Novel functional PI 3-kinase antagonists inhibit cell growth and tumorigenicity in human cancer cell lines.

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.     

Small peptides derived from somatotropin domain-containing proteins inhibit blood and lymphatic endothelial cell proliferation, migration, adhesion and tube formation

Angiogenesis is thoroughly balanced and regulated in health; however, it is dysregulated in many diseases including cancer, age-related macular degeneration, cardiovascular diseases such as coronary and peripheral artery diseases and stroke, abnormal embryonic development, and abnormal wound healing. In addition to angiogenesis, lymphangiogenesis is pivotal for maintaining the immune system, homeostasis of body fluids and lymphoid organs; dysregulated lymphangiogenesis may cause inflammatory diseases and lymph node mediated tumor metastasis. Anti-angiogenic or anti-lymphangiogenic small peptides may play an important role as therapeutic agents normalizing angiogenesis or lymphangiogenesis in disease conditions. Several novel endogenous peptides derived from proteins containing a conserved somatotropin domain have been previously identified with the help of our bioinformatics-based methodology. These somatotropin peptides were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. We found that the peptides have the potential for inhibiting both angiogenesis and lymphangiogenesis. Focusing the study on the inhibition of lymphangiogenesis, we found that a peptide derived from the somatotropin conserved domain of transmembrane protein 45A human was the most potent lymphangiogenesis inhibitor, blocking lymphatic endothelial cell migration, adhesion, and tube formation.     

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.     

Design,synthesis, and characterization of novel apigenin analogues that suppress pancreatic stellate cell proliferation in vitro and associated pancreatic fibrosis in vivo

Accumulating evidence suggests that activated pancreatic stellate cells (PSC) play an important role in chronic pancreatitis (CP), and inhibition of the activated PSC is considered as a potential strategy for the treatment and prevention of CP. Herein, we disclose our findings that apigenin and its novel analogues suppress the proliferation and induce apoptosis in PSC, which reduce the PSC-mediated fibrosis in CP. Chemical modifications of apigenin have been directed to build a focused library of O-alkylamino-tethered apigenin derivatives at 4′-O position of the ring C with the attempt to enhance the potency and drug-like properties including aqueous solubility. A number of compounds such as 14, 16, and 24 exhibited potent antiproliferative effects as well as improved aqueous solubility. Intriguingly, apigenin, new analogues 23 and 24 displayed significant efficacy to reduce pancreatic fibrosis even at a low dose of 0.5 mg/kg in our proof-of-concept study using a preclinical in vivo mouse model of CP.     

Zinc(II) complexes with potent cyclin-dependent kinase inhibitors derived from 6-benzylaminopurine: synthesis, characterization, X-ray structures and biological activity

The synthesis, characterization and biological activity of the first zinc(II) complexes with potent inhibitors of cyclin-dependent kinases (CDKs) derived from 6-benzylaminopurine are described. Based on the results following from elemental analyses, infrared, NMR and ES+MS (electrospray mass spectra in the positive ion mode) spectroscopies, conductivity data, thermal analysis and X-ray structures, the tetrahedral Zn(II) complexes of the compositions [Zn(Olo)Cl(2)](n) (1), [Zn(iprOlo)Cl(2)](n) (2), [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been prepared, where Olo=2-(2-hydroxyethylamino)-6-benzylamino-9-methylpurine (Olomoucine), iprOlo=2-(2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (i-propyl-Olomoucine), Boh=2-(3-hydroxypropylamino)-6-benzylamino-9-isopropylpurine (Bohemine). The 1D-polymeric chain structure for [Zn(Olo)Cl(2)](n) (1) as well as the monomeric one for [Zn(BohH(+))Cl(3)] x H(2)O (3) and [Zn(iprOloH(+))Cl(3)] x H(2)O (4) have been revealed unambiguously by single crystal X-ray analyses. The 1D-polymeric chain of 1 consists of Zn(Olo)Cl(2) monomeric units in which the Zn(II) ion is coordinated by two chlorine atoms and one oxygen atom of the 2-hydroxyethylamino group of Olomoucine. The next monomeric unit is bonded to Zn(II) through the N7 atom of a purine ring. Thus, each of Zn(II) ions is tetrahedrally coordinated and a ZnCl(2)NO chromophore occurs in the complex 1. The complexes 3 and 4 are mononuclear species with a distorted tetrahedral arrangement of donor atoms around the Zn(II) ion with a ZnCl(3)N chromophore. The corresponding CDK inhibitor, i.e., both Boh and iprOlo, is coordinated to Zn(II) via the N7 atom of the purine ring in 3 and 4. The cytotoxicity of the zinc(II) complexes against human melanoma, sarcoma, leukaemia and carcinoma cell lines has been determined as well as the inhibition of the CDK2/cyclin E kinase. A relationship between the structure and biological activity of the complexes is also discussed.     

