Protein Tyrosine Kinase Blockers Research Articles

Regulation of K–Cl cotransport in erythrocytes of frog Rana temporaria by commonly used protein kinase and protein phosphatase inhibitors

Recently (Agalakova and Gusev in J Comp Physiol 179:443-450, 2009), we demonstrated that the activity of K-Cl cotransport (KCC) in frog red blood cells is inhibited under stimulation of protein kinase C (PKC) with phorbol ester PMA (12-myristate-13-acetate). Present work was performed to uncover possible implication of protein kinases and protein phosphatases (PPs) in the regulation of baseline and volume-dependent KCC activity in these cells. K+ influx was estimated as 86Rb uptake by the cells in isotonic or hypotonic media in the presence of ouabain, K+ efflux was determined as the difference between K+ loss by the cells incubated in parallel in isotonic or hypotonic K(+)-free Cl(-)- and NO(3)(-)-media. Swelling of the cells in hypotonic medium was accompanied by approximately 50% activation of Cl-dependent K+ influx and efflux. Protein tyrosine kinase (PTK) inhibitor genistein (0.1 mM) stably and considerably (up to 89%) suppressed both baseline and volume-dependent KCC activity in each direction. Other PTK blockers (tyrphostin 23 and quercetin) had no influence on KCC activity in frog erythrocytes. PKC inhibitor chelerythrine (20 microM) and both PP inhibitors, fluoride (5 mM) and okadaic acid (1 microM), reduced KCC activity by 25-70%. Neither basal nor swelling-activated KCC in frog erythrocytes was affected by PKC inhibitor staurosporine (1 microM). Based on the previous and present results, we can suggest that the main role in the maintenance of basal and volume-dependent KCC activity in frog erythrocytes belongs to PTKs and PPs, whereas PKC is a negative regulator of this ion system.

Pharmacological modulation of monovalent cation currents through the epithelial Ca2+ channel ECaC1.

1. The recent identification of the epithelial Ca(2+) channel, ECaC1, represents a major step forward in our knowledge of renal Ca(2+) handling. ECaC1 constitutes the rate-limiting apical Ca(2+) entry mechanism of active, transcellular Ca(2+) reabsorption. This unique highly selective Ca(2+) channel shares a low but significant homology with transient receptor potential (TRP) channels and vanilloid receptors (VR). 2. We have studied the pharmacological modulation of currents through ECaC1 heterologously expressed in HEK 293 cells. Monovalent cation currents were measured by use of the whole cell patch clamp technique in cells dialysed with 10 mM BAPTA or 10 mM EGTA to prevent the fast Ca(2+) dependent inactivation of ECaC1. 3. Several modulators were tested, including inorganic cations, putative store-operated Ca(2+) entry (SOC) blockers, the vanilloid receptor (VR-1) blocker capsazepine, protein tyrosine kinase blockers, calmodulin antagonists and ruthenium red. 4. Ruthenium red and econazole appeared to be the most effective inhibitors of currents through ECaC1, with IC(50) values of 111 nM and 1.3 microM, respectively, whereas the selective SOC inhibitor, SKF96365, was nearly ineffective. 5. The divalent cation current block profile for ECaC1 is Pb(2+)=Cu(2+) >Zn(2+) >Co(2+) >Fe(2+) with IC(50) values between 1 and approximately 10 microM. 6. In conclusion, ECaC activity is effectively inhibited by various compounds including ruthenium red, antimycotic drugs and divalent cations, which might be useful tools for pharmacological manipulation and several disorders related to Ca(2+) homeostasis could benefit from such developments.

The p15 matrix protein of moloney murine leukemia virus is a phosphotyrosine protein

Retroviruses have been etiologically implicated with leukemia in humans and animals. Understanding the virus life cycle, the proteins and enzymes involved in its replication, is essential for developing potent anti-viral drugs. Phosphorylation of retroviral proteins may alter their shape in such a way as to increase or inhibit their biological activities and thus influence the replication and pathogenic potential of the retroviruses. In our previous work, we demonstrated that non-cytotoxic doses of tyrphostins (protein tyrosine kinase blockers) inhibit moloney murine leukemia virus (Mo-MuLV) replication in acutely and chronically infected cells. In an attempt to understand their mode of action as anti-MoMuLV drugs, we examined the possibility that a viral protein is phosphorylated in tyrosine. Indeed, in our present work, we show that the p15 matrix protein (MA) of MoMuLV is a phosphotyrosine protein and is the only viral protein which is phosphorylated on tyrosine. Moreover, treatment of Mo-MuLV/NIH/ 3T3 chronically infected cells with tyrphostin AG-555 specifically inhibits the synthesis of p15 and other viral proteins but does not affect the synthesis of cellular proteins. Our results suggest that tyrosine phosphorylation of p15 MA protein may play a pivotal role in Mo-MuLV replication.

Effect of selective PDGF-receptor versus non-selective protein tyrosine kinase blockers on aortic smooth muscle cells (SMC's) and endothelial cells proliferation

Effect of selective PDGF-receptor versus non-selective protein tyrosine kinase blockers on aortic smooth muscle cells (SMC's) and endothelial cells proliferation

Vanadium salts as insulin substitutes: mechanisms of action, a scientific and therapeutic tool in diabetes mellitus research.

