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Nanomaterials (NMs) inevitably adsorb proteins in blood and form "protein corona" upon intravenous administration as drug carriers, potentially changing the biological properties and intended functions. Inspired by anti-adhesion properties of natural proteins, herein, we employed the one-bead one-compound (OBOC) combinatorial peptide library method to screen anti-adhesion peptides (AAPs) against proteins. The library beads displaying random peptides were screened with three fluorescent-labeled plasma proteins. The nonfluorescence beads, presumed to have anti-adhesion property against the proteins, were isolated for sequence determination. These identified AAPs were coated on gold nanorods (GNRs), enabling significant extension of the blood circulating half-life of these GNRs in mice to 37.8 h, much longer than that (26.6 h) of PEG-coated GNRs. In addition, such AAP coating was found to alter the biodistribution profile of GNRs in mice. The bioinspired screening strategy and resulting peptides show great potential for enhancing the delivery efficiency and targeting ability of NMs.

Amiloride and its derivatives have long attracted attention as potential anticancer therapeutic agents. Several original studies characterized amilorides as inhibitors of sodium-proton antiporter tumor growth and urokinase plasminogen activator-mediated metastasis. However, more recent observations indicate that amiloride derivatives are specifically cytotoxic toward tumor cells relative to normal cells and have the capacity to target tumor cell populations resistant to currently-employed therapies. A major barrier to clinical translation of the amilorides is their modest cytotoxic potency, with EC50 values in the high micromolar to low millimolar range. Here we report structure-activity relationship observations that underscore the importance of the guanidinium group and the presence of lipophilic substituents at the C(5) position of the amiloride pharmacophore in promoting cytotoxicity. Moreover, we demonstrate that our most potent derivative called LLC1 is cytotoxic toward mouse mammary tumor organoids and drug-resistant populations of various breast cancer cell lines, and induces lysosomal membrane permeabilization as a prelude to lysosome-dependent cell death. Our observations offer a roadmap for the future development of amiloride-based cationic amphiphilic drugs that engage the lysosome to specifically kill breast tumor cells.

This work describes the discovery of a bead-bound membrane-active peptide (MAP), LBF127, that selectively binds fungal giant unilamellar vesicles (GUVs) over mammalian GUVs. LBF127 was re-synthesized in solution form and demonstrated to have antifungal activity with limited hemolytic activity and cytotoxicity against mammalian cells. Through systematic structure-activity relationship studies, including N- and C-terminal truncation, alanine-walk, and d-amino acid substitution, an optimized peptide, K-oLBF127, with higher potency, less hemolytic activity, and cytotoxicity emerged. Compared to the parent peptide, K-oLBF127 is shorter by three amino acids and has a lysine at the N-terminus to confer an additional positive charge. K-oLBF127 was found to have improved selectivity toward the fungal membrane over mammalian membranes by 2-fold compared to LBF127. Further characterizations revealed that, while K-oLBF127 exhibits a spectrum of antifungal activity similar to that of the original peptide, it has lower hemolytic activity and cytotoxicity against mammalian cells. Mice infected with

