Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvβ₆

The integrin α(v)β(6) is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α(v)β(6) and displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to actively deliver paclitaxel to α(v)β(6)-positive cells. We synthesized a water soluble paclitaxel-H2009.1 peptide conjugate in which the 2'-position of paclitaxel is attached to the tetrameric peptide via an ester linkage. The conjugate maintains its specificity for α(v)β(6)-expressing NSCLC cells, resulting in selective cytotoxicity. Treatment of α(v)β(6)-positive cells with the conjugate results in cell cycle arrest followed by induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.     

Synthesis and in vitro evaluation of a novel radioligand for αvβ3 integrin receptor imaging: [18F]FPPA-c(RGDfK)

The development of RGD-based antagonist of αvβ3 integrin receptor has enhanced the interest in PET probes to image this receptor for the early detection of cancer, to monitor the disease progression and the response to therapy. In this work, a novel prosthetic group (N-(4-fluorophenyl)pent-4-ynamide or FPPA) for the ¹⁸F-labeling of an αvβ3 selective RGD-peptide was successfully prepared. [¹⁸F]FPPA was obtained in three steps with a radiochemical yield of 44% (decay corrected). Conjugation to c(RGDfK(N₃)) by the Cu(II) catalyzed Huisgen azido alkyne cycloaddition provided the [¹⁸F]FPPA-c(RGDfK) with a radiochemical yield of 29% (decay corrected), in an overall synthesis time of 140 min.     

Synthesis and initial evaluation of novel,non-peptidic antagonists of the αv-integrins αvβ3 and αvβ5

The discovery, synthesis and preliminary SAR of a novel class of non-peptidic antagonists of the α_v-integrins α_vβ₃ and α_vβ₅ is described. High-throughput screening of an extensive series of ECLiPS? compound libraries led to the identification of compound 1 as a dual inhibitor of the α_v-integrins α_vβ₃ and α_vβ₅. Optimization of compound 1 involving, in part, introduction of two novel constraints led to the discovery of compounds 15a and 15b with reduced PSA and much improved potency for both the α_vβ₃ and α_vβ₅ integrins. Compounds 15a and 15b were shown to have promising activity in functional cellular assays and compound 15a also exhibited a promising Caco-2 permeability profile.     

βPix plays a dual role in cerebral vascular stability and angiogenesis, and interacts with integrin αvβ8

The growth of new blood vessels by angiogenesis and their stabilization by the recruitment of perivascular mural cells are thought to be two sequential, yet independent events. Here we identify molecular links between both processes through the βPix and integrin α(v)β(8) proteins. Bubblehead (bbh) mutants with a genetic mutation in βPix show defective vascular stabilization. βPix is a guanine nucleotide exchange factor and scaffold protein that binds many proteins including Git1, which bridges βPix to integrins at focal adhesions. Here we show that the ability of βPix to stabilize vessels requires Git1 binding residues. Knockdown of Git1 leads to a hemorrhage phenotype similar to loss of integrin α(v), integrin β(8) or βPix, suggesting that vascular stabilization through βPix involves interactions with integrins. Furthermore, double loss of function of βPix and integrin α(v) shows enhanced hemorrhage rates. Not only is vascular stability impaired in these embryos, but we also uncover a novel role of both βPix and integrin α(v)β(8) in cerebral angiogenesis. Downregulation of either βPix or integrin α(v)β(8) results in fewer and morphologically abnormal cerebral arteries penetrating the hindbrain. We show that this is coupled with a significant reduction in endothelial cell proliferation in bbh mutants or integrin α(v)β(8) morphants. These data suggest that a complex involving βPix, GIT1 and integrin α(v)β(8) may regulate vascular stability, cerebral angiogenesis and endothelial cell proliferation in the developing embryo.     

