Synthesis and exploration of novel radiolabeled bombesin peptides for targeting receptor positive tumor

Increasing evidence of peptide receptor overexpression in various cancer cells, warrant the development of receptor specific radiolabeled peptides for molecular imaging and therapy in nuclear medicine. Gastrin-releasing-peptide (GRP) receptor, are overexpressed in a variety of human cancer cells. The present study report the synthesis and biological evaluation of new bombesin (BBN) analogs, HYNIC-Asp-[Phe¹³]BBN(7–13)-NH-CH₂-CH₂-CH3:BA1, HYNIC-Pro-[Tyr¹³Met¹⁴]BBN(7–14)NH₂:BA2 as prospective tumor imaging agent with compare to BBN(7–14)NH₂:BS as standard. The pharmacophores were radiolabeled in high yields with ^99mTc, characterized for their stability in serum and saline, cysteine/histidine and were found to be substantially stable. Internalization/externalization and receptor binding studies were assessed using MDA-MB-231 cells and showed high receptor binding-affinity and favourable internalization. Fluorescence studies revealed that BA1 changed the morphology of the cells and could localize in the nucleus more effectively than BA2/BS. Cell-viability studies displayed substantial antagonistic and nuclear-internalization effect of BA1. BA1 also exhibited antiproliferative effect on MDA-MB-231 cell by inducing apoptosis. In vivo behaviour of the radiopeptides was evaluated in GRP receptor positive tumor bearing mice. The ^99mTc-BA1/^99mTc-BA2 demonstrated rapid blood/urinary clearance through the renal pathway and comparatively more significant tumor uptake image and favourable tumor-to-non-target ratios provided by ^99mTc-BA1. The specificity of the in vivo uptake was confirmed by co-injection with BS. Moreover, ^99mTc-BA1 provided a much clearer tumor image in scintigraphic studies than others. Thus the combination of favourable in vitro and in vivo properties renders BA1 as more potential antagonist bombesin-peptide for targeting GRP-receptor positive tumor. These properties are encouraging to carry out further experiments for non-invasive receptor targeting potential diagnostinc and therapeutic agent for tumors.     

The Effect of Molecular Weight,PK, and Valency on Tumor Biodistribution and Efficacy of Antibody-Based Drugs

Poor drug delivery and penetration of antibody-mediated therapies pose significant obstacles to effective treatment of solid tumors. This study explored the role of pharmacokinetics, valency, and molecular weight in maximizing drug delivery. Biodistribution of a fibroblast growth factor receptor 4 (FGFR4) targeting CovX-body (an FGFR4-binding peptide covalently linked to a nontargeting IgG scaffold; 150 kDa) and enzymatically generated FGFR4 targeting F(ab)₂ (100 kDa) and Fab (50 kDa) fragments was measured. Peak tumor levels were achieved in 1 to 2 hours for Fab and F(ab)₂versus 8 hours for IgG, and the percentage injected dose in tumors was 0.45%, 0.5%, and 2.5%, respectively, compared to 0.3%, 2%, and 6% of their nontargeting controls. To explore the contribution of multivalent binding, homodimeric peptides were conjugated to the different sized scaffolds, creating FGFR4 targeting IgG and F(ab)₂ with four peptides and Fab with two peptides. Increased valency resulted in an increase in cell surface binding of the bivalent constructs. There was an inverse relationship between valency and intratumoral drug concentration, consistent with targeted consumption. Immunohistochemical analysis demonstrated increased size and increased cell binding decreased tumor penetration. The binding site barrier hypothesis suggests that limited tumor penetration, as a result of high-affinity binding, could result in decreased efficacy. In our studies, increased target binding translated into superior efficacy of the IgG instead, because of superior inhibition of FGFR4 proliferation pathways and dosing through the binding site barrier. Increasing valency is therefore an effective way to increase the efficacy of antibody-based drugs.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

Multivalent Ligand System Carrying Enkephalin and Neurotensin Coimmobilized on Liposomes