The methods of OB2C library and LLS compounds synthesis

Purpose: The majority of patients with prostate cancer who are treated with androgen-deprivation therapy (ADT) will eventually develop fatal metastatic castration-resistant prostate cancer (mCRPC). Currently, there are no effective durable therapies for patients with mCRPC. High expression of galectin-1 (Gal-1) is associated with prostate cancer progression and poor clinical outcome. The role of Gal-1 in tumor progression is largely unknown. Here, we characterized Gal-1 functions and evaluated the therapeutic effects of a newly developed Gal-1 inhibitor, LLS30, in mCRPC.Experimental Design: Cell viability, colony formation, migration, and invasion assays were performed to examine the effects of inhibition of Gal-1 in CRPC cells. We used two human CRPC xenograft models to assess growth-inhibitory effects of LLS30. Genome-wide gene expression analysis was conducted to elucidate the effects of LLS30 on metastatic PC3 cells.Results: Gal-1 was highly expressed in CRPC cells, but not in androgen-sensitive cells. Gal-1 knockdown significantly inhibited CRPC cells' growth, anchorage-independent growth, migration, and invasion through the suppression of androgen receptor (AR) and Akt signaling. LLS30 targets Gal-1 as an allosteric inhibitor and decreases Gal-1–binding affinity to its binding partners. LLS30 showed in vivo efficacy in both AR-positive and AR-negative xenograft models. LLS30 not only can potentiate the antitumor effect of docetaxel to cause complete regression of tumors, but can also effectively inhibit the invasion and metastasis of prostate cancer cells in vivo.Conclusions: Our study provides evidence that Gal-1 is an important target for mCRPC therapy, and LLS30 is a promising small-molecule compound that can potentially overcome mCRPC. Clin Cancer Res; 24(17); 4319–31. ©2018 AACR.

Increasing literature suggests that cell adhesion molecule α4β1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of α4β1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies. Due to the lack of high-affinity targeting ligands, molecular imaging of α4β1 integrin is much less explored than that of αvβ3 and αvβ5 integrins. We have recently reported using the one bead–one compound combinatorial library method to identify a peptidomimetic, LLP2A, that preferentially binds to activated α4β1 integrin. Here, we described the use of LLP2A-Cy5.5 conjugate as an in vivo optical imaging probe in a human lymphoma xenograft model. This univalent LLP2A-Cy5.5 conjugate retained the binding activity and specificity to α4β1 integrin as shown by cell binding assays using α4β1-positive Molt-4 T-leukemia cells. The subcutaneous Molt-4 tumor was clearly visualized from 1 to 24 h after tail vein injection of the conjugate. Direct imaging and confocal microscopic examination of excised tumors and organs confirmed the accumulation of LLP2A in tumors and revealed very little or no uptake in normal organs except for lymph nodes. Kidney uptake was high when the whole organ was scanned but it was negative when examined microscopically, suggesting that LLP2A bound to the renal tubules loosely. Tumor uptake of LLP2A-Cy5.5 conjugate was blocked by excess unlabeled LLP2A. This study showed that the combinatorial chemical library-derived peptidomimetic LLP2A can be easily developed into an optical imaging probe for noninvasively monitoring of activated α4β1 integrin in vivo. [Mol Cancer Ther 2008;7(2):432–7]

Supplementary tables

Supplementary Methods from In vivo optical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integrin

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68 ± 0.08 μmol/L) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. Mol Cancer Ther; 9(10); 2714–23. ©2010 AACR.

Supplementary materials and methods

Supplementary Data from The Use of One-Bead One-Compound Combinatorial Library Technology to Discover High-Affinity αvβ3 Integrin and Cancer Targeting Arginine-Glycine-Aspartic Acid Ligands with a Built-in Handle

Supplementary Figures

Diabetic ischemic wound treatment remains a critical clinical challenge. Neovascularization plays a significant role in wound healing during all stages of the tissue repair process. Strategies that enhance angiogenesis and neovascularization and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic conditions. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates vascular endothelial growth factor (VEGF) receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a multi-functional pro-angiogenic molecule by grafting LXW7 and collagen-binding peptides (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY, and further employed this multi-functional molecule to functionalize collagen-based extracellular matrix (ECM) scaffolds. We confirmed that LXW7-DS-SILY modification significantly promoted EPC attachment and growth on the ECM scaffolds in vitro and supported EPC survival in vivo in the ischemic environment. When applied in an established Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model, LXW7-DS-SILY-functionalized ECM scaffolds loaded with EPCs significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients., Graphical abstract Schematics for the fabrication of ligand modified scaffolds.Image 1, Highlights • Diabetic foot ulcers represent a significant healthcare burden. • Neovascularization plays a significant role in all stages of the wound healing process. • Endothelial progenitor cells (EPCs) have the potential to facilitate neovascularization. • LXW7-DS-SILY promoted EPC attachment and growth on ECM scaffolds in vitro. • LXW7-DS-SILY improved neovascularization and wound healing in ZDF rat ischemic model.

