Cep-1347 (KT7515), a semisynthetic inhibitor of the mixed lineage kinase family.

CEP-1347 (KT7515) promotes neuronal survival at dosages that inhibit activation of the c-Jun amino-terminal kinases (JNKs) in primary embryonic cultures and differentiated PC12 cells after trophic withdrawal and in mice treated with 1-methyl-4-phenyl tetrahydropyridine. In an effort to identify molecular target(s) of CEP-1347 in the JNK cascade, JNK1 and known upstream regulators of JNK1 were co-expressed in Cos-7 cells to determine whether CEP-1347 could modulate JNK1 activation. CEP-1347 blocked JNK1 activation induced by members of the mixed lineage kinase (MLK) family (MLK3, MLK2, MLK1, dual leucine zipper kinase, and leucine zipper kinase). The response was selective because CEP-1347 did not inhibit JNK1 activation in cells induced by kinases independent of the MLK cascade. CEP-1347 inhibition of recombinant MLK members in vitro was competitive with ATP, resulting in IC(50) values ranging from 23 to 51 nm, comparable to inhibitory potencies observed in intact cells. In addition, overexpression of MLK3 led to death in Chinese hamster ovary cells, and CEP-1347 blocked this death at doses comparable to those that inhibited MLK3 kinase activity. These results identify MLKs as targets of CEP-1347 in the JNK signaling cascade and demonstrate that CEP-1347 can block MLK-induced cell death.

Quantitative, high-throughput cell-based assays for inhibitors of trkA receptor.

Two quantitative, high-throughput cell-based assays for evaluating inhibitors of NGF-stimulated trkA phosphorylation in trkA-transfected NIH3T3 cells have been established. Both assays involve capture of the trkA receptor from cell lysates in microtiter plates coated with an anti-trk antibody. The amount of trkA phosphorylation is then measured using either an anti-phosphotyrosine antibody with a colorimetric readout or a lanthanide (europium)-labeled anti-phosphotyrosine antibody with a fluorometric detection. The two assay formats exhibited at least a fivefold increase in phosphorylated trkA signal in trkA-transfected cells compared to vector control. Inhibition plots generated for trkA kinase inhibitors using the two detection systems yielded comparable IC(50) values. Overall, the two assays represent a marked improvement over the standard gel-based/western blot method in terms of throughput, quantitation, and amenability to automation.

Sustained in vivo regression of Dunning H rat prostate cancers treated with combinations of androgen ablation and Trk tyrosine kinase inhibitors, CEP-751 (KT-6587) or CEP-701 (KT-5555).

The indolocarbazole analogue CEP-751 is a potent and selective tyrosine kinase inhibitor of the neurotrophin-specific trk receptors that has demonstrated antitumor activity in nine different models of prostate cancer growth in vivo. In the slow-growing, androgen-sensitive Dunning H prostate cancers, which express trk receptors, CEP-751 induced transient regressions independent of effects on cell cycle. Because androgen ablation is the most commonly used treatment for prostate cancer, we examined whether the combination treatment of CEP-751 with castration would lead to better antitumor efficacy than either treatment alone. For a 60-day period, H tumor-bearing rats received treatment with either castration, CEP-751 (10 mg/kg once a day s.c. for 5 days every 2 weeks), a combination of both, or vehicle. Castration caused tumor regression, followed by tumor regrowth in 4-6 weeks, whereas intermittent CEP-751 treatments resulted in tumor regressions during each treatment, which were followed by a period of regrowth between intermittent drug treatment cycles. Overall, both monotherapies significantly inhibited tumor growth compared with the vehicle-treated control group. However, the combination of castration and concomitant CEP-751 produced the most dramatic results: sigificantly greater tumor regression than either therapy alone, with no signs of regrowth. A related experiment using an orally administered CEP-751 analogue (CEP-701), as the trk inhibitor, and a gonadotrophin-releasing hormone agonist, Leuprolide, to induce androgen ablation demonstrated similar results, indicating that these effects could be generalized to other forms of androgen ablation and other trk inhibitors within this class. In addition, when CEP-701 was given sequentially to rats bearing H tumors, which were progressing in the presence of continuous androgen ablation induced by Leuprolide, regression of the androgen-independent tumors occurred. In summary, these data demonstrate that CEP-751 or CEP-701, when combined with surgically or chemically induced androgen ablation, offer better antitumor efficacy than either monotherapy and suggest that each therapy produces prostate cancer cell death through complementary mechanisms.