A multivalent ligand system was constructed by coimmobilization of two kinds of peptide ligands, enkephalin and neurotensin derivatives having a dioctadecyl group, on dimyristoylphosphatidylcholine (DMPC) liposomes. The enkephalin derivatives are Tyr-D -Ala-Gly-Trp-Leu- (Sar-Sar-Pro)_n-[N(C₁₈H₃₇)₂] (Enk3nD, n=0, 1, 2), where a dioctadecyl group was connected to the C-terminal side of enkephalin directly or through a hydrophilic and flexible spacer chain of different lengths. The neurotensin derivatives are Ac-Glu[N(C₁₈H₃₇)₂]-(Sar-Sar-Pro)_n-Arg-Arg-Pro-Tyr-Ile-Leu-OH (D3nNT, n=0, 1, 2, 3). The derivatives were spontaneously immobilized on DMPC liposomes by overnight incubation. The receptor affinity of the enkephalin derivatives became significantly higher upon immobilization on liposomes. The highest affinity was obtained for the δ receptor by Enk6D immobilized on DMPC liposomes. This affinity is higher than that of enkephalinamide. Neurotensin derivatives coimmobilized with large amounts of Enk3D on DMPC liposomes show higher affinity than the neurotensin derivatives immobilized alone. The effect of Enk3D on the receptor affinity of the coimmobilized neurotensin derivative disappeared by the addition of [Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAGO). Therefore, the receptor affinity of a peptide hormone is altered by immobilization on DMPC liposomes and by coimmobilization with other peptide hormones. It was confirmed by fluorescent microscopy that the multivalent ligand system binds to receptors without release of the bound ligands from DMPC liposomes.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

A novel series of pyrazole-platinum(II) complexes as potential anti-cancer agents that induce cell cycle arrest and apoptosis in breast cancer cells

Six novel compounds of platinum(II) with pyrazole derivatives PtPz1–PtPz6 were synthesised and characterised (PtPz1 - [Pt₂N-hydroksymethyl-3,5-dimethylpyrazole₄(berenil)₂]Cl₄; PtPz2 - [Pt₂3,5-dimethylpyrazole₄(berenil)₂]Cl₄; PtPz3 - [Pt₂3,4-dimethylpyrazole₄(berenil)₂]Cl₄; PtPz4 - [Pt₂pyrazole₄(berenil)₂]Cl₄; PtPz5- [Pt₂5-methylpyrazole₄(berenil)₂]Cl₄; PtPz6 - [Pt₂N-ethylpyrazole₄(berenil)₂]Cl₄). The cytotoxic activity of these complexes against MCF-7 and MDA-MB-231 breast cancer cell lines was determined using the MTT assay. Evaluation of apoptosis induction was done with the Annexin V-fluorescein isothiocyanate/propidium iodide assay. In addition, using a flow cytometer, we determined the influence of test compounds on the cell cycle and caspase-3, -8, and -9 activity. The obtained results of caspase activity were confirmed by cell imaging. Moreover, using the flow cytometer, the effects of the test compounds on mitochondrial potential change were assessed. The test results showed that novel pyrazole-platinum(II) complexes exhibited stronger anti-proliferative activity against two breast cancer cell lines than reference cisplatin. Compounds PtPz1, PtPz2, and PtPz3 with methyl substituents at the pyrazole ring showed stronger activity than pyrazole or ethylpyrazole containing complexes. Studies have shown that inhibition of cell survival occurs by arresting the G1 cell cycle and inducing apoptosis. Our analysis associated with the response of MCF-7 and MDA-MB-231 cells to treatment with PtPz1–PtPz6 showed that it leads the cells through the external and intrinsic (mitochondrial) apoptotic pathway via indirect DNA damage.     