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It plays a critical role in the native transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a noncovalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). Site-specific bioconjugation strategies for HSA only would greatly expand its role as the biocompatible, non-toxic platform for theranostics purposes. Using the enabling one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets, and an acrylamide. We then used HSA as a probe to screen the OBOC myristylated peptide libraries for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with a high yield within 1 h at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional lysine labeling strategy with N-hydroxysuccinimide ester. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs the promising leads to further develop in vitro and in vivo HSA bioconjugation strategies for numerous biomedical applications.

Gamete fusion is a critical event of mammalian fertilization. A random one-bead one-compound combinatorial peptide library represented synthetic human egg mimics and identified a previously unidentified ligand as Fc receptor–like 3, named MAIA after the mythological goddess intertwined with JUNO. This immunoglobulin super family receptor was expressed on humanoolemmaand played a major role during sperm-egg adhesion and fusion. MAIA forms a highly stable interaction with the known IZUMO1/JUNO sperm-egg complex, permitting specific gamete fusion. The complexity of the MAIA isotype may offer a cryptic sexual selection mechanism to avoid genetic incompatibility and achieve favorable fitness outcomes.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective infiltration of T effector (Teff) cells to tumors and the immunosuppressive tumor microenvironment (TME). Here, we report a programmable tumor cells/Teff cells bispecific nano-immunoengager (NIE) that can circumvent these limitations to improve ICB therapy. The peptidic nanoparticles (NIE-NPs) bind tumor cell surface α3β1 integrin and undergo in situ transformation into nanofibrillar network nanofibers (NIE-NFs). The prolonged retained nanofibrillar network at the TME captures Teff cells via the activatable α4β1 integrin ligand and allows sustained release of resiquimod for immunomodulation. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

Immune checkpoint blockade (ICB) therapy has revolutionized clinical oncology. However, the efficacy of ICB therapy is limited by the ineffective homing of T effector (Teff) cells to tumours and the immunosuppressive tumour microenvironment (TME). Here, we report a programmable tumour cells/Teff cells bispecific nano-immuno-engager (NIE) that can circumvent these limitations to improve ICB therapy. We have developed 28 nm non-toxic peptidic micellar nanoparticles (NIE-NPs) that bind α3β1 integrin on tumour cells membrane and undergo in situ transformation on surface of tumour cells into nanofibrillar network (NIE-NFs). The nanofibrillar network persistently facilitates cytotoxic T cells’ homing to the proximity of tumour cells via activatable α4β1 integrin ligands, and also allows sustained release of resiquimod to reprogram the TME. This bispecific NIE eliminates syngeneic 4T1 breast cancer and Lewis lung cancer models in mice, when given together with anti-PD-1 antibody. The in vivo structural transformation-based supramolecular bispecific NIE represents an innovative class of programmable receptor-mediated targeted immunotherapeutics to greatly enhance ICB therapy against cancers.

This study investigated the feasibility and efficiency of neuron-targeting hybrid placental mesenchymal stromal cell-derived extracellular vesicles (PMSC-EVs), engineered by membrane fusion with Targeted Axonal Import (TAxI) peptide modified, TrkB agonist 7,8-DHF-loaded liposomes for treatment of myelomeningocele (MMC) via intra-amniotic cavity administration. The prepared TAxI modified liposomes with 7,8-DHF were used to fuse with PMSC-EVs. Different fusion approaches were investigated and freeze-thaw-extrude method was found to be the optimal. The engineered PMSC-EVs had a uniform particle size and efficiently loaded 7,8-DHF. It also had typical markers of native EVs. Freeze-thaw-extrude process did not change the release profile of 7,8-DHF from engineered EVs compared to TAxI modified, 7,8-DHF loaded liposomes. The engineered EVs could elicit TrkB phosphorylation depending on the incorporation of 7,8-DHF while native EVs did not. The engineered EVs increased neurite outgrowth of apoptotic cortical neurons induced by staurosporine, suggesting that they exhibited neuroprotective function. In a rodent model of MMC, neuron-targeting, engineered EVs became an active targeting delivery system to MMC defect sites. Pups treated with engineered EVs had the lowest density of apoptotic cells and displayed a therapeutic outcome. The study suggests the potential use of engineered hybrid, active neuron-targeting EVs for the in utero treatment of MMC.

