Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array.

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.

Dual G1 and G2/M phase inhibition by SC-alpha alpha delta 9, a combinatorially derived Cdc25 phosphatase inhibitor.

The Cdc25 dual specificity phosphatase family has a central role in controlling cell cycle progression and has been implicated in the etiology of cancer. One compound, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alpha alpha delta 9), was previously identified as the most potent reported synthetic inhibitor of Cdc25 phosphatases in vitro. In the present study, we demonstrate that SC-alpha alpha delta 9 inhibited Cdc25-dependent cell cycle progression at both G1 and G2/M phase using tsFT210 cells, which express a temperature-sensitive Cdc2 mutant. SC-alpha alpha delta 9 blocked both G2/M transition and dephosphorylation of Cdc2 in a concentration-dependent manner. SC-alpha alpha delta 9 also enhanced tyrosine phosphorylation of both Cdk2 and Cdk4, and decreased Cdk4 kinase activity. Both of the kinases are potent regulators of G1 transition. Furthermore, closely related chemical analogs that lacked Cdc25 inhibitory activity failed to block cell cycle progression at both G1 and G2/M, and did not affect Cdc2 phosphorylation or Cdk4 kinase activity. SC-alpha alpha delta 9 did not alter p53, p21 or p16 levels. Our results support the hypothesis that the disruption in cell cycle transition caused by SC-alpha alpha delta 9 was due to intracellular Cdc25 inhibition. We propose that the SC-alpha alpha delta 9 pharmacophore could be useful in further clarifying the role of Cdc25 phosphatase-dependent pathways in checkpoint control, oncogenesis, and apoptosis.

Cell cycle dependent subcellular distribution of Cdc25B subtypes.

The dual specificity phosphatase and oncogene Cdc25B has been implicated in the G2/M cell cycle checkpoint, but the mode by which it is regulated remains poorly understood. Regional subcellular redistribution of proteins represents a unique potential regulatory mechanism. Thus, we examined in live cells the subcellular localization characteristics of Cdc25B2 and Cdc25B3 fused to green fluorescent protein. Cdc25B2 partitioned primarily in the cytoplasm during G1 and progressively migrated to the nucleus as cells transited from S to G2/M phase. In contrast, Cdc25B3 maintained a homogeneously staining diffuse phenotype irrespective of cell cycle phase. Treatment of the Cdc25B2-green fluorescent protein stable transfectants with vanadate inhibited the cell cycle dependency of intracellular distribution, while okadaic acid had little effect except in G1, suggesting regulation by at least one phosphorylation-dependent pathway. The DNA topoisomerase II poison and DNA damaging agent, etoposide, inhibited nuclear localization of Cdc25B2 in S phase, possibly by invoking a sequestration cascade. Thus, differences in the spatial distribution of Cdc25B subtypes exist within cells and the 41 amino acid insert in the N-terminus of the Cdc25B3 splice variant encodes an important inhibitory determinant for such regulation. The subcellular redistribution of Cdc25B2 could be functionally important for G2/M checkpoint regulation.

Disruption of insulin-like growth factor-1 signaling and down-regulation of cdc2 by SC-alphaalphadelta9, a novel small molecule antisignaling agent identified in a targeted array library.

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.

Evaluation of physiologic indices in mice vaccinated with protein-ergotamine conjugates and fed an endophyte-infected fescue diet.

OBJECTIVES: To assess effects of vaccination against fescue toxicosis on weight gain, serum prolactin and cholesterol concentrations, and alkaline phosphatase (ALP) activity in mice fed an endophyte-infected (EI) or endophyte-free (EF) fescue diet. ANIMALS: 50 six-week-old male BALB/c mice. PROCEDURE: Mice were randomly allocated to the following 5 groups: 1, vaccinated intraperitoneally with a bovine serum albumin-ergotamine (EG) conjugate and fed an EI fescue diet; 2, orally vaccinated with cholera toxin (CT) subunit B-EG conjugate mixed with free CT and fed an EI fescue diet; 3, not vaccinated and fed an EI fescue diet; 4, passively vaccinated with monoclonal antibodies specific for ergovaline (EV) and fed an EI fescue diet; and 5, not vaccinated and fed an EF fescue diet. RESULTS: Antibodies against EG and EV were in serum of mice of groups 1 and 4, respectively. Secretory IgA and IgG coproantibodies against EG were induced in mice of group 2. Weight increased in groups 1 and 2 and tended to be increased in group 4 versus group 3. Prolactin concentration was similar in all groups; cholesterol concentration was decreased in groups 1, 3, and 4, compared with group 5. Compared with that in group 5, serum ALP activity decreased in groups 1 and 4 and was further decreased in group 1, compared with that in groups 2 and 3; it was negatively correlated with anti-EG titer. CONCLUSIONS AND CLINICAL RELEVANCE: Induction of anti-EG antibodies and administration of EV monoclonal antibodies tended to increase short-term weight gain in this murine model of fescue toxicosis. However, systemic IgG antibodies against EG or EV antibodies were not protective against decreases in serum ALP activity and cholesterol concentrations. Clinical significance of decreased ALP activity associated with vaccination is unknown, but represents a worsening of a response often associated with fescue toxicosis in cattle.