Contributory Role of BLT2 in the Production of Proinflammatory Cytokines in Cecal Ligation and Puncture-Induced Sepsis

BLT2 is a low-affinity receptor for leukotriene B₄, a potent lipid mediator of inflammation generated from arachidonic acid via the 5-lipoxygenase pathway. The aim of this study was to investigate whether BLT2 plays any role in sepsis, a systemic inflammatory response syndrome caused by infection. A murine model of cecal ligation and puncture (CLP)-induced sepsis was used to evaluate the role of BLT2 in septic inflammation. In the present study, we observed that the levels of ligands for BLT2 (LTB₄ [leukotriene B₄] and 12(S)-HETE [12(S)-hydroxyeicosatetraenoic acid]) were significantly increased in the peritoneal lavage fluid and serum from mice with CLP-induced sepsis. We also observed that the levels of BLT2 as well as 5-lipoxygenase (5-LO) and 12-LO, which are synthesizing enzymes for LTB₄ and 12(S)-HETE, were significantly increased in lung and liver tissues in the CLP mouse model. Blockade of BLT2 markedly suppressed the production of sepsis-associated cytokines (IL-6 [interleukin-6], TNF-α[[tumor necrosis factor alpha], and IL-1β [interleukin-1β] as well as IL-17 [interleukin-17]) and alleviated lung inflammation in the CLP group. Taken together, our results suggest that BLT2 cascade contributes to lung inflammation in CLP-induced sepsis by mediating the production of inflammatory cytokines. These findings suggest that BLT2 may be a potential therapeutic target for sepsis patients.     

Treatment of Peritoneal Carcinomatosis by Targeted Delivery of the Radio-Labeled Tumor Homing Peptide 213Bi-DTPA-[F3]2 into the Nucleus of Tumor Cells

Background

α-particle emitting isotopes are effective novel tools in cancer therapy, but targeted delivery into tumors is a prerequisite of their application to avoid toxic side effects. Peritoneal carcinomatosis is a widespread dissemination of tumors throughout the peritoneal cavity. As peritoneal carcinomatosis is fatal in most cases, novel therapies are needed. F3 is a tumor homing peptide which is internalized into the nucleus of tumor cells upon binding to nucleolin on the cell surface. Therefore, F3 may be an appropriate carrier for α-particle emitting isotopes facilitating selective tumor therapies.

Principal Findings

A dimer of the vascular tumor homing peptide F3 was chemically coupled to the α-emitter ²¹³Bi (²¹³Bi-DTPA-[F3]₂). We found ²¹³Bi-DTPA-[F3]₂ to accumulate in the nucleus of tumor cells in vitro and in intraperitoneally growing tumors in vivo. To study the anti-tumor activity of ²¹³Bi-DTPA-[F3]₂ we treated mice bearing intraperitoneally growing xenograft tumors with ²¹³Bi-DTPA-[F3]₂. In a tumor prevention study between the days 4–14 after inoculation of tumor cells 6×1.85 MBq (50 µCi) of ²¹³Bi-DTPA-[F3]₂ were injected. In a tumor reduction study between the days 16–26 after inoculation of tumor cells 6×1.85 MBq of ²¹³Bi-DTPA-[F3]₂ were injected. The survival time of the animals was increased from 51 to 93.5 days in the prevention study and from 57 days to 78 days in the tumor reduction study. No toxicity of the treatment was observed. In bio-distribution studies we found ²¹³Bi-DTPA-[F3]₂ to accumulate in tumors but only low activities were found in control organs except for the kidneys, where ²¹³Bi-DTPA-[F3]₂ is found due to renal excretion.

Conclusions/Significance

In conclusion we report that ²¹³Bi-DTPA-[F3]₂ is a novel tool for the targeted delivery of α-emitters into the nucleus of tumor cells that effectively controls peritoneal carcinomatosis in preclinical models and may also be useful in oncology.     

Design of a Tumor Homing Cell-Penetrating Peptide for Drug Delivery

The major drawbacks with conventional cancer chemotherapy are the lack of satisfactory specificity towards tumor cells and poor antitumor activity. In order to improve these characteristics, chemotherapeutic drugs can be conjugated to targeting moieties e.g. to peptides with the ability to recognize cancer cells. We have previously reported that combining a tumor homing peptide with a cell-penetrating peptide yields a chimeric peptide with tumor cell specificity that can carry cargo molecules inside the cells. In the present study, we have used a linear breast tumor homing peptide, CREKA, in conjunction with a cell-penetrating peptide, pVEC. This new chimeric peptide, CREKA–pVEC, is more convenient to synthesize and moreover it is better in translocating cargo molecules inside cancer cells as compared to previously published PEGA–pVEC peptide. This study demonstrates that CREKA–pVEC is a suitable vehicle for targeted intracellular delivery of a DNA alkylating agent, chlorambucil, as the chlorambucil–peptide conjugate was substantially better at killing cancer cells in vitro than the anticancer drug alone.     