Self-assembling peptides have shown tremendous potential in the fields of material sciences, nanoscience, and medicine. Because of the vast combinatorial space of even short peptides, identification of self-assembling sequences remains a challenge. Herein, we develop an experimental method to rapidly screen a huge array of peptide sequences for self-assembling property, using the one-bead one-compound (OBOC) combinatorial library method. In this approach, peptides on beads are N-terminally capped with nitro-1,2,3-benzoxadiazole, a hydrophobicity-sensitive fluorescence molecule. Beads displaying self-assembling peptides would fluoresce under aqueous environment. Using this approach, we identify eight pentapeptides, all of which are able to self-assemble into nanoparticles or nanofibers. Some of them are able to interact with and are taken up efficiently by HeLa cells. Intracellular distribution varied among these non-toxic peptidic nanoparticles. This simple screening strategy has enabled rapid identification of self-assembling peptides suitable for the development of nanostructures for various biomedical and material applications., Self-assembling peptides have a range of potential applications but developing self-assembling sequences can be challenging. Here, the authors report on a one-bead one-compound combinatorial library where fluorescence is used to detect the potential for self-assembly and identified candidates are evaluated.

A maytansin derivative, DM1, is a promising therapeutic compound for treating tumors, but is also a highly poisonous substance with various side effects. For clinical expansion, we tried to develop novel peptide–drug conjugates (PDCs) with DM1. In the study, a one-bead one-compound (OBOC) platform was used to screen and identify a novel, highly stable, non-natural amino acid peptide targeting the tyrosine receptor FGFR2. Then, the identified peptide, named LLC2B, was conjugated with the cytotoxin DM1. Our results show that LLC2B has high affinity for the FGFR2 protein according to an isothermal titration calorimetry (ITC) test. LLC2B-Cy5.5 binding to FGFR2-positive cancer cells was confirmed by fluorescent microscopic imaging and flow cytometry in vitro. Using xenografted nude mouse models established with breast cancer MCF-7 cells and esophageal squamous cell carcinoma KYSE180 cells, respectively, LLC2B-Cy5.5 was observed to specifically target tumor tissues 24 h after tail vein injection. Incubation assays, both in aqueous solution at room temperature and in human plasma at 37 °C, suggested that LLC2B has high stability and strong anti-proteolytic ability. Then, we used two different linkers, one of molecular disulfide bonds and another of a maleimide group, to couple LLC2B to the toxin DM1. The novel peptide–drug conjugates (PDCs) inhibited tumor growth and significantly increased the maximum tolerated dose of DM1 in xenografted mice. In brief, our results suggest that LLC2B–DM1 can be developed into a potential PDC for tumor treatment in the future.

Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2650.

