Optimization of RGD-Containing Cyclic Peptides against ?v?3 Integrin.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC4747864 | biostudies-literature | 2016 Feb
REPOSITORIES: biostudies-literature
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Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin αvβ3/αvβ5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin αvβ3/αvβ5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin αvβ3/αvβ5 binding affinity followed a general trend: FITC-Galacto-RGD2 ∼ FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin αvβ3/αvβ5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin αvβ3/αvβ5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin αvβ3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin β3 antibody, their tumor localization and tumor cell binding are integrin αvβ3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin αvβ3/αvβ5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin β3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin αvβ3/αvβ5-targeted radiotracer) and the relative integrin αvβ3/αvβ5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin αvβ3/αvβ5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin αvβ3/αvβ5 expression levels in tumor tissues.
| S-EPMC4240344 | biostudies-literature
Project description:Integrin ligands containing the tripeptide sequences Arg-Gly-Asp (RGD) and iso-Asp-Gly- Arg (isoDGR) were actively investigated as inhibitors of tumor angiogenesis and directing unit in tumor-targeting drug conjugates. Reported herein is the synthesis, of two RGD and one isoDGR cyclic peptidomimetics containing (1S,2R) and (1R,2S) cis-2-amino-1-cyclopentanecarboxylic acid (cis-β-ACPC), using a mixed solid phase/solution phase synthetic protocol. The three ligands were examined in vitro in competitive binding assays to the purified αvβ3 and α5β1 receptors using biotinylated vitronectin (αvβ3) and fibronectin (α5β1) as natural displaced ligands. The IC50 values of the ligands ranged from nanomolar (the two RGD ligands) to micromolar (the isoDGR ligand) with a pronounced selectivity for αvβ3 over α5β1. In vitro cell adhesion assays were also performed using the human skin melanoma cell line WM115 (rich in integrin αvβ3). The two RGD ligands showed IC50 values in the same micromolar range as the reference compound (cyclo[RGDfV]), while for the isoDGR derivative an IC50 value could not be measured for the cell adhesion assay. A conformational analysis of the free RGD and isoDGR ligands by NMR (VT-NMR and NOESY experiments) and computational studies (MC/EM and MD), followed by docking simulations performed in the αVβ3 integrin active site, provided a rationale for the behavior of these ligands toward the receptor.
| S-EPMC7766232 | biostudies-literature
Project description:Cyclic RGD peptides are well-known ligands of integrins. The integrins αV β3 and α5 β1 are involved in angiogenesis, and integrin αV β3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on-resin intramolecular Suzuki-Miyaura cross-coupling (SMC) between l- or d-7-bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine-indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin αV β3 . The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range.
| S-EPMC7540173 | biostudies-literature
Project description:Integrin alpha(v)beta(3) plays a significant role in tumor angiogenesis and is a receptor for the extracellular matrix proteins with the exposed arginine-glycine-aspartic (RGD) tripeptide sequence. These include vitronectin, fibronectin, fibrinogen, lamin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles. Integrin alpha(v)beta(3) is expressed at low levels on epithelial cells and mature endothelial cells, but it is overexpressed on the activated endothelial cells of tumor neovasculature and some tumor cells. The restricted expression of integrin alpha(v)beta(3) during tumor growth, invasion, and metastasis presents an interesting molecular target for both early detection and treatment of rapidly growing solid tumors. Over the past decade, many radiolabeled linear and cyclic RGD peptide antagonists have been evaluated as integrin alpha(v)beta(3)-targeted radiotracers. Significant progress has been made on their use for imaging integrin alpha(v)beta(3)-positive tumors by SPECT or PET. Among the radiotracers evaluated in preclinical tumor-bearing models, [18F]Galacto-RGD (2-[18F]fluoropropanamide c(RGDfK(SAA); SAA = 7-amino-L-glyero-L-galacto-2,6-anhydro-7-deoxyheptanamide) and [18F]-AH111585 are currently under clinical investigation for visualization of integrin alpha(v)beta(3) expression in cancer patients. However, their low tumor uptake, high cost, and lack of preparative modules for routine radiosynthesis will limit their continued clinical application. Thus, there is a continuing need for more efficient integrin alpha(v)beta(3)-targeted radiotracers that are readily prepared from a kit formulation without further postlabeling purification. This article will focus on different approaches to maximize the targeting capability of cyclic RGD peptides and to improve the radiotracer excretion kinetics from noncancerous organs. Improvement of tumor uptake and tumor-to-background ratios is important for early detection of integrin alpha(v)beta(3)-positive tumors and/or noninvasive monitoring of therapeutic efficacy of antiangiogenic therapy.