Polysaccharides from Lycium barbarum leaves: Isolation, characterization and splenocyte proliferation activity

The polysaccharides from the fruits of Lycium barbarum have received considerable attention in previous publication, but the polysaccharides from the leaves were rarely reported. In the present work, four water-soluble polysaccharide fractions: LBP-I, LBP-II, LBP-III and LBP-IV isolated from L. barbarum leaves were purified through DEAE-Sephadex A-25. LBP-II and LBP-IV respectively showed one symmetrical peak on HPGPC with average molecular weight of 9.39×10(4)Da and 4.18×10(5)Da. UV and IR analysis of the two fractions showed the characteristics of acidic polysaccharides combined with polypeptides or proteins. GC analysis showed LBP-IV was mainly composed of rhamnose, arabinose, xylose, glucose and galactose with molar ratio of 1.61:3.82:3.44:7.54:1.00, and the uronic acid content was 47.68% (w/w) determined by sulfuric acid-carbazole method. (1)H and (13)C NMR spectra of LBP-IV also showed the presence of carboxyl carbon and five anomeric carbons, and suggested there may be both α- and β-anomeric configurations in this fraction. Moreover, splenocyte proliferation activity assay showed that LBP-IV significantly enhanced the proliferation of splenocyte stimulated by ConA or LPS, indicating the fraction has the beneficial effect on immunostimulating activity.     

In vitro and in vivo characterization of novel nonsteroidal progesterone receptor antagonists derived from the fungal metabolite PF1092C

We studied the pharmacological effects of novel nonsteroidal progesterone receptor antagonists CP8661 and CP8754, which were synthesized from the fungal metabolite PF1092C. CP8661 possess a tetrahydrobenzindolone skeleton and CP8754 possess a tetrahydronaphthofuranone skeleton. In binding assays for steroid receptors, CP8661 and CP8754 inhibited [(3)H]-progesterone binding to human progesterone receptors (hPR), though they are less potent than RU486. CP8661 also showed moderate affinity to rat androgen receptors (rAR), although CP8754 did not. Neither compound showed affinity to human glucocorticoid receptors (hGR) or human estrogen receptors (hER). In exogeneous and endogeneous PR-dependent enzyme expression assays using human mammary carcinoma T47D, CP8661 and CP8754 showed pure antagonistic activity. In a rabbit endometrial transformation test, CP8661 and CP8754 showed anti-progestational activity by s.c. administration in a dose-dependent manner; meanwhile, these compounds showed no progestational activity at the same dose. These results suggested that CP8661 and CP8754 are in vivo effective pure progesterone receptor antagonists and presented the possibility of synthesizing pure progesterone receptor antagonists from both tetrahydronaphthofuranone and tetrahydrobenzindolone skeletons.     

Identification of an imidazopyridine scaffold to generate potent and selective TYK2 inhibitors that demonstrate activity in an in vivo psoriasis model

Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.     

In vitro and in vivo protein phosphorylation in Avena sativa L. coleoptiles: effects of Ca2+, calmodulin antagonists, and auxin

In vitro and in vivo protein phosphorylations in oat (Avena sativa L.) coleoptile segments were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. In vitro phosphorylation of several polypeptides was distinctly promoted at 1 to 15 micromolar free Ca2+ concentrations. Ca2(+)-stimulated phosphorylation was markedly reduced by trifluoperazine, chlorpromazine, and naphthalene sulfonamide (W7). Two polypeptides were phosphorylated both under in vitro and in vivo conditions, but the patterns of phosphorylation of several other polypeptides were different under the two conditions indicating that the in vivo phosphorylation pattern of proteins is not truly reflected by in vitro phosphorylation studies. Trifluoperazine, W7, or ethylene glycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) + calcium ionophore A23187 treatments resulted in reduced levels of in vivo protein phosphorylation of both control and auxin-treated coleoptile segments. Analysis by two-dimensional electrophoresis following in vivo phosphorylation revealed auxin-dependent changes of certain polypeptides. A general inhibition of phosphorylation by calmodulin antagonists suggested that both control and auxin-treated coleoptiles exhibited Ca2+, and calmodulin-dependent protein phosphorylation in vivo.     