Currently, no morphological characteristics have been described in clinical specimens to delineate an androgen receptor (AR)-active from an AR-inactive phenotype in AR-expressing aggressive variant prostate cancer (AVPC). 36 localized and 51 metastatic prostate cancer (CaP) samples were analyzed - 19 (37.3%) of the metastases were in the bone, 20 (39.2%) were in the lung, while the rest were in other areas such as brain, lymph node, bladder, etc). Expression level of Gal-1 was graded individually in the nucleus and the cytoplasm of both primary and metastatic tissues. Very low Gal-1 expression was observed in epithelial cells of benign prostate, while stromal staining was strong. Localized prostate tumors obtained from prostatectomy samples showed strong gal-1 expression in the epithelial cells, but remarkably the Gal-1 staining was confined to the cytoplasm. In contrast, in metastatic tumors many nuclei stained for Gal-1. Among localized tumors, Kaplan-Meier curves showed a significant increase in metastasis formation (p=0.022) and decreased overall survival (p=0.015) among those expressing nuclear Gal-1 (n=16) compared to those with no nuclear Gal-1 (n=20). Whereas cytoplasmic Gal-1 negatively correlated with patient age (R=-0.51, p=0.008) and PSA (R=-0.41, p=0.006), and positively correlated with Ki67 (R=0.35, p=0.014), the expression of nuclear Gal-1 correlated strongly with the expression of NE markers including chromogranin A (R=0.32, p=0.031). Significantly, nuclear Gal-1 was expressed in AVPC models but not in those derived from localized tissues. Significantly, androgen deprivation, including treatment with AR antagonists, resulted in nuclear localization of Gal-1. We adapted the powerful one-bead-two-compound (OB2C) combinatorial library method for the discovery of synthetic small molecule death ligands against cancer cells in an ultra-high throughput fashion. One of these benzimidazole-based ligands, LLS80, was active against tumor cells at low micromolar IC50. Pull-down assay followed by LC MS/MS indicated that galectin-1 is the target protein of LLS80, which induced apoptosis preferentially in AVPC cells expressing high level of Gal-1. LLS80 has all the indicators of being a good therapeutic agent: (i) LLS80 has a low in vitro IC50 in AVPC cells, (ii) it shows significant in vivo anti-tumor efficacy using xenograft models, (iii) it is a novel small molecule that can penetrate AVPC cells, (iv) it can synergize with enzalutamide in vivo and in vitro, (v) it inhibits a known therapeutic target, Gal-1, and (vi) low toxicity. Treatment with LLS80 (2 µM) prevented AR antagonist-induced Gal-1 nuclear localization, and also downregulated NE markers in AVPC models of CaP progression. We conclude that nuclear Gal-1 is a novel marker of AVPC. Citation Format: Maria Malvina Tsamouri, Salma Siddiqui, Blythe P. Durbin-Johnson, Kit S. Lam, Ruiwu Liu, Paramita M. Ghosh. New targets in prostate cancer: Nuclear localization of galectin-1 in androgen receptor indifferent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 818.

Diabetic ischemic wound treatment remains a critical clinical challenge. Strategies that enhance angiogenesis and improve ischemic pathology may promote the healing of poor wounds, particularly diabetic wounds in highly ischemic condition. We previously identified a cyclic peptide LXW7 that specifically binds to integrin αvβ3 on endothelial progenitor cells (EPCs) and endothelial cells (ECs), activates VEGF receptors, and promotes EC growth and maturation. In this study, we designed and synthesized a pro-angiogenic molecule LXW7-DS-SILY by conjugating LXW7 to a collagen-binding proteoglycan mimetic DS-SILY and further employed this novel bifunctional ligand to functionalize extracellular matrix (ECM) scaffolds, promote neovascularization and accelerate ischemic wound healing. We established a Zucker Diabetic Fatty (ZDF) rat ischemic skin flap model and found the wounds treated by LXW7-DS-SILY-functionalized ECM scaffolds, with or without EPCs, significantly improved wound healing, enhanced neovascularization and modulated collagen fibrillogenesis. These functionalized ECM scaffolds also significantly promoted EPC attachment, growth and survival in the ischemic environment. Altogether, this study provides a promising novel treatment to accelerate diabetic ischemic wound healing, thereby reducing limb amputation and mortality of diabetic patients.