| S-EPMC2795072 | biostudies-literature
Project description:αvβ3 integrins play an important role in angiogenesis and cell migration in cancer and are highly expressed on the activated endothelial cells of newly formed blood vessels. Here, we compare the targeting characteristics of 4 68Ga-labeled multimeric cyclic arginine-glycine-aspartate (RGD)-based tracers in an αvβ3 integrin-expressing tumor model and a tumor model in which αvβ3 integrin is expressed solely on the neovasculature. Methods: Female BALB/c nude mice were subcutaneously injected with SK-RC-52 (αvβ3 integrin-positive) or FaDu (αvβ3 integrin-negative) tumor cells. 68Ga-labeled DOTA-(RGD)2, TRAP-(RGD)3, FSC-(RGD)3, or THP-(RGD)3 was intravenously administered to the mice (0.5 nmol per mouse, 10-20 MBq), followed by small-animal PET/CT imaging and ex vivo biodistribution studies 1 h after injection. Nonspecific uptake of the tracers in both models was determined by coinjecting an excess of unlabeled DOTA-(RGD)2 (50 nmol) along with the radiolabeled tracers. Results: Imaging and biodistribution data showed specific uptake in the tumors for each tracer in both models. Tumor uptake of 68Ga-FSC-(RGD)3 was significantly higher than that of 68Ga-DOTA-(RGD)2, 68Ga-TRAP-(RGD)3, or 68Ga-THP-(RGD)3 in the SK-RC-52 model but not in the FaDu model, in which 68Ga-FSC-(RGD)3 showed significantly higher tumor uptake than 68Ga-TRAP-(RGD)3 Most importantly, differences were also observed in normal tissues and in tumor-to-blood ratios. Conclusion: All tracers showed sufficient targeting of αvβ3 integrin expression to allow for tumor detection. Although the highest tumor uptake was found for 68Ga-FSC-(RGD)3 and 68Ga-THP-(RGD)3 in the SK-RC-52 and FaDu models, respectively, selection of the optimal tracer for specific diagnostic applications also depends on tumor-to-blood ratio and uptake in normal tissues; these factors should therefore also be considered.
| S-EPMC6175042 | biostudies-literature
Project description:RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.
| S-EPMC7388056 | biostudies-literature
Project description:The main objective of this study is to explore the impact of cyclic RGD peptides and (99m)Tc chelates on biological properties of (99m)Tc radiotracers. Cyclic RGD peptide conjugates, HYNIC-K(NIC)-RGD(2) (HYNIC = 6-hydrazinonicotinyl; RGD(2) = E[c(RGDfK)](2) and NIC = nicotinyl), HYNIC-K(NIC)-3G-RGD(2) (3G-RGD(2) = Gly-Gly-Gly-E[Gly-Gly-Gly-c(RGDfK)](2)), and HYNIC-K(NIC)-3P-RGD(2) (3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2)), were prepared. Macrocyclic (99m)Tc complexes [(99m)Tc(HYNIC-K(NIC)-RGD(2))(tricine)] (1), [(99m)Tc(HYNIC-K(NIC)-3G-RGD(2))(tricine)] (2), and [(99m)Tc(HYNIC-K(NIC)-3P-RGD(2))(tricine)] (3) were evaluated for their biodistribution and tumor-targeting capability in athymic nude mice bearing MDA-MB-435 human breast tumor xenografts. It was found that 1, 2, and 3 could be prepared with high specific activity (?111 GBq/?mol). All three (99m)Tc radiotracers have two major isomers, which show almost identical uptake in tumors and normal organs. Replacing the bulky and highly charged [(99m)Tc(HYNIC)(tricine)(TPPTS)] (TPPTS = trisodium triphenylphosphine-3,3',3?-trisulfonate) with a smaller [(99m)Tc(HYNIC-K(NIC))(tricine)] resulted in less uptake in the kidneys and lungs for 3. Surprisingly, all three (99m)Tc radiotracers shared a similar tumor uptake (1, 5.73 ± 0.40%ID/g; 2, 5.24 ± 1.09%ID/g; and 3, 4.94 ± 1.71%ID/g) at 60 min p.i. The metabolic stability of (99m)Tc radiotracers depends on cyclic RGD peptides (3P-RGD(2) > 3G-RGD(2) ? RGD(2)) and (99m)Tc chelates ([(99m)Tc(HYNIC)(tricine)(TPPTS)] > [(99m)Tc(HYNIC-K(NIC))(tricine)]). Immunohistochemical studies revealed a linear relationship between the ?(v)?(3) expression levels and tumor uptake or tumor/muscle ratios of 3, suggesting that 3 is useful for monitoring the tumor ?(v)?(3) expression. Complex 3 is a very attractive radiotracer for detection of integrin ?(v)?(3)-positive tumors.