Absence of Dap12 and the αvβ3 integrin causes severe osteopetrosis

In vitro, ligand occupancy of αvβ3 integrin induces phosphorylation of Dap12, which is essential for osteoclast function. Like mice deleted of only αvβ3, Dap12^−/− mice exhibited a slight increase in bone mass, but Dap12^−/− mice, lacking another ITAM protein, FcRγ, were severely osteopetrotic. The mechanism by which FcRγ compensates for Dap12 deficiency is unknown. We find that co-deletion of FcRγ did not exacerbate the skeletal phenotype of β3^−/− mice. In contrast, β3/Dap12 double-deficient (DAP/β3^−/−) mice (but not β1/Dap12 double-deficient mice) were profoundly osteopetrotic, reflecting severe osteoclast dysfunction relative to those lacking αvβ3 or Dap12 alone. Activation of OSCAR, the FcRγ co-receptor, rescued Dap12^−/− but not DAP/β3^−/−osteoclasts. Thus, the absence of αvβ3 precluded compensation for Dap12 deficiency by FcRγ. In keeping with this, Syk phosphorylation did not occur in OSCAR-activated DAP/β3^−/− osteoclasts. Thus, FcRγ requires the osteoclast αvβ3 integrin to normalize the Dap12-deficient skeleton.     

Synthesis and evaluation of a radioiodinated peptide probe targeting αvβ6 integrin for the detection of pancreatic ductal adenocarcinoma

Introduction

Pancreatic ductal adenocarcinoma (PDAC) remains a major cause of cancer-related death. Since significant upregulation of αvβ6 integrin has been reported in PDAC, this integrin is a promising target for PDAC detection. In this study, we aimed to develop a radioiodinated probe for the imaging of αvβ6 integrin-positive PDAC with single-photon emission computed tomography (SPECT).

Methods

Four peptide probes were synthesized and screened by competitive and saturation binding assays using 2 PDAC cell lines (AsPC-1, αvβ6 integrin-positive; MIA PaCa-2, αvβ6 integrin-negative). The probe showing the best affinity was used to study the biodistribution assay, an in vivo blocking study, and SPECT imaging using tumor bearing mice. Autoradiography and immunohistochemical analysis were also performed.

Results

Among the 4 probes examined in this study, ¹²⁵I-IFMDV2 showed the highest affinity for αvβ6 integrin expressed in AsPC-1 cells and no affinity for MIA PaCa-2 cells. The accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft was 3–5 times greater than that in the MIA PaCa-2 xenograft, consistent with the expression of αvβ6 integrin in each xenograft, and confirmed by immunohistochemistry. Pretreatment with excess amounts of A20FMDV2 significantly blocked the accumulation of ¹²⁵I-IFMDV2 in the AsPC-1 xenograft, but not in the MIA PaCa-2 xenograft. Furthermore, ¹²³I-IFMDV2 enabled clear visualization of the AsPC-1 xenograft.

Conclusion

¹²³I-IFMDV2 is a potential SPECT probe for the imaging of αvβ6 integrin in PDAC.     

Identification of inhibitors against interaction between pro-inflammatory sPLA2-IIA protein and integrin αvβ3

Increased concentrations of secreted phospholipase A2 type IIA (sPLA2-IIA), have been found in the synovial fluid of patients with rheumatoid arthritis. It has been shown that sPLA2-IIA specifically binds to integrin αvβ3, and initiates a signaling pathway that leads to cell proliferation and inflammation. Therefore, the interaction between integrin and sPLA2-IIA could be a potential therapeutic target for the treatment of proliferation or inflammation-related diseases. Two one-bead-one-compound peptide libraries were constructed and screened, and seven target hits were identified. Herein we report the identification, synthesis, and biological testing of two pyrazolylthiazole-tethered peptide hits and their analogs. Biological assays showed that these compounds were able to suppress the sPLA2-IIA–integrin interaction and sPLA2-IIA-induced migration of monocytic cells and that the blockade of the sPLA2-IIA–integrin binding was specific to sPLA2-IIA and not to the integrin.     

Design and synthesis of novel dual-cyclic RGD peptides for αvβ3 integrin targeting

The specific binding of RGD cyclic peptide with integrin α_vβ₃ attracts great research interest for tumor-targeting drug delivery. Herein, we designed and synthesized a series of dual-ring RGD-peptide derivatives as a drug carrier for α_vβ₃ targeting. Three novel peptides showed excellent cell adhesion inhibition effect, in which, P3 exhibited 7-fold enhancement in IC₅₀ compared with cyclo(RGDfK). Drug-loaded cytotoxicity experiment and imaging experiment indicated that such dual-cyclic RGD peptides have good tumor targeting effects. This work provides a new strategy for the design of novel RGD peptides.     