12(S)-hydroxyeicosatetraenoic acid increases the actin microfilament content in B16a melanoma cells: a protein kinase-dependent process.

12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], a lipoxygenase metabolite of arachidonic acid, has been shown to be involved in a wide variety of cellular activities (i.e., adhesion, spreading, motility, invasion) which promote metastasis to occur in tumor cells. In this study, several techniques (Western blotting, flow cytometry and DNase I assay) were performed to examine the alterations in the distribution of G- and F-actin expressed in B16a melanoma cells. Each of these methods independently revealed that 12(S)-HETE treatment (0.1 mM, 15 min) resulted in an increase in the F-actin content in the cytoskeletal preparations. Since the integrity of cytoskeletal networks (i.e., actin filaments) can be dynamically regulated through protein phosphorylation, we investigated the potential role of several protein kinases in the 12(S)-HETE-induced actin polymerization. By flow cytometric analysis, 12(S)-HETE was found to increase the actin filament contents. This effect could be inhibited by protein kinase C (PKC) inhibitors (calphostin C and staurosporine) as well as by protein tyrosine kinase (PTK) inhibitor (genistein) but not by protein kinase A inhibitor (H8), suggesting that the 12(S)-HETE effect involves PKC and PTK. This conclusion is consistent with the observations that phorbol 12-myristate-13-acetate (PMA) mimics the biological effect of 12(S)-HETE in promoting the F-actin formation in B16a cells. As a final analysis, direct protein phosphorylation studies indicate that 12(S)-HETE treatment led to enhanced phosphorylation of myosin light chain, which may contribute to the increased stress fiber formation following 12(S)-HETE stimulation.

Actin cleavage in various tumor cells is not a critical requirement for executing apoptosis.

Actin is a major cytoskeletal protein which is involved in many physiological cellular functions such as motility, cell shape, and adhesion. Recently, actin has also been reported to be cleaved by apoptotic proteases (i.e., caspases) and this cleavage is thought to contribute to the apoptotic process. However, conflicting data also exists as to whether actin represents a true caspase substrate during apoptosis induction in vivo (i.e., inside the cells). In this study, we critically examined the actin cleavage patterns during apoptosis of several tumor cell lines derived from three different species (i.e., mouse, rat, and human). Our findings demonstrate that: 1) actin cleavage in vivo is not a common phenomenon since apoptosis caused by multiple inducers in most cell types examined occurs without evidence of actin degradation; and 2) in certain cell types (e.g., U937), spontaneous, actin cleavage is observed which is not prevented by various specific chemical/peptide inhibitors of proteases such as caspases or serine proteases although apoptosis per se is retarded by some of these inhibitors. Our results conclude that actin is not a critical substrate for apoptotic proteases in vivo during apoptosis.

Oral and parenteral vaccination of mice with protein-ergotamine conjugates and evaluation of protection against fescue toxicosis.

Acremonium coenophialum produces ergopeptide alkaloids in tall fescue (Festuca arundinacea Schreb.). These ergot alkaloids decrease serum alkaline phosphatase (ALP) activity, serum cholesterol and prolactin concentrations, as well as average daily gains (ADG) in cattle. The objective of this study was to evaluate the protection of anti-ergotamine antibodies induced by either oral or parenteral vaccination with protein-ergotamine conjugates or passive vaccination with anti-ergovaline, monoclonal antibodies in a murine model of fescue toxicosis. Ergotamine (EG) was conjugated to bovine serum albumin (BSA) and cholera toxin subunit B (CTB) by the Mannich reaction. Mice were blocked based on weight and randomly allocated into five groups of 10 mice each. Treatment groups were as follows: (1) group vaccinated intraperitoneally (ip) with a BSA-EG conjugate and fed an endophyte-infected (EI) fescue diet (BSA-EG group); (2) group orally vaccinated with a CTB-EG conjugate mixed with free cholera toxin (CT) and fed an EI fescue diet (CTB-EG group); (3) nonvaccinated group fed an EI fescue diet (EI group); (4) group passively vaccinated with anti-ergovaline, monoclonal antibodies and fed an EI fescue diet (MoAB group); and (5) nonvaccinated group fed an endophyte-free (EF) fescue diet (EF group). The EI diet contained 1.5 ppm of Ergovaline (EV), whereas no EV was detected in the EF diet.Respective diets were similar upon nutritional analysis. Unvaccinated mice in the EI group exhibited features of fescue toxicosis as indicated by decreased serum ALP activity and cholesterol, and decreased weight gain as compared to mice in the EF group. Antibodies against EG and EV were present in sera of mice in the BSA-EG and MoAB groups, respectively. Mice orally vaccinated with the CTB-EG conjugate developed secretory IgA (sIgA) antibodies and short-lived, systemic IgG responses against EG. Weight gains were increased in the BSA-EG and CTB-EG groups and tended to be increased in the MoAB group vs. the unvaccinated EI group. Serum ALP activity was decreased in the BSA-EG and MoAB groups as compared to the EF group. Serum ALP activity was further decreased in the BSA-EG vaccinated group as compared to the EI group. Cholesterol concentrations were decreased in the EI, BSA-EG and MoAB groups as compared to the EF group. Prolactin concentrations were similar in all groups.