In malignancies, fibroblast growth factor receptors (FGFRs) signaling is reinforced through overexpression of fibroblast growth factors (FGFs) or their receptors. FGFR2 has been proposed as a target for cancer therapy, because both the expression and activation of FGFR2 are boosted in various malignant carcinomas. Although several chemicals have been designed against FGFR2, they did not exhibit enough specificity and might bring potential accumulated toxicity. In this study, we developed an epitope peptide (P5) and its cyclic derivative (DcP5) based on the structure of FGF2 to limit the activation of FGFR2. The anticancer activities of P5 and DcP5 were examined in vitro and in vivo. Our results demonstrated that P5 significantly inhibited the cell proliferation in FGFR2‐dependent manner in DU145 cells and retarded tumor growth in DU145 xenograft model with negligible toxicity toward normal organs. Further investigations found that the Gln4 and Glu6 residues of P5 bind to FGFR2 to abolish its activation. Moreover, we developed the P5 cyclic derivative, DcP5, which achieved reinforced stability and anticancer activity in vivo. Our findings suggest P5 and its cyclic derivative DcP5 as potential candidates for anticancer therapy., In various malignancies, fibroblast growth factor receptors (FGFRs) signaling is reinforced, resulting in tumor progression and poor prognosis. Herein, we developed an FGFR2 competitive inhibitory peptide (P5) to limit the overactivation of FGFR2 signaling. Moreover, this P5 peptide was upgraded to DcP5 through cyclization to achieve improved in vivo stability and FGFR2‐binding affinity.

Self-assembling peptides have shown tremendous potential in the fields of material sciences, nanoscience, and medicine. Because of the vast combinatorial space of even short peptides, identification of self-assembling sequences remains a challenge. Herein, we develop an experimental method to rapidly screen a huge array of peptide sequences for self-assembling property, using the one-bead one-compound (OBOC) combinatorial library method. In this approach, peptides on beads are N-terminally capped with nitro-1,2,3-benzoxadiazole, a hydrophobicity-sensitive fluorescence molecule. Beads displaying self-assembling peptides would fluoresce under aqueous environment. Using this approach, we identified eight pentapeptides, all of which were able to self-assemble into nanoparticles or nanofibers. Some of them were able to interact with and taken up efficiently by HeLa cells. Intracellular distribution varied among these non-toxic peptidic nanoparticles. This novel but very simple screening strategy, has enabled rapid identification of self-assembling peptides suitable for the development of nanostructures for various biomedical and material applications.

Background Currently, there are no effective medications to reverse periodontal disease (PD)-induced bone loss. The objective of this study was to test a new anabolic compound, LLP2A-Ale, or with the combination treatment of mesenchymal stromal cell (MSC), in the treatment of bone loss secondary to PD. Methods PD was induced in mice by placing a ligature around the second right molar. At one week after disease induction, the mice were treated with placebo, LLP2A-Ale, MSCs, or combination of LLP2A-Ale + MSCs, and euthanized at week 4. Results We found that PD induced alveolar bone loss that was associated with reduced bone formation. LLP2A-Ale alone or in combination with MSCs sustained alveolar bone formation and reversed alveolar bone loss. Additionally, PD alone caused systemic inflammation and increased the circulating levels of G-CSF, IP-10, MIP-1a, and MIP2, which were suppressed by LLP2A-Ale +/− MSCs. LLP2A-Ale +/− MSCs increased bone formation at the peripheral skeletal site (distal femur), which was otherwise suppressed by PD. Conclusion Our findings indicated that LLP2A-Ale treatment rescued alveolar bone loss caused by PD, primarily by increasing bone formation. LLP2A-Ale also attenuated the circulating levels of a series of inflammatory cytokines and reversed the PD-induced suppression of systemic bone formation.

