwent a G₁ arrest. However, the EGFR monoclonal antibody, cetuximab had no effect on cell proliferation and consistently, had no effect on cell cycle either. Similarly, cetuximab did not inhibit proliferation of U87 ΔEGFR cells or primary glioblastoma cell cultures, whereas small-molecule EGFR inhibitors did. Activity of downstream signaling molecules of EGFR such as Akt and especially ERK1/2 was interrupted with EGFR tyrosine kinase inhibitors, whereas cetuximab treatment could not sustain this blockade over time. Small-molecule EGFR inhibitors were able to prevent phosphorylation of erbB3 and erbB4, whereas cetuximab only hindered EGFR phosphorylation, suggesting that EGFR tyrosine kinase inhibitors may mediate their anti-proliferative effects through other erbB family members. We can conclude that small-molecule EGFR inhibitors may be a therapeutic approach for the treatment of glioblastoma patients.     

Protein kinases 1988: a current perspective

This review focuses on several recent developments in the field of protein kinases. In the area of protein serine/threonine kinases, much has been learned recently about protein kinase C structure and function. Novel lipid mediators, both stimulatory and inhibitory, have been discovered, and kinase has been shown to be an increasingly large family of gene products. Heterogeneity of cellular localization and function has been documented. Calcium/calmodulin-dependent protein kinases are now believed to consist of at least five enzymes, which range from those with extreme substrate specificity such as phosphorylase kinase and myosin light-chain kinases to calcium calmodulin kinase II, with several known substrates. Several of these enzymes appear to be important in synaptic transmission and, for calcium/calmodulin kinase III, in the regulation of protein synthesis. Several new examples of pseudosubstrate prototopes as endogenous kinase inhibitors have been described, including regions intrinsic to kinase primary sequences, which could serve as constitutive inhibitors of enzyme activity. In the field of protein tyrosine kinases, new enzyme species are being discovered at a rapid rate. There are several well-documented examples of kinase autophosphorylation on tyrosine leading to stimulation of catalytic activity. For the growth factor receptors with intrinsic protein tyrosine kinase activity, it now seems clear that kinase catalytic activity is necessary for most hormone effects on cells, with the general exceptions of ligand binding and, possibly, receptor cycling. Finally, several groups have recently described a close association between protein tyrosine kinases and a phosphatidylinositol kinase activity, a link that might eventually explain some of the initial steps in signal transduction that occur after kinase activation.     

Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors

Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70k Da (ZAP-70) are members of the Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell activation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production. To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents, compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reaction and Concanavalin A-induced IL-2 production in a mouse model.     

Analysis of styryl-based inhibitors of the lymphocyte tyrosine protein kinase p56lck.

Several styryl-based compounds were evaluated for their capacity to act as inhibitors of the non-receptor tyrosine protein kinase p56lck. Our results demonstrate that alpha-cyanocinnamamide compounds can inhibit both the in vitro tyrosine autophosphorylation of p56lck as well as p56lck phosphorylation of exogenous substrates. Compound 67B-83-A was found to inhibit p56lck protein kinase activity with a calculated IC50 of 7 to 10 microM. This compound did not significantly inhibit the tyrosine protein kinase activity of the epidermal growth factor receptor and was found to be a less effective tyrosine protein kinase inhibitor for other members of the src family of protein kinases.     

Calorimetric investigation of phosphorylated and non-phosphorylated peptide ligand binding to the human Grb7-SH2 domain

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20-30% of breast cancers. In general, growth factor receptor bound (Grb) proteins bind to activated membrane-bound receptor tyrosine kinases (RTKs; e.g., the epidermal growth factor receptor, EGFR) through their Src homology 2 (SH2) domains. In particular, Grb7 binds to erbB2 (a.k.a. EGFR2) and may be involved in cell signaling pathways that promote the formation of metastases and inflammatory responses. In previous studies, we reported the solution structure and the backbone relaxation behavior of the Grb7-SH2/erbB2 peptide complex. In this study, isothermal titration calorimetry studies have been completed by measuring the thermodynamic binding parameters of several phosphorylated and non-phosphorylated peptides representative of natural Grb7 receptor ligands as well as ligands developed through combinatorial peptide screening methods. The entirety of these calorimetric studies is interpreted in an effort to describe the specific ligand binding characteristics of the Grb7 protein.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

Specific structural features of heparan sulfate proteoglycans potentiate neuregulin-1 signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.     