Synthesis and evaluation of bivalent, peptidomimetic antagonists of the αvβ3 integrins

Targeting the integrin α(v)β(3) by directly interfering with its function is considered to be an effective and non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidomimetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to inhibit the integrin α(v)β(3). All the bivalent ligands exhibited increased potency compared to that of their monomeric counterparts for the integrin α(v)β(3) with low nanomolar range binding affinity. The best bivalent ligand 6 tested in the series has an IC(50)=0.09 nM evaluated by ELISA assay. We conclude that multivalency is providing a useful template for the development novel integrin α(v)β(3) antagonists as potential therapeutics.     

CCL2 increases αvβ3 integrin expression and subsequently promotes prostate cancer migration

Background

Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family which is associated with the disease status and outcomes of cancers. Prostate cancer is the most commonly diagnosed malignancy in men and shows a predilection for metastasis to the bone. However, the effect of CCL2 on human prostate cancer cells is largely unknown. The aim of this study was to examine the role of CCL2 in integrin expression and migratory activity in prostate cancers.

Methods

Prostate cancer migration was examined using Transwell, wound healing, and invasion assay. The PKCδ and c-Src phosphorylations were examined by using western blotting. The qPCR was used to examine the mRNA expression of integrins. A transient transfection protocol was used to examine AP-1 activity.

Results

Stimulation of prostate cancer cell lines (PC3, DU145, and LNCaP) induced migration and expression of integrin αvβ3. Treatment of cells with αvβ3 antibody or siRNA abolished CCL2-increased cell migration. CCL2-increased migration and integrin expression were diminished by CCR2 but not by CCR4 inhibitors, suggesting that the CCR2 receptor is involved in CCL2-promoted prostate cancer migration. CCL2 activated a signal transduction pathway that includes PKCδ, c-Src, and AP-1. Reagents that inhibit specific components of this pathway each diminished the ability of CCL2 to effect cell migration and integrin expression.

Conclusions

Interaction between CCL2 and CCR2 enhances migration of prostate cancer cells through an increase in αvβ3 integrin production.

General significance

CCL2 is a critical factor of prostate cancer metastasis.     

Dexamethasone increases αvβ3 integrin expression and affinity through a calcineurin/NFAT pathway

The purpose of this study was to determine how dexamethasone (DEX) regulates the expression and activity of αvβ3 integrin. FACS analysis showed that DEX treatment induced expression of an activated αvβ3 integrin. Its expression remained high as long as DEX was present and continued following DEX removal. FACS analysis showed that the upregulation of αvβ3 integrin was the result of an increase in the expression of the β3 integrin subunit. By real time qPCR, DEX treatment induced a 6.2-fold increase (p < 0.04) in β3 integrin mRNA by day 2 compared to control and remained elevated for 6 days of treatment and then an additional 10 days once the DEX was removed. The increase in β3 integrin mRNA levels required only 1 day of DEX treatment to increase levels for 4 days in the absence of DEX. In contrast, DEX did not alter β1 integrin mRNA or protein levels. The DEX-induced upregulation of β3 integrin mRNA was partly due to an increase in its half-life to 60.7 h from 22.5 h in control cultures (p < 0.05) and could be inhibited by RU486 and cycloheximide, suggesting that DEX-induced de novo protein synthesis of an activation factor was needed. The calcineurin inhibitors cyclosporin A (CsA) and FK506 inhibited the DEX induced increase in β3 integrin mRNA. In summary, the DEX-induced increase in β3 integrin is a secondary glucocorticoid response that results in prolonged expression of αvβ3 integrin and the upregulation of the β3 integrin subunit through the calcineurin/NFAT pathway.     