Lysophosphatidylethanolamine utilizes LPA1 and CD97 in MDA-MB-231 breast cancer cells

Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca^2 + ([Ca^2 +]_i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca^2 +]_i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA₁ and LPA₃) inhibited LPE-induced [Ca^2 +]_i increases, and knockdown of LPA₁ by transfection with LPA₁ siRNA completely inhibited LPE-induced [Ca^2 +]_i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA₁ in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of G_i/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP₃ receptor) completely inhibited LPE-induced [Ca^2 +]_i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA₁-CD97/G_i/o proteins/phospholipase C/IP₃/Ca^2 + rise in MDA-MB-231 breast cancer cells.     

Role of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancer cells into cancer stem-like cells

Previous studies have demonstrated that a small subset of cancer cells is capable of tumor initiation. The existence of tumor initiating cancer stem cells (CSCs) has several implications in terms of future cancer treatment and therapies. However, recently, several researchers proposed that differentiated cancer cells (non-CSCs) can convert to stem-like cells to maintain equilibrium. These results imply that removing CSCs may prompt non-CSCs in the tumor to convert into stem cells to maintain the equilibrium. Interleukin-6 (IL-6) has been found to play an important role in the inducible formation of CSCs and their dynamic equilibrium with non-stem cells. In this study, we used CSC-like human breast cancer cells and their alternate subset non-CSCs to investigate how IL-6 regulates the conversion of non-CSCs to CSCs. MDA-MB-231 and MDA-MB-453 CSC-like cells formed mammospheres well, whereas most of non-stem cells died by anoikis and only part of the remaining non-stem cells produced viable mammospheres. Similar results were observed in xenograft tumor formation. Data from cytokine array assay show that IL-6 was secreted from non-CSCs when cells were cultured in ultra-low attachment plates. IL-6 regulates CSC-associated OCT-4 gene expression through the IL-6-JAK1-STAT3 signal transduction pathway in non-CSCs. Inhibiting this pathway by treatment with anti-IL-6 antibody (1 μg/ml) or niclosamide (0.5–2 μM)/LLL12 (5–10 μM) effectively prevented OCT-4 gene expression. These results suggest that the IL-6-JAK1-STAT3 signal transduction pathway plays an important role in the conversion of non-CSCs into CSCs through regulation of OCT-4 gene expression.     

Synthesis of Tc-99m labeled 1,2,3-triazole-4-yl c-met binding peptide as a potential c-met receptor kinase positive tumor imaging agent

The mesenchymal-epithelial transition factor (c-Met), which is related to tumor cell growth, angiogenesis and metastases, is known to be overexpressed in several tumor types. In this study, we synthesized technetium-99m labeled 1,2,3-triazole-4-yl c-Met binding peptide (cMBP) derivatives, prepared by solid phase peptide synthesis and the ‘click-to-chelate’ protocol for the introduction of tricarbonyl technetium-99m, as a potential c-Met receptor kinase positive tumor imaging agent, and evaluated their in vitro c-Met binding affinity, cellular uptake, and stability. The ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12, [^99mTc(CO)₃]13, and [^99mTc(CO)₃]14) were prepared in 85-90% radiochemical yields. The cold surrogate cMBP derivatives, [Re(CO)₃]12, [Re(CO)₃]13, and [Re(CO)₃]14, were shown to have high binding affinities (0.13 μM, 0.06 μM, and 0.16 μM, respectively) to a purified cMet/Fc chimeric recombinant protein. In addition, the in vitro cellular uptake and inhibition studies demonstrated the high specific binding of these ^99mTc labeled cMBP derivatives ([^99mTc(CO)₃]12–14) to c-Met receptor positive U87MG cells.     