Pyrimidine-based antagonists of h-MCH-R1 derived from ATC0175: In vitro profiling and in vivo evaluation

A series of pyrimidine analogues derived from ATC0175 were potent antagonists of human MCH-R1 in vitro. Significantly improved receptor selectivity was achieved with several analogues from this series, but no improvement in brain partitioning was noted. One example from this series was shown to inhibit food intake and decrease body weight in a chronic study. However no clear correlation between the pharmacodynamic effect and the pharmacokinetic data with respect to brain concentration was discernible leading us to conclude that the observed effect was most likely not due to interaction with the MCH-R1.     

Novel Aza-resveratrol analogs: Synthesis,characterization and anticancer activity against breast cancer cell lines

Novel Aza-resveratrol analogs were synthesized, structurally characterized and evaluated for cytotoxic activity against MDA-MB-231 and T47D breast cancer cell lines, which exhibited superior inhibitory activity than parent resveratrol compound. The binding mechanism of these compounds with estrogen receptor-α was rationalized by molecular docking studies which indicated additional hydrogen binding interactions and tight binding in the protein cavity. Induction of Beclin-1 protein expression in breast cancer cell lines after treatment with newly synthesized resveratrol analogs indicated inhibition of growth of these cell lines through autophagy. The study highlighted the advantage of introducing the imino-linkage in resveratrol motif in enhancing the anticancer potential of resveratrol suggesting that these analogs can serve as better therapeutic agents against breast cancer and can provide starting point for building more potent analogs in future.     

Polysialylated insulin: synthesis,characterization and biological activity in vivo

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96–99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12–48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60–1.74 for the 22-kDa CA and 2.37–2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.     

Polysialylated insulin: synthesis,characterization and biological activity in vivo

Polysialic acids (PSA) (colominic acid; CA) of 22 and 39 kDa average molecular weight were oxidized with sodium periodate at carbon 7 of the nonreducing end to form an aldehyde group. The oxidized CAs (96-99% oxidation) were then reacted with the amino groups of recombinant human insulin at various CA/insulin molar ratios (25:1 to 150:1 range) for up to 48 h in the presence of sodium cyanoborohydride (reductive amination). Polysialylated insulin conjugates were precipitated (together with intact nonreacted insulin, if any) at time intervals from the reaction mixtures with ammonium sulfate, further purified by size exclusion chromatography and/or ion exchange chromatography (IEC), and the final conjugates assayed for PSA and protein. Results showed an initial rapid conjugation rate peaking at about 12 h, to form a plateau over a period of 12-48 h. Moreover, the extent of polysialylation (CA/insulin molar ratios in the conjugate) was dependent on the PSA used, the initial CA/insulin molar ratios in the reaction mixture and the time of the coupling reaction. Thus at 48 h of incubation, CA/insulin molar ratios in the conjugates were 1.60-1.74 for the 22-kDa CA and 2.37-2.45 for the 39-kDa CA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of intact insulin and insulin reacted with non-oxidized CA for 48 h revealed well-resolved single bands which migrated similar distances in the gel. On the other hand, polysialylated (22-kDa CA) insulin yielded multiple diffused bands suggesting heterogenicity as a result of differential polysialylation. The pharmacological activity of polysialylated insulin was compared with that of intact insulin in normal female outbred T/O mice. After subcutaneous injection of intact insulin (0.3 units per mouse), blood glucose levels were reduced to nadir values at 1 h to return to normal at 3 h. In contrast, blood glucose levels in animals injected with polysialylated insulin (0.3 units or protein equivalence for polysialylated insulin), having attained nadir values also at 1 h, returned to normal levels after 6 h (39 kDa) and 9 h (22 kDa CA-insulin). It is concluded that polysialylation offers a promising strategy for the enhancement of the therapeutic value of insulin and other pharmacologically active peptides.     