Tyrosine kinase inhibition effects of novel Pyrazolo[1,5-a]pyrimidines and Pyrido[2,3-d]pyrimidines ligand: Synthesis,biological screening and molecular modeling studies

Tyrosine kinases are one of the most critical mediators in the signaling path way. Late studies have proved the part of tyrosine kinases in the pathophysiology of cancer diseases. This current research paper has focused on investigating the novel Pyrazolo[1,5-a]pyrimidines and Pyrido[2,3-d]pyrimidines as a small molecules that can inhibit tyrosine kinase in cancer cells. NCI protocol was applied to test the antitumor activity of such compounds. Leukemia and renal cancer cell lines proved to be sensitive to some derivatives such as 6b–d, 9a and 11 with GI% values ranging from 30.4 to 41.3%. In addition, compound 11 proved to be the most active against MCF-7 with GI% 62.5. The synthesized compounds were also evaluated for their inhibitory effects against EGFR kinase enzyme. Compound 9b proved to be the most active one among the synthesized series with inhibition % value of 81.72 at 25 nM concentration and IC₅₀ 8.4 nM which is very close to the reference drug Sorafenib. In vitro cytotoxicity test was also performed using the MCF-7 breast cell line. Computer modeling using the active site of tyrosine kinase as a template and the most active tyrosine kinase inhibitors were calculated. Docking studies of the synthesized compounds into the active site of EGFR kinase domain showed good agreement with the obtained biological results.     

The Src module: an ancient scaffold in the evolution of cytoplasmic tyrosine kinases

Tyrosine kinases were first discovered as the protein products of viral oncogenes. We now know that this large family of metazoan enzymes includes nearly one hundred structurally diverse members. Tyrosine kinases are broadly classified into two groups: the transmembrane receptor tyrosine kinases, which sense extracellular stimuli, and the cytoplasmic tyrosine kinases, which contain modular ligand-binding domains and propagate intracellular signals. Several families of cytoplasmic tyrosine kinases have in common a core architecture, the “Src module,” composed of a Src-homology 3 (SH3) domain, a Src-homology 2 (SH2) domain, and a kinase domain. Each of these families is defined by additional elaborations on this core architecture. Structural, functional, and evolutionary studies have revealed a unifying set of principles underlying the activity and regulation of tyrosine kinases built on the Src module. The discovery of these conserved properties has shaped our knowledge of the workings of protein kinases in general, and it has had important implications for our understanding of kinase dysregulation in disease and the development of effective kinase-targeted therapies.     

N-arginine dibasic convertase is a specific receptor for heparin-binding EGF-like growth factor that mediates cell migration.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a mitogen and chemotactic factor, binds to two receptor tyrosine kinases, erbB1 and erbB4. Now we demonstrate that HB-EGF also binds to a novel 140 kDa receptor on MDA-MB 453 cells. Purification of this receptor showed it to be identical to N-arginine dibasic convertase (NRDc), a metalloendopeptidase of the M16 family. Binding to cell surface NRDc and NRDc in solution was highly specific for HB-EGF among EGF family members. When overexpressed in cells, NRDc enhanced their migration in response to HB-EGF but not to EGF. Conversely, inhibition of endogenous NRDc expression in cells by antisense morpholino oligonucleotides inhibited HB-EGF-induced cell migration. Anti-erbB1 neutralizing antibodies completely abrogated the ability of NRDc to enhance HB-EGF-dependent migration, demonstrating that this NRDc activity was dependent on erbB1 signaling. Although NRDc is a metalloproteinase, enzymatic activity was not required for HB-EGF binding or enhancement of cell migration; neither did NRDc cleave HB-EGF. Together, these results suggest that NRDc is a novel specific receptor for HB-EGF that modulates HB-EGF-induced cell migration via erbB1.     

Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.     

Targeting Eph receptors with peptides and small molecules: progress and challenges

The Eph receptors are a large family of receptor tyrosine kinases. Their kinase activity and downstream signaling ability are stimulated by the binding of cell surface-associated ligands, the ephrins. The ensuing signals are bidirectional because the ephrins can also transduce signals (known as reverse signals) following their interaction with Eph receptors. The ephrin-binding pocket in the extracellular N-terminal domain of the Eph receptors and the ATP-binding pocket in the intracellular kinase domain represent potential binding sites for peptides and small molecules. Indeed, a number of peptides and chemical compounds that target Eph receptors and inhibit ephrin binding or kinase activity have been identified. These molecules show promise as probes to study Eph receptor/ephrin biology, as lead compounds for drug development, and as targeting agents to deliver drugs or imaging agents to tumors. Current challenges are to find (1) small molecules that inhibit Eph receptor-ephrin interactions with high binding affinity and good lead-like properties and (2) selective kinase inhibitors that preferentially target the Eph receptor family or subsets of Eph receptors. Strategies that could also be explored include targeting additional Eph receptor interfaces and the ephrin ligands.