Rania Soudy

Engineered breast tumor targeting peptide ligand modified liposomal doxorubicin and the effect of peptide density on anticancer activity.

This study aimed to develop actively targeted liposomal formulations of doxorubicin (DOX) using an engineered breast tumor targeting peptide ligand, p18-4 (WxEAAYQrFL). Towards this goal, stealth liposomes bearing different molar ratios of p18-4 peptide (1.5 and 0.3 peptide/total lipid mol %), namely HD and LD liposomes, were successfully prepared. The effect of p18-4 peptide modification and density on breast cancer cell uptake, selective cytotoxicity as well as inhibition of tumor growth and the tissue disposition of encapsulated DOX in breast tumor xenograft models in mice were assessed. The results showed a 2.4 and 5 folds decrease in the IC50 of HD liposomes in MDA-MB-435 and MCF-7 breast tumor cells, respectively. Although LD liposomes showed less (1.6 and 2.2 folds) decrease in the IC50 of DOX in the same breast cancer cell lines, they were more selective in their cytotoxic effect and uptake towards breast cancer over normal breast epithelial cells, MCF10A. Evaluation of the anticancer activity in NOD-SCID mice bearing MDA-MB-435 xenografts after receiving six i.v. injections of 2.5mg/kg/week DOX equivalent showed a superior anticancer activity for LD liposomal DOX compared to HD and unmodified liposomal formulations. Mice treated with LD liposomal DOX illustrated 4.8 folds reduction in the mean relative tumor volume compared to non-targeted DOX liposomes. This was despite similar tumor accumulation of DOX as part of LD liposomes compared to that for unmodified liposomes 24h following the last injection. In contrast, HD liposomes showed decreased DOX accumulation in the tumor and preferential uptake by liver and spleen. Treatment with unmodified and LD liposomes did not have any adverse impact on the activity level and mean body weight of live animals during the study period. In conclusion, surface modification of liposomal DOX with engineered p18-4 peptide at an optimum density can improve the antitumor efficacy and selectivity of liposomal DOX.

Proteolytically Stable Cancer Targeting Peptides with High Affinity for Breast Cancer Cells

Cancer cell targeting peptides have emerged as a highly efficient approach for selective delivery of chemotherapeutics and diagnostics to different cancer cells. However, the use of α-peptides in pharmaceutical applications is hindered by their enzymatic degradation and low bioavailability. Starting with a 10-mer α-peptide 18 that we developed previously, here we report three novel analogues of 18 that are proteolytically stable and display better (up to 3.5-fold) affinity profiles for breast cancer cells compared to 18. The design strategy involved replacement of two or three amino acids in the sequence of 18 with d-residues or β(3)-amino acids. Such replacement maintained the specificity for cancer cells (MDA-MB-435, MDA-MB-231, and MCF-7) with low affinity for control noncancerous cells (MCF-10A and HUVEC), showed an increase in secondary structure, and rendered the analogues completely stable to human serum and liver homogenate from mice. The three analogues are potentially safe with minimal cellular toxicity and are efficient targeting moieties for specific drug delivery to breast cancer cells. The strategy used here may be adapted to develop peptide analogues that will target other cancer cell types.

Engineered peptides for the development of actively tumor targeted liposomal carriers of doxorubicin

Chemotherapy is still the treatment of choice for many types of cancer; but its effectiveness is hampered by dose limiting toxicity. Properly designed delivery systems can overcome this shortcoming by reducing the non-specific distribution and toxicity of chemotherapeutics in healthy organs and at the same time increasing drug concentrations at tumor tissue. In this study, we developed stealth liposomal formulations of doxorubicin (DOX) having a novel stable engineered peptide ligand, namely p18-4, that binds specifically to breast cancer cell line MDA-MB-435 on its surface. The coupling of p18-4 to liposomes was carried out through conventional, post insertion and post conjugation techniques and prepared liposomes were characterized for their size and level of peptide modification. The p18-4 decorated liposomal DOX formulations were then evaluated for their cellular uptake as well as cytotoxicity against the