Discovery and characterization of an antibody, anti-egressin, that is able to inhibit Trypanosoma cruzi egress in vitro

Trypanosoma cruzi, a protozoan parasite, is the etiologic agent of Chagas disease. The disease is characterized by acute and chronic phases, with high and low parasitemia, respectively. A strong immune activation is necessary for the host to enter the chronic phase; however, immune mechanisms participating in the reduction of parasites between the acute and chronic phases of the disease have been very difficult to elucidate. We report here the discovery of anti-egressin, an antibody present in serum from chronically infected BALB/c mice that is able to inhibit parasite egress from infected BALB/c fibroblast cultures in vitro. The antibody is very concentrated in serum from these mice; chronic serum may be diluted 1:20 while still maintaining functional activity. Isotype analysis of anti-egressin has suggested it to be IgG2a. Further analysis revealed that anti-egressin was specific for a component expressed on the surface of infected host cells. The specificity of anti-egressin toward the extracellular portion of infected host cells was demonstrated both by using a quantitative assay measuring released trypomastigotes and through immunocytochemical staining. The novel role of anti-egressin in the inhibition of parasite egress from infected host cells has not been described in the literature to date. We believe that anti-egressin plays an important role in achieving the low parasitemia characteristic of chronic Chagas disease.     

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor

Summary Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors—releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47±7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69±9 d). Line RM3 showed tumor induction in 40% of the mice after 186±16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.     

Chemical characterization and opioid activity of an exorphin isolated from in vivo digests of casein

The in vivo formation of an opioid peptide (exorphin) derived from beta-casein has been proved for the first time. It was isolated from duodenal chyme of minipigs after feeding with the milk protein casein. The exorphin has been identified as a beta-casein fragment by end-group determinations and qualitative amino acid analysis of the purified peptide. This peptide, named beta-casomorphin-11, displayed substantial opioid activity in an opiate receptor-binding assay.     

Cloning and characterization of human Lnk, an adaptor protein with pleckstrin homology and Src homology 2 domains that can inhibit T cell activation

Lnk was originally cloned from a rat lymph node cDNA library and shown to participate in T cell signaling. Human Lnk (hLnk) was cloned by screening a Jurkat cell cDNA library. hLnk has a calculated molecular mass of 63 kDa, and its deduced amino acid sequence indicates the presence of an N-terminal proline-rich region, a pleckstrin homology domain, and a Src homology 2 domain. When expressed in COS cells, hLnk migrates with an apparent molecular mass of 75 kDa. Confocal fluorescence microscope analysis indicates that in COS cells transfected with an expression vector encoding a chimeric Lnk-green fluorescent protein, hLnk is found at the juxtanuclear compartment and also appears to be localized at the plasma membrane. Lnk is tyrosine-phosphorylated by p56lck. Following phosphorylation, p56lck binds to tyrosine-phosphorylated hLnk through its Src homology 2 domain. In COS cells cotransfected with hLnk, p56lck, and CD8-zeta, hLnk associated with tyrosine-phosphorylated TCR zeta-chain through its Src homology 2 domain. The overexpression of Lnk in Jurkat cells led to an inhibition of anti-CD3 mediated NF-AT-Luc activation. Our study reveals a potentially new mechanism of T cell-negative regulation.     

Establishment and characterization of a cell line, HS-Os-1 derived from an osteoblastic type of human osteosarcoma

A new human cell line, HS-Os-1, derived from a case of osteoblastic osteosarcoma arising in the humerus of an 11-year-old girl was established. Light microscopically, HS-Os-1 cells growing in a monolayer (in vitro) were pleomorphic, intermingled with a few multinucleated giant ones, and positive with alkaline phosphatase reaction. In the transplanted tumors in athymic nude mice (in vivo), atypical spindle or polygonal cells densely proliferated with prominent osteoid formation and even calcification. HS-Os-1 cells, both in vitro and in vivo, were mostly positive for vimentin and a few for S-100 protein. Ultrastructurally, HS-Os-1 cells in vitro and in vivo also revealed essentially the same features as the eccentrically located, euchromatin-rich nuclei with prominent nucleoli, a lot of well-developed, irregularly-dilated rough endoplasmic reticula, polysomes and microfilaments in the cytoplasm. Namely, HS-Os-1 cells fully expressed and possessed morphological characteristics as osteoblastic nature during the cultivation and heterotransplantation. This cell line, therefore, proved to be extremely useful to search for human osteosarcomas.