Suppression of W256 carcinosarcoma cell apoptosis by arachidonic acid and other polyunsaturated fatty acids.

Serum-cultured rat W256 carcinosarcoma cells of the monocytoid origin undergo rapid apoptosis in response to the lipoxygenase inhibitor NDGA (nordihydroguaiaretic acid). Exogenous arachidonic acid (AA), in a time- and dose-dependent fashion, suppressed NDGA-induced W256 cell apoptosis as well as DNA fragmentation, with the maximal effect observed at approximately 25 microM. Mobilization of endogenous AA by calcium ionophore A23187 provided an even stronger and longer-lasting protection against NDGA-caused cell death. The A23187 effect on AA release as well as W256 cell death can be blocked by bromophenacyl bromide, thus suggesting involvement of phospholipase A2 activation. Serum withdrawal similarly caused W256 cells to undergo typical apoptosis, which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation-induced W256 cell apoptosis and significantly extended cell survival in a dose-dependent manner. Lipoxygenase products, 12(S)- and 15(S)-, but not 5(S)-hydroxyeicosatetraenoic acid (HETE), in a dose-dependent fashion, also prevented both NDGA- and serum-starvation-induced W256 cell apoptosis. AA appears to suppress W256 cell apoptosis via distinct signaling pathway(s) since it does not prevent cell death triggered by several other inducers. Examination of a panel of polyunsaturated fatty acids revealed that alpha-linolenic and linoleic acid can also suppress NDGA-induced W256 cell apoptosis. Our data suggest that AA and other polyunsaturated fatty acids and/or their metabolites may enhance tumor growth not only by promoting cell proliferation but also by suppressing apoptosis.

Combinatorial synthesis and biological evaluation of library of small-molecule Ser/Thr-protein phosphatase inhibitors.

In eukaryotes, phosphorylation of serine, threonine, and tyrosine residues on proteins is a fundamental posttranslational regulatory process for such functions as signal transduction, gene transcription, RNA splicing, cellular adhesion, apoptosis, and cell cycle control. Based on functional groups present in natural product serine/threonine protein phosphatase (PSTPase) inhibitors, we have designed pharmacophore model 1 and demonstrated the feasibility of a combinatorial chemistry approach for the preparation of functional analogues of 1. Preliminary biological testing of 18 structural variants of 1 has identified two compounds with growth inhibitory activity against cultured human breast cancer cells. In vitro inhibition of the PSTPase PP2A was demonstrated with compound 1d. Using flow cytometry we observed that compound f1 caused prominent inhibition in the G1 phase of the cell cycle. Thus, the combinatorial modifications of the minimal pharmacophore 1 can generate biologically interesting antiproliterative agents.

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation.

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.

Evaluation of humoral immune responses in cattle grazing endophyte-infected or endophyte-free fescue.

Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination and thus, experience increased incidence of Bovine Respiratory Disease Complex (BRDC) when shipped to feedlots. Fescue toxicosis causes hypoprolactemia in cattle. Hypoprolactemia decreases humoral immune responses in mice. Therefore, a study was conducted to compare the magnitude of primary and secondary humoral immune responses against specific antigens in cattle grazing endophyte-infected or endophyte-free fescue. Angus steers were blocked by weight and allocated into four groups. Two groups grazed endophyte-infected (EI) fescue and the other two groups grazed endophyte-free (EF) fescue. All steers were injected IM on d 0 and 21 with lysozyme without adjuvant and concanavalin. A (Con A) with sheep red blood cells (SRBC) in incomplete adjuvant of Freund. Steers were bled on days 0, 21 and 35 post-vaccination. Average daily gains (ADG), alkaline phosphatase (ALP) activity, cholesterol concentrations, rectal temperatures, and serum prolactin concentrations were measured to confirm fescue toxicosis in steers grazing EI fescue. Antibodies to Con A and SRBC were determined by ELISA and hemagglutination assay, respectively. The ADG were decreased for the EI group during the first month. Rectal temperature were elevated and serum prolactin concentrations were decreased in the EI group. Cholesterol and ALP concentrations also were decreased in the EI group. Primary and secondary immune responses against Con A tended to be increased and were increased against SRBC in the EI group. Antibodies against lysozyme were not induced in either group. In conclusion, cattle grazing EI fescue mounted similar humoral immune responses to vaccination, despite hypoprolactemia, as cattle grazing EF fescue. Increases in bovine respiratory disease in cattle maintained on EI fescue probably is not associated with lack of humoral immune response to vaccination protocols as a result of fescue toxicosis.