Activation and migration of endogenous mesenchymal stromal cells (MSCs) are critical for bone regeneration. Here, we report a combinational peptide screening strategy for rapid discovery of ligands that not only bind strongly to osteogenic progenitor cells (OPCs) but also stimulate osteogenic cell Akt signaling in those OPCs. Two lead compounds are discovered, YLL3 and YLL8, both of which increase osteoprogenitor osteogenic differentiation in vitro. When given to normal or osteopenic mice, the compounds increase mineral apposition rate, bone formation, bone mass, and bone strength, as well as expedite fracture repair through stimulated endogenous osteogenesis. When covalently conjugated to alendronate, YLLs acquire an additional function resulting in a “tri-functional” compound that: (i) binds to OPCs, (ii) targets bone, and (iii) induces “pro-survival” signal. These bone-targeted, osteogenic peptides are well suited for current tissue-specific therapeutic paradigms to augment the endogenous osteogenic cells for bone regeneration and the treatment of bone loss., Activation of osteogenic cells is essential for bone regeneration. Here, the authors screen a peptide library and identify 2 compounds that promote osteogenic progenitor cell differentiation in vitro, and show that they increase bone formation and fracture repair in mice.

Establishing and maintaining a healthy endothelium on vascular and intravascular devices is crucial for the prevention of thrombosis and stenosis. Generating a biofunctional surface on vascular devices to recruit endothelial progenitor cells (EPCs) and endothelial cells (ECs) has proven efficient in promoting in situ endothelialization. However, molecules conventionally used for EPC/EC capturing generally lack structural stability, capturing specificity, and biological functionalities, which have limited their applications. Discovery of effective, specific, and structurally stable EPC/EC capturing ligands is desperately needed. Using the high-throughput One-Bead One-Compound combinatorial library screening technology, we recently identified a disulfide cyclic octa-peptide LXW7 (cGRGDdvc), which possesses strong binding affinity and functionality to EPCs/ECs, weak binding to platelets, and no binding to inflammatory cells. Because LXW7 is cyclic and 4 out of the 8 amino acids are unnatural D-amino acids, LXW7 is highly proteolytically stable. In this study, we applied LXW7 to modify small diameter vascular grafts using a Click chemistry approach. In vitro studies demonstrated that LXW7-modified grafts significantly improved EPC attachment, proliferation and endothelial differentiation and suppressed platelet attachment. In a rat carotid artery bypass model, LXW7 modification of the small diameter vascular grafts significantly promoted EPC/EC recruitment and rapidly achieved endothelialization. At 6 weeks after implantation, LXW7-modified grafts retained a high patency of 83%, while the untreated grafts had a low patency of 17%. Our results demonstrate that LXW7 is a potent EPC/EC capturing and platelet suppressing ligand and LXW7-modified vascular grafts rapidly generate a healthy and stable endothelial interface between the graft surface and the circulation to reduce thrombosis and improve patency. STATEMENT OF SIGNIFICANCE: In this study, One-Bead One-Compound (OBOC) technology has been applied for the first time in discovering bioactive ligands for tissue regeneration applications. Current molecules used to modify artificial vascular grafts generally lack EPC/EC capturing specificity, biological functionalities and structural stability. Using OBOC technology, we identified LXW7, a constitutionally stable disulfide cyclic octa-peptide with strong binding affinity and biological functionality to EPCs/ECs, very weak binding to platelets and no binding to inflammatory cells. These characteristics are crucial for promoting rapid endothelialization to prevent thrombosis and improve patency of vascular grafts. LXW7 coating technology could be applied to a wide range of vascular and intravascular devices, including grafts, stents, cardiac valves, and catheters, where a "living" endothelium and healthy blood interface are needed.