Near-infrared fluorescent divalent RGD ligand for integrin α(v)β(3)-targeted optical imaging

A new near-infrared fluorescent compound containing two cyclic RGD motifs, cypate-[c(RGDfK)](2) (1), was synthesized based on a carbocyanine fluorophore bearing two carboxylic acid groups (cypate) for integrin α(v)β(3)-targeting. Compared with its monovalent counterpart cypate-c(RGDfK) (2), 1 exhibited remarkable improvements in integrin α(v)β(3) binding affinity and tumor uptake in nude mice of A549. The results suggest that cypate-linked divalent ligands can serve as an important molecular platform for exploring receptor-targeted optical imaging and treatment of various diseases.     

Synthesis and in vivo evaluation of Tc-99m-labeled cyclic CisoDGRC peptide conjugates for targeting αvβ3 integrin expression

Two α_vβ₃ integrin-binding peptide conjugates containing the cyclic CisoDGRC motif, a linker, and a chelator to enable Tc-99m labeling via the fac-[^99mTc(CO)₃]+ core were synthesized. In vivo biodistribution studies in U87MG tumor-bear nude mice at 1 h post-injection revealed a profound effect of the linker on the clearance of the radiotracer from the blood stream. In vivo blocking studies demonstrated the selective binding to the tumors expressing α_vβ₃-integrin and other tissues. The HPLC analysis of urine samples collected upon necropsy showed no degradation indicating their metabolic stability. These results suggest that cyclic CisoDGRC motif could be exploited as a new α_vβ₃-targeting vector by an appropriate selection of a linker between the peptide and the payload to obtain optimum pharmacokinetic properties.     

αv integrin processing interferes with the cross-talk between αvβ5/β6 and α2β1 integrins

Background information. Previous studies have reported that cross‐talk between integrins may be an important regulator of integrin—ligand binding and subsequent signalling events that control a variety of cell functions in many tissues. We previously demonstrated that αvβ5/β6 integrin represses α2β1‐dependent cell migration. The αv subunits undergo an endoproteolytic cleavage by protein convertases, whose role in tumoral invasion has remained controversial. Results. Inhibition of convertases by the convertase inhibitor α1‐PDX (α1‐antitrypsin Portland variant), leading to the cell‐surface expression of an uncleaved form of the αv integrin, stimulated cell migration toward type I collagen. Under convertase inhibition, α2β1 engagement led to enhanced phosphorylation of both FAK (focal adhesion kinase) and MAPK (mitogen‐activated protein kinase). This outside‐in signalling stimulation was associated with increased levels of activated β1 integrin located in larger than usual focal‐adhesion structures and a cell migration that was independent of the PI3K (phosphoinositide 3‐kinase)/Akt (also called protein kinase B) pathway. Conclusions. The increase in cell migration observed upon convertases inhibition appears to be due to the up‐regulation of β1 integrins and to their location in larger focal‐adhesion structures. The endoproteolytic cleavage of αv subunits is necessary for αvβ5/β6 integrin to control α2β1 function and could thus play an essential role in colon cancer cell migration.     

Novel inhibitors of the αvβ3 integrin—lead identification strategy

A novel approach to inhibition of the αvβ3 integrin is described, which uses compounds designed to generate nM potency without using the arginine binding site.     

Photoaffinity labeling the agonist binding domain of α4β4 and α4β2 neuronal nicotinic acetylcholine receptors with [I]epibatidine and 5[I]A-85380

The development of nicotinic acetylcholine receptor (nAChR) agonists, particularly those that discriminate between neuronal nAChR subtypes, holds promise as potential therapeutic agents for many neurological diseases and disorders. To this end, we photoaffinity labeled human α4β2 and rat α4β4 nAChRs affinity-purified from stably transfected HEK-293 cells, with the agonists [¹²⁵I]epibatidine and 5[¹²⁵I]A-85380. Our results show that both agonists photoincorporated into the β4 subunit with little or no labeling of the β2 and α4 subunits respectively. [¹²⁵I]epibatidine labeling in the β4 subunit was mapped to two overlapping proteolytic fragments that begin at β4V102 and contain Loop E (β4I109-P120) of the agonist binding site. We were unable to identify labeled amino acid(s) in Loop E by protein sequencing, but we were able to demonstrate that β4Q117 in Loop E is the principal site of [¹²⁵I]epibatidine labeling. This was accomplished by substituting residues in the β2 subunit with the β4 homologs and finding [¹²⁵I]epibatidine labeling in β4 and β2F119Q subunits with little, if any, labeling in α4, β2, or β2S113R subunits. Finally, functional studies established that the β2F119/β4Q117 position is an important determinant of the receptor subtype-selectivity of the agonist 5I-A-85380, affecting both binding affinity and channel activation.     