Vanadium and its compounds exhibit a wide variety of insulin-like effects. In this review, these effects are discussed with respect to the treatment of type I and type II diabetes in animal models, in vitro actions, antineoplastic role, treatment of IDDM and NIDDM patients, toxicity, and the possible mechanism(s) involved. Newly established CytPTK plays a major role in the bioresponses of vanadium. It has a molecular weight of approximately 53 kDa and is active in the presence of Co2+ rather than Mn2+. Among the protein-tyrosine kinase blockers, staurosporine is found to be a potent inhibitor of CytPTK but a poor inhibitor of InsRTK. Vanadium inhibits PTPase activity, and this in turn enhances the activity of protein tyrosine kinases. Our data show that inhibition of PTPase and protein tyrosine kinase activation has a major role in the therapeutic efficacy of vanadium in treating diabetes mellitus.

Tyrphostin-Induced Inhibition of p210bcr-abl Tyrosine Kinase Activity Induces K562 to Differentiate

AbstractWe report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

Tyrphostin-induced inhibition of p210bcr-abl tyrosine kinase activity induces K562 to differentiate

We report on the potency of two Tyrphostin tyrosine kinase blockers, AG 1112 and AG 568, to inhibit p210bcr-abl tyrosine kinase activity in K562 cells, concomitant with the induction of erythroid differentiation. AG 568 and especially AG 1112 represent a specific group of nontoxic protein tyrosine kinase blockers among more than 1,400 tested. These compounds possess therapeutic potential for purging Philadelphia chromosome-positive cells in preparation for autologous bone marrow transplantation in chronic myelogenous leukemia.

Selective interactions of transforming and normal abl proteins with ATP, tyrosine-copolymer substrates, and tyrphostins.

The transforming abl proteins p160gag-abl, p185bcr-abl, and p210bcr-abl and the normal protein p140c-abl have identical catalytic sites, but differ in their N-terminal domains. Previous studies have indicated that the transforming abl proteins possess higher tyrosine kinase activity than the normal abl proto-oncogene product. In the present study, we demonstrate that two transforming abl proteins, p210bcr-abl and p160gag-abl, exhibit a higher affinity toward ATP and synthetic tyrosine containing substrates than p140c-abl. Furthermore, protein tyrosine kinase blockers from the tyrphostin family can discriminate between normal abl and transforming abl proteins of both human and mouse origin. These results suggest that the transforming potency of the abl proteins may result from their higher affinities toward intracellular signal transducers and demonstrate for the first time that oncogene products can differ from their homologous proto-oncogene product in substrate specificity. The ability of tyrphostins to discriminate between normal and transforming abl proteins suggests that it may be possible to design specific abl kinase inhibitors to combat abl-associated human leukemias.

The inhibition of EGF-dependent proliferation of keratinocytes by tyrphostin tyrosine kinase blockers.

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.

Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors

A novel class of low molecular weight protein tyrosine kinase inhibitors is described. These compounds constitute a systematic series of molecules with a progressive increase in affinity toward the substrate site of the EGF receptor kinase domain. These competitive inhibitors also effectively block the EGF-dependent autophosphorylation of the receptor. The potent EGF receptor kinase blockers examined were found to competitively inhibit the homologous insulin receptor kinase at 10(2)-10(3) higher inhibitor concentrations in spite of the significant homology between these protein tyrosine kinases. These results demonstrate the ability to synthesize selective tyrosine kinase inhibitors. The most potent EGF receptor kinase inhibitors also inhibit the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on EGF independent cell growth. These results demonstrate the potential use of protein tyrosine kinase inhibitors as selective antiproliferative agents for proliferative diseases caused by the hyperactivity of protein tyrosine kinases. We have suggested the name "tyrphostins" for this class of antiproliferative compounds which act as protein tyrosine kinase blockers.

Inhibition of insulin-dependent lipogenesis and anti-lipolysis by protein tyrosine kinase inhibitors.

Protein tyrosine kinase (PTK) blockers which competitively inhibit the kinase activity of insulin receptors were synthesized and their properties examined. The best insulin receptor kinase (IRK) inhibitors possess either one hydroxyphenyl ring and two carboxyl groups or two phenyl rings and one carboxyl group. All the inhibitors, except tBoc-tyrosine aminomalonate, effectively block the IRK-catalyzed phosphorylation of exogenous substrate, but only partially block receptor autophosphorylation. These PTK blockers inhibit the insulin induced [14C]glucose assimilation into lipids (lipogenesis), but fail to inhibit the anti-lipolytic effect of the hormone. Only tBocTyr-aminomalonate was found to inhibit all the effects of insulin measured: insulin-stimulated phosphorylation of exogenous substrate, IRK autophosphorylation, insulin-dependent lipogenesis and the insulin-dependent anti-lipolytic effect. This inhibitor is the first blocker which is reported to block insulin-dependent anti-lipolysis. The inhibitors examined are devoid of general adverse effects since they have no effect on insulin-independent lipolysis, on [U14C]fructose assimilation or on (-)isoproterenol-stimulated lipolysis. These studies suggest that insulin-dependent lipogenesis and anti-lipolysis may be mediated by two distinguishable signalling pathways. This study also suggests that PTK inhibitors may become useful tools in the investigation of the signalling pathways of PTKs.