| S-EPMC3310279 | biostudies-literature
Project description:BACKGROUND:Integrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvβ3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvβ3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvβ3/VEGFR2 cross-talk and its downstream pathways. METHODS:Human umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvβ3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy. RESULTS:DisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvβ3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvβ3 integrin and VEGFR2 in treated cells. CONCLUSIONS:Disintegrin inhibition of αvβ3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.
| S-EPMC6425665 | biostudies-literature
Project description:Of the various WD40 family proteins, WDR5 is a particularly important multifunctional adaptor protein that can bind to several protein complexes to regulate gene activation, so it was considered as a promising epigenetic target in anti-cancer drug development. Despite many inhibitors having been discovered directing against the arginine-binding cavity in WDR5 called the WIN site, the side hydrophobic cavity called the WBM site receives rather scant attention. Herein, we aim to obtain novel WBM-targeted peptidic inhibitors with high potency and selectivity. We employed two improved biopanning approaches with a disulfide-constrained cyclic peptide phage library containing 7 randomized residues and identified several peptides with micromole binding activity by docking and binding assay. To further optimize the stability and activity, 9 thiol-reactive chemical linkers were utilized in the cyclization of the candidate peptide DH226027, which had good binding affinity. This study provides an effective method to discover potent peptides targeting protein-protein interactions and highlights a broader perspective of peptide-mimic drugs.
| S-EPMC10583817 | biostudies-literature
Project description:The ?v?3 integrin, a receptor for many extracellular matrix proteins with RGD-sequence motif, is involved in multiple physiological processes and highly expressed in tumor cells, therefore making it a target for cancer therapy and tumor imaging. Several RGD-containing cyclic octapeptide (named LXW analogs) were screened as ?v?3 antagonists with dramatically different binding affinity, and their structure-activity relationship (SAR) remains elusive. We performed systematic SAR studies and optimized LXW analogs to improve antagonistic potency. The NMR structure of LXW64 was determined and docked to the integrin. Structural comparison and docking studies suggested that the hydrophobicity and aromaticity of the X7 amino acid are highly important for LXW analogs binding to the integrin, a potential hydrophobic pocket on the integrin surface was proposed to play a role in stabilizing the peptide binding. To develop a cost-efficient and fast screening method, computational docking was performed on LXW analogs and compared with in vitro screening. A consistency within the results of both methods was found, leading to the continuous optimization and testing of LXW mutants via in silico screening. Several new LXW analogs were predicted as the integrin antagonists, one of which-LXZ2-was validated by in vitro examination. Our study provides new insight into the RGD recognition specificity and valuable clues for rational design of novel ?v?3 antagonists.
| S-EPMC7246635 | biostudies-literature