Expression of integrin subunits αv and β3 in acute lung inflammation

Integrin subunits v and 3 form a dimer, v3, which is expressed on normal neutrophils and endothelium. We investigated the expression of integrin subunits v and 3 in acute lung inflammation in Sprague-Dawley rats (n=5 each) following intratracheal challenge with Escherichia coli or Streptococcus pneumoniae, which induce neutrophil recruitment through different mechanisms. Control rats (n=5) were given endotoxin-free saline. Both bacterial challenges induced similar levels of recruitment of neutrophils in lungs. Western blots showed lower expression of integrin subunits v and 3 in lungs challenged with E. coli compared to those given S. pneumoniae. Immunohistochemistry and immunogold electron microscopy localized both integrin subunits in neutrophils and endothelium in the control and treated rat lungs. Quantitative immunohistochemistry showed that E. coli-challenged rat lungs contained a lower percentage of neutrophils expressing integrin subunits v and 3 compared to those challenged with S. pneumoniae (P<0.05). We conclude that E. coli infection decreased the percentage of neutrophils expressing integrin subunits v and 3 compared to S. pneumoniae infection. These data lay the foundation for further characterization of these integrin subunits in neutrophil migration specifically in S. pneumoniae infection that utilizes molecules other than 2 integrins for neutrophil recruitment.     

A novel series of [3.2.1] azabicyclic biaryl ethers as α3β4 and α6/4β4 nicotinic receptor agonists

We report the synthesis of a series of [3.2.1]azabicyclic biaryl ethers as selective agonists of α3- and α6-containing nicotinic receptors. In particular, compound 17a from this series is a potent α3β4 and α6/4β4 receptor agonist in terms of both binding and functional activity. Compound 17a also shows potent in vivo activity in CNS-mediated animal models that are sensitive to antipsychotic drugs. Compound 17a may thus be a useful tool for studying the role of α3β4 and α6/4β4 nicotinic receptors in CNS pharmacology.     

Identification of a negative allosteric site on human α4β2 and α3β4 neuronal nicotinic acetylcholine receptors

Acetylcholine-based neurotransmission is regulated by cationic, ligand-gated ion channels called nicotinic acetylcholine receptors (nAChRs). These receptors have been linked to numerous neurological diseases and disorders such as Alzheimer's disease, Parkinson's disease, and nicotine addiction. Recently, a class of compounds has been discovered that antagonize nAChR function in an allosteric fashion. Models of human α4β2 and α3β4 nicotinic acetylcholine receptor (nAChR) extracellular domains have been developed to computationally explore the binding of these compounds, including the dynamics and free energy changes associated with ligand binding. Through a blind docking study to multiple receptor conformations, the models were used to determine a putative binding mode for the negative allosteric modulators. This mode, in close proximity to the agonist binding site, is presented in addition to a hypothetical mode of antagonism that involves obstruction of C loop closure. Molecular dynamics simulations and MM-PBSA free energy of binding calculations were used as computational validation of the predicted binding mode, while functional assays on wild-type and mutated receptors provided experimental support. Based on the proposed binding mode, two residues on the β2 subunit were independently mutated to the corresponding residues found on the β4 subunit. The T58K mutation resulted in an eight-fold decrease in the potency of KAB-18, a compound that exhibits preferential antagonism for human α4β2 over α3β4 nAChRs, while the F118L mutation resulted in a loss of inhibitory activity for KAB-18 at concentrations up to 100 μM. These results demonstrate the selectivity of KAB-18 for human α4β2 nAChRs and validate the methods used for identifying the nAChR modulator binding site. Exploitation of this site may lead to the development of more potent and subtype-selective nAChR antagonists which may be used in the treatment of a number of neurological diseases and disorders.