Assessment of cytosolic free calcium changes during ceramide-induced cell death in MDA-MB-231 breast cancer cells expressing the calcium sensor GCaMP6m

Alterations in Ca²⁺ signaling can regulate key cancer hallmarks such as proliferation, invasiveness and resistance to cell death. Changes in the regulation of intracellular Ca²⁺ and specific components of Ca²⁺ influx are a feature of several cancers and/or cancer subtypes, including the basal-like breast cancer subtype, which has a poor prognosis. The development of genetically encoded calcium indicators, such as GCaMP6, represents an opportunity to measure changes in intracellular free Ca²⁺ during processes relevant to breast cancer progression that occur over long periods (e.g. hours), such as cell death. This study describes the development of a MDA-MB-231 breast cancer cell line stably expressing GCaMP6m. The cell line retained the key features of this aggressive basal-like breast cancer cell line. Using this model, we defined alterations in relative cytosolic free Ca²⁺ ([Ca²⁺]_CYT) when the cells were treated with C2-ceramide. Cell death was measured simultaneously via assessment of propidium iodide permeability. Treatment with ceramide produced delayed and heterogeneous sustained increases in [Ca²⁺]_CYT. Where cell death occurred, [Ca²⁺]_CYT increases preceded cell death. The sustained increases in [Ca²⁺]_CYT were not related to the rapid morphological changes induced by ceramide. Silencing of the plasma membrane Ca²⁺ ATPase isoform 1 (PMCA1) was associated with an augmentation in ceramide-induced increases in [Ca²⁺]_CYT and also cell death. This work demonstrates the utility of GCaMP6 Ca²⁺ indicators for investigating [Ca²⁺]_CYT changes in breast cancer cells during events relevant to tumor progression, which occur over hours rather than minutes.     

Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

Nature-inspired pyrrolo[2,3-d]pyrimidines targeting the histamine H3 receptor

Inspired by marine compounds the derivatization of the natural pyrrolo[2,3-d]pyrimidine lead scaffold led to a series of novel compounds targeting the histamine H₃ receptor. The focus was set on improved binding towards the receptor and to establish an initial structure-activity relationship for this compound class based on the lead structure (compound V, K_i value of 126 nM). As highest binding affinities were found with 1,4-bipiperidines as basic part of the ligands, further optimization was focused on 4-([1,4′-bipiperidin]-1′-yl)-pyrrolo[2,3-d]pyrimidines. Related pyrrolo[2,3-d]pyrimidines that were isolated from marine sponges like 4-amino-5-bromopyrrolo[2,3-d]pyrimidine (compound III), showed variations in halogenation pattern, though in a next step the impact of halogenation at 2-position was evaluated. The chloro variations did not improve the affinity compared to the dehalogenated compounds. However, the simultaneous introduction of lipophilic cores with electron-withdrawing substitution patterns in 7-position and dehalogenation at 2-position (11b, 12b) resulted in compounds with significantly higher binding affinities (K_i values of 7 nM and 6 nM, respectively) than the initial lead structure compound V. The presented structures allow for a reasonable structure-activity relationship of pyrrolo[2,3-d]pyrimidines as histamine H₃ receptor ligands and yielded novel lead structures within the natural compound library against this target.     

Synthesis,characterization and cytotoxicity studies of 1,2,3-triazoles and 1,2,4-triazolo [1,5-a] pyrimidines in human breast cancer cells

Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) is essential for physiological functions of tissues and neovasculature. VEGFR signaling is associated with the progression of pathological angiogenesis in various types of malignancies, making it an attractive therapeutic target in cancer treatment. In the present work, we report the synthesis of 1,4-disubstituted 1,2,3-triazoles and 1,2,4-triazolo[1, 5-a]pyrimidine derivatives via copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and screened for their anticancer activity against MCF7 cells. We identified 1-(2′-ethoxy-4′-fluoro-[1,1′-biphenyl]-4-yl)-4-phenyl-1H-1,2,3-triazole (EFT) as lead cytotoxic agent against MCF7 cell lines with an IC₅₀ value of 1.69?µM. Further evaluation revealed that EFT induces cytotoxicity on Ishikawa, MDA-MB-231 and BT474 cells with IC₅₀ values of 1.97, 4.81 and 4.08?µM respectively. However, EFT did not induce cytotoxicity in normal lung epithelial (BEAS-2B) cells. Previous reports suggested that 1,2,3-triazoles are the inhibitors of VEGFR1 and therefore, we evaluated the effect of EFT on the expression of VEGFR1. The results demonstrated that EFT downregulates the expression of VEGFR1 in MCF7 cells. In summary, we identified a potent cytotoxic agent that imparts its antiproliferative activity by targeting VEGFR1 in breast cancer cells. The novel compound could serve as a lead structure in developing VEGFR1 inhibitors.     

[Ca2+]i Oscillations and IL-6 Release Induced by α-Hemolysin from Escherichia coli Require P2 Receptor Activation in Renal Epithelia

Urinary tract infections are commonly caused by α-hemolysin (HlyA)-producing Escherichia coli. In erythrocytes, the cytotoxic effect of HlyA is strongly amplified by P2X receptors, which are activated by extracellular ATP released from the cytosol of the erythrocytes. In renal epithelia, HlyA causes reversible [Ca²⁺]_i oscillations, which trigger interleukin-6 (IL-6) and IL-8 release. We speculate that this effect is caused by HlyA-induced ATP release from the epithelial cells and successive P2 receptor activation. Here, we demonstrate that HlyA-induced [Ca²⁺]_i oscillations in renal epithelia were completely prevented by scavenging extracellular ATP. In accordance, HlyA was unable to inflict any [Ca²⁺]_i oscillations in 132-1N1 cells, which lack P2R completely. After transfecting these cells with the hP2Y₂ receptor, HlyA readily triggered [Ca²⁺]_i oscillations, which were abolished by P2 receptor antagonists. Moreover, HlyA-induced [Ca²⁺]_i oscillations were markedly reduced in medullary thick ascending limbs isolated from P2Y₂ receptor-deficient mice compared with wild type. Interestingly, the following HlyA-induced IL-6 release was absent in P2Y₂ receptor-deficient mice. This suggests that HlyA induces ATP release from renal epithelia, which via P2Y₂ receptors is the main mediator of HlyA-induced [Ca²⁺]_i oscillations and IL-6 release. This supports the notion that ATP signaling occurs early during bacterial infection and is a key player in the further inflammatory response.     

The Ca2+ influx induced by β-amyloid peptide 25–35 in cultured hippocampal neurons results from network excitation

Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca²⁺]_i elevations and electrical activity were enhanced by removal of extracellular Mg²⁺, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca²⁺ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca²⁺]_i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca²⁺]_i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca²⁺ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.     

Design,synthesis, and structure–activity relationships of pyrimido[4,5-b]indole-4-amines as microtubule depolymerizing agents that are effective against multidrug resistant cells

To identify the structural features of 9H-pyrimido[4,5-b]indoles as microtubule depolymerizers, pyrimido[4,5-b]indoles 2–8 with varied substituents at the 2-, 4- and 5-positions were designed and synthesized. Nucleophilic displacement of 2,5-substituted-4-chloro-pyrimido[4,5-b]indoles with appropriate arylamines was the final step employed in the synthesis of target compounds 2–8. Compounds 2 and 6 had two-digit nanomolar potency (IC₅₀) against MDA-MB-435, SK-OV-3 and HeLa cancer cells in vitro. Compounds 2 and 6 also depolymerized microtubules comparable to the lead compound 1. Compounds 2, 3, 6 and 8 were effective in cells expressing P-glycoprotein or the βIII isotype of tubulin, mechanisms that are associated with clinical drug resistance to microtubule targeting drugs. Proton NMR and molecular modeling studies were employed to identify the structural basis for the microtubule depolymerizing activity of pyrimido[4,5-b]indoles.