Kinetics of trkA tyrosine kinase activity and inhibition by K-252a.

The kinetic mechanism of the trk receptor-linked tyrosine kinase was determined using a baculovirus expressed trk kinase domain and a bacterially expressed phospholipase C-gamma/glutathione S-transferase (PLC-gamma/ GST) fusion protein as substrate. Product and dead-end inhibition studies indicate an ordered association of substrates to trkA kinase with the nucleotide ATP binding prior to the exogenous substrate PLC-gamma/GST, followed by release of the phosphorylated PLC-gamma/GST product prior to release of ADP (sequential ordered bi-bi mechanism). This is in contrast to the reported kinetic mechanisms of closely related EGF receptor and insulin receptor kinases which appear to proceed via a rapid equilibrium random mechanism. The indolocarbazole K-252a, which was previously shown to be a potent and relatively selective inhibitor of trk kinase activity, acts as a competitive inhibitor with respect to ATP. The data suggest that potent and selective kinase inhibitors can be rationally designed by exploring subtle variations surrounding the nucleotide binding sites of receptor tyrosine kinases.

Motoneuron apoptosis is blocked by CEP-1347 (KT 7515), a novel inhibitor of the JNK signaling pathway.

Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

CEP-751 inhibits TRK receptor tyrosine kinase activity in vitro exhibits anti-tumor activity.

The present report describes the in vitro and in vivo profile of CEP-751, a novel receptor tyrosine kinase inhibitor. CEP-751 at 100 nM inhibits the receptor tyrosine kinase activity of the neurotrophin receptors trkA, trkB and trkC. CEP-751 has no effect on activity of receptors for EGF, IGF-I, insulin or on erbB2; inhibition of receptors for PDGF and bFGF was observed but occurred with lesser potency than inhibition of trk. CEP-751 exhibited anti-tumor efficacy against tumors derived from NIH3T3 cells transfected with trkA. Inhibition of trk phosphorylation could also be measured in these tumors, suggesting that anti-tumor efficacy of CEP-751 is related to inhibition of trk receptor tyrosine kinase activity. CEP-751 was found to be without effect when administered to nude mice bearing SK-OV-3 tumors, which overexpress erbB2 receptors, providing further evidence that inhibition of tumor growth may be related to inhibition of trk receptor tyrosine kinase activity. Our data indicate that CEP-751 is a potent trk inhibitor which possesses anti-tumor activity.

Neurotrophic 3,9-bis[(alkylthio)methyl]-and-bis(alkoxymethyl)-K-252a derivatives.

A series of 3,9 disubstituted [(alkylthio)methyl]- and (alkoxymethyl)-K-252a derivatives was synthesized with the aim of enhancing and separating the neurotrophic properties from the undesirable NGF (trk A kinase) and PKC inhibitory activities of K-252a. Data from this series reveal that substitution in the 3- and 9-positions of K-252a with these groups reduces trk A kinase inhibitory properties approximately 100- to > 500-fold while maintaining or in certain cases enhancing the neurotrophic activity. From this research, 3,9-bis[(ethylthio)methyl]-K-252a (8) was identified as a potent and selective neurotrophic agent in vitro as measured by enhancement of choline acetyltransferase activity in embryonic rat spinal cord and basal forebrain cultures. Compound 8 was found to have weak kinase inhibitory activity for trk A, protein kinase C1 protein kinase A, and myosin light chain kinase. On the basis of the in vitro profile, 8 was evaluated in in vivo models suggestive of neurological diseases. Compound 8 was active in preventing degeneration of cholinergic neurons of the nucleus basalis magnocellularis (NBM) and reduced developmentally programmed cell death (PCD) of female rat spinal nucleus of the bulbocavernosus motoneurons and embryonic chick lumbar motoneurons.

A radioactive binding assay for inhibitors of trkA kinase.

The high-affinity receptor for nerve growth factor (NGF), trkA, is a receptor-linked tyrosine kinase. The binding of NGF to trkA, depending on the context of its environment, can cause beneficial or deleterious responses in the target cells. For example, the activation of trkA in sympathetic and sensory neurons causes the subsequent survival and differentiation of these cells. On the other hand, the activation of trkA by NGF in other cells has been implicated in several pathologies including inflammation-induced hyperalgesia and several cancers. A radioactive binding assay to evaluate inhibitors of the kinase domain of trkA has been developed and validated. The assay monitors the specific binding of an inhibitor of trkA kinase activity, the indolocarbazole K-252a, to the trkA receptor. [3H]K-252a binds with high affinity to one site on the cytoplasmic kinase domain of the trkA receptor. Binding is saturable and reversible with a dissociation constant (Kd) of 1.5 nM. The binding assay has been used in competition binding experiments to determine the inhibition constants for other indolocarbazole compounds. The IC50 values for compounds obtained in the binding assay correlate very well with the IC50 values obtained in an enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.

Enzyme-linked immunosorbent assay for trkA tyrosine kinase activity.

A 96-well microtiter enzyme-linked immunosorbent assay was developed to assay the activity of the cytoplasmic domain of trkA tyrosine kinase. The assay involves immobilization of phospholipase C- gamma/glutathione S-transferase fusion protein on a microtiter plate, addition of the kinase reaction mixture, and detection by an antibody to phosphotyrosine followed by an alkaline phosphatase-conjugated second antibody. The substrate used in this system, phospholipase C-gamma, is one of several biologically important substrates for the phosphorylation reaction of receptor-linked tyrosine kinases. The assay was then used to characterize kinase inhibitory activities of various small molecules including analogs of K-252a.

The role of conditioned taste aversion in the hypophagia induced by intraperitoneal epinephrine and glucose.

It has been repeatedly shown that relatively high doses of epinephrine (E) and glucose (G) injected intraperitoneally (ip) produce hypophagia in fasted rats. In the present work we used a conditioned taste aversion (CTA) paradigm in order to test whether this effect could be due to "malaise." We determined the effect on food intake and saccharin preference with the following treatments: (a) E ip 100 and 250 micrograms/kg; (b) E ip 250 micrograms/kg with or without previous alpha 1 plus beta adrenergic blockade; (c) G ip 3.5 and 4 g/kg. Both doses of E significantly reduced food intake more than 75% but only the high dose produced a significant (50%) reduction in saccharin preference. Blockade of alpha 1 and beta adrenergic receptors completely suppressed the E-induced hypophagia but attenuated only slightly the taste aversivon effect. Both doses of G decreased food intake but only the high dose reduced saccharin preference; part of these effects would appear to be due to the high osmolarity of the solution. The present results indicate that E and G may induce CTA in our experimental conditions. However, their hypophagic and aversive effects seem to be elicited by different mechanisms.

Aspects of antibody-catalyzed primary amide hydrolysis.

Because there are many known C-terminally amidated peptides of biological importance, there is great potential in medicine and organic synthesis for antibodies that catalyze primary amide bond hydrolysis or formation. We characterized a catalytic antibody, 13D11, raised to a phosphinate hapten, that hydrolyzed the primary amide of a dansyl-alkylated derivative of (R)-phenylalaninamide (DNS-(R)F-NH2). At pH 9.0, 13D11 hydrolyzed DNS-(R)F-NH2 with a kcat of 1.65 x 10(-7) s-1 (kcat/kuncat = 132) and a Km of 432 microM, and was stereospecifically hapten-inhibited (Ki = 14.0 microM). Control experiments indicated that the catalytic activity was not the result of a contaminating protease. In accordance with the hapten being a transition-state analog of base hydrolysis, the rate of DNS-(R)F-NH2 hydrolysis increased with hydroxide concentration to an optimum pH of 9.5. Above pH 9.5, activity declined rapidly suggesting the antibody was inactivated during the long incubation period. This work demonstrates the feasibility of generating catalytic antibodies to hydrolyze unactivated amide bonds without cofactor assistance.

Use of steady state kinetic methods to elucidate the kinetic and chemical mechanisms of retroviral proteases.

Despite the current plethora of structural data of HIV-1 protease and the availability of potent inhibitors, whose structures are based in part on the presumed mechanism of action of this enzyme, our actual understanding of its chemical mechanism has been until now based largely on the precedents of the mammalian and fungal aspartic proteases and static three-dimensional data. The available steady state kinetic data of the protease, as reviewed here, constitute a first step in a detailed description of the mechanism of the enzyme to complement the structural data.

Mechanism-based catalytic antibody inactivation.

Studies of alternative substrates of the catalytic monoclonal antibodies 18H4, 7D4, and 45A11 provided us with a better understanding of the mechanism of ester hydrolysis employed by these isoabzymes. The antibodies were studied with analogs of the substrate, phenyl acetate; N-acetylglycine phenyl ester 3 and N-carbobenzoxy-glycine phenyl ester 4. All three antibodies catalyzed 3 hydrolysis with kinetic constants similar to those seen with phenyl acetate hydrolysis. However, 4 was found to be a mechanism-based (suicide) inactivator of 18H4 with a kinact of 0.29 min-1 and a K' of 64 microM. Antibody 18H4 was inactivated by 4 after 3.6 turnovers resulting in the acylation of 1.6 tyrosines per combining site. The data conform to a mechanism in which an inactive O-ZGly-tyrosyl-antibody is formed via a Michaelis complex.

Use of nitrogen-15 kinetic isotope effects to elucidate details of the chemical mechanism of human immunodeficiency virus 1 protease.

We have used 15N kinetic isotope effects of the HIV-1 protease-catalyzed peptidolysis of Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 to characterize the chemical mechanism of this enzyme. In addition, the multiple isotope effects have been determined by measuring the 15N kinetic isotope effects in both H2O and D2O. The isotope effects, measured on values of V/K, were determined by the incorporation of a radiolabel (tritium and 14C in peptides bearing the heavy and light isotopes, respectively) at a position remote from the isotopically labeled scissile peptide bond, such that the isotope effect was determined by measurement of the change in the 14C/3H ratio in recovered substrates at various fractions of reaction. At pH = 6.0 (37 degrees C), the nitrogen isotope effects were slightly, but significantly, inverse in both solvents: 15(V/K)H2O = 0.995 +/- 0.002, and 15(V/K)D2O = 0.992 +/- 0.003. The observation of an inverse nitrogen kinetic isotope effect implies that bonding to the nitrogen atom is becoming stiffened in a reaction transition state, and since this inverse isotope effect is enhanced in D2O, this isotope effect likely arises from protonation of the proline nitrogen atom.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoabzymes: structurally and mechanistically similar catalytic antibodies from the same immunization.

Mechanistic and structural comparisons of five catalytic monoclonal antibodies generated from the same hybridoma fusion indicated that all five hydrolyze phenyl acetate by subtle variations of the same mechanism. All of the antibodies showed a pre-steady-state multi-turnover burst in which kcat and Km declined but kcat/Km did not change. The burst of one of the antibodies, 20G9, has previously been found to result from inhibition by the product, phenol. Although all of the antibodies showed the burst, their individual values for kcat, Km, and hapten Ki differed substantially. Three of the antibodies that were investigated for the effect of pH on kcat showed an acid limb pK of 9.5-9.6. Substrate inhibition was seen in four of the five antibodies. Variable region nucleotide sequencing of the heavy and light chains confirmed that all five antibodies were structurally similar and also revealed several potentially critical tyrosines. Despite their structural similarities, analysis of their sequences suggested that the antibodies are products of distinct, independent rearrangements of immunoglobulin gene segments that took place in different progenitor B cells. A plot of Ki for hapten inhibition vs Km/kcat for substrate hydrolysis for the mechanistically related antibodies ("isoabzymes") gave a linear relationship suggesting a catalytic role for transition-state complementarity. Taken together with previous work [Martin et al. (1991) Biochemistry 30, 9757-9761], the data conform to a mechanism in which the antibodies exploit both transition-state complementarity and an acyl-tyrosyl intermediate during phenyl acetate hydrolysis.