An analysis of group versus individual child health supervision.

This study compares the effectiveness of group health supervision (three or four families counseled simultaneously) with traditional visits in conveying knowledge of child health and development, increasing perceived maternal support, and mitigating maternal depression. Subjects were recruited from a predominantly white, middle-class, suburban/rural pediatric practice. Twenty-five families were allocated to group health supervision and 25 to individual visits. A questionnaire covering knowledge of child health and development (CHDQ), the Maternal Social Support Index (MSSI), and the Center for Epidemiologic Studies Depression Scale (CESD) were administered to both groups before their 2-month and after their 10-month visits. A subset of these charts was reviewed for problem visits between 2 and 6 months. As compared with families having traditional visits, families who received the group intervention did at least as well in acquiring knowledge of child care and development and, although not statistically significant, tended to recover from postpartum depression faster and deal better with minor illnesses. The investigators found group child health supervision to be a pleasant and effective method of health care delivery.

Effect of feedback on resource use and morbidity in hip and knee arthroplasty in an integrated group practice setting.

OBJECTIVE: To assess the effect of a structured program of feedback about resource utilization and morbidity on resource consumption and complications in an orthopedic surgical practice. DESIGN: We prospectively analyzed use and outcomes before and after an intervention (departmental data presentation). MATERIAL AND METHODS: Feedback on resource utilization and morbidity for 2,820 patients who underwent a primary total hip or knee arthroplasty for a diagnosis of osteoarthritis between Jan. 1, 1990, and Dec. 31, 1992, was provided to members of the orthopedic department of an academic medical center. Data were adjusted for severity of disease. RESULTS: On reassessment 18 months after the beginning of the feedback program, total charges and length of hospital stay for hip or knee arthroplasty were significantly reduced. Interpractitioner variability was also reduced but not significantly. The feedback process was instrumental in identifying a specific complication--pulmonary embolism after bilateral total knee replacement--which was significantly reduced by addition of warfarin prophylaxis. CONCLUSION: The intervention was successful in reducing resource use (length of hospital stay) and complications (pulmonary embolism). In addition, total charges for hip and knee arthroplasty declined significantly at a time when medical center charges overall were increasing. Efforts to maintain continuous improvement will primarily focus on the development of critical pathways.

The effect of the initial family interview on a pediatric practice.

To examine the effect of initial family interviews by health care providers on patients' use of health services, 177 patients and their families were randomly assigned to an interviewed (I) or a non-interviewed (NI) subgroup. Initial interviews would encourage families to bring their children in for health supervision visits, but discourage families from making after-hour telephone calls, using the emergency room and bringing the children to the clinic frequently for problem visits. The I families had an initial interview, attended by all family members. Both a physician and a nurse elicited patient histories and explained use of the emergency room, when to make after-hour calls, how to schedule appointments and other information about the clinic. If I families failed to have an initial interview, they were deleted from the study. In the NI subgroup, patient histories were elicited during a routine health supervision visit without the entire family in attendance, and information about emergency room visits, after-hour calls and appointment scheduling was provided during the same visit. After one year (1987) into the study and two years (1988) into the study, all patient charts were examined. Data analysis was performed using analysis of variance for repeated measures (ANOVA) and step-wise multiple regression of Statistical Analysis System. For 1987, the interview intervention explained a significant (p = 0.01) amount of variance in the number of problem visits (less in I) after controlling for months in the study and age of the child.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial line associated with pseudophakic bullous keratopathy.

Three patients undergoing uncomplicated cataract extraction with intraocular lens implantation developed an endothelial line, clinically appearing identical to that seen in allograft rejection following corneal transplantation. Both intracapsular and extracapsular surgery were involved. Three different intraocular lens types were implanted (pupillary supported, anterior chamber, and posterior chamber). Each case was associated with postoperative iritis and glaucoma. They were nonresponsive to steroid therapy and eventually resulted in total corneal decompensation. This clinical entity may represent an autoimmune phenomenon stimulated by chronic inflammation induced by the intraocular lens.