Cell–biomaterial interactions are primarily governed by cell adhesion, which arises from the binding of cellular integrins to the extracellular matrix (ECM). Integrins drive the assembly of focal contacts that serve as mechanotransducers and signaling nexuses for stem cells, for example integrin α4β1 plays pivotal roles in regulating mesenchymal stem cell (MSC) homing, adhesion, migration and differentiation. The strategy to control the integrin-mediated cell adhesion to bioinspired, ECM-mimicking materials is essential to regulate cell functions and tissue regeneration. Previously, using one-bead one-compound (OBOC) combinatorial technology, we discovered that LLP2A was a high-affinity peptidomimetic ligand (IC50 = 2 pM) against integrin α4β1. In this study, we identified that LLP2A had a strong binding to human early gestation chorionic villi-derived MSCs (CV-MSCs) via integrin α4β1. To improve CV-MSC seeding, expansion and delivery for regenerative applications, we constructed artificial scaffolds simulating the structure of the native ECM by immobilizing LLP2A onto the scaffold surface as cell adhesion sites. LLP2A modification significantly enhanced CV-MSC adhesion, spreading and viability on the polymeric scaffolds via regulating signaling pathways including phosphorylation of focal adhesion kinase (FAK), and AKT, NF-kB and Caspase 9. In addition, we also demonstrated that LLP2A had strong binding to MSCs of other sources, such as bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs). Therefore, LLP2A and its derivatives not only hold great promise for improving CV-MSC-mediated treatment of fetal diseases, but they can also be widely applied to functionalize various biological and medical materials, which are in need of MSC recruitment, enrichment and survival, for regenerative medicine applications.

Membrane active peptides (MAPs) have gained wide interest due to their far reaching applications in drug discovery and drug delivery. The search for new MAPs, however, has been largely skewed with bias selecting for physicochemical parameters believed to be important for membrane activity, such as alpha helicity, cationicity and hydrophobicity. Here we carry out a search-and-find strategy to screen a 100,000-membered one-bead-one-compound (OBOC) combinatorial peptide library for lead compounds, agnostic of those physicochemical constraints. Such a synthetic strategy also permits expansion of our peptide repertoire to include unnatural amino acids. Using this approach, we discovered a structurally unique lead peptide LBF14, a linear 14-mer peptide, that induces gross morphological disruption of membranes, irrespective of membrane composition. Further, we demonstrate that the unique insertion mechanism of the peptide, visualized by spinning disc confocal microscopy and further analyzed by electron paramagnetic resonance measurements, may be the cause of this large scale membrane deformation. We also demonstrate the robustness, reproducibility, and potential application of this technique to discover and characterize new membrane active peptides that display activity by local insertion and subsequent allosteric effects leading to global membrane disruption.

Aim: Targeted diagnosis and therapy for colorectal cancer (CRC) is limited by the lack of specific biomarkers. Our aim was to discover CRC-specific targeting ligands using a one-bead one-compound (OBOC) combinatorial library. Method: Samples of OBOC peptide libraries were color coded, mixed and incubated with commercially available human CRC cells (HT-29 and DLD-1). Libraries with compound beads that bound to CRC cells were selected for further screening. Compound beads that bound to both CRC cells were screened with human colonic epithelial cells to select beads that bound only to CRC cells but not to human colonic epithelial cells. Chemical structures of the positive peptides were determined by Edman chemistry. CRC-targeted imaging agents were developed by conjugation of CRC binding peptide with biotin through a hydrophilic linker and then complexed with streptavidin–Cy5.5. Immunohistochemistry studies were used to evaluate CRC detection efficacy. Targeting specificity was further tested with subcutaneous CRC xenografts in nude mice. Results: Two cyclic peptides, CRC-6 and CRC-9, composed of natural and unnatural amino acids, bind specifically to CRC cells with moderately high affinity and specificity. CRC-9 is able to detect CRC cells grown on chamber slides at the concentration of 1 µM after 30 min incubation. Tail vein injection of 1.8 nmol biotinylated peptide CRC-9, complexed with streptavidin–Cy5.5 (SA–Cy5.5), is able to target the subcutaneous CRC xenograft implants in nude mice. None of the two peptides showed cytotoxic effect on human blood cells, up to the concentration of 500 µM. Conclusion: CRC-9 has the potential to be developed as an effective biomarker for improving the management of CRC patients by enhancing the efficiency of detection and efficacy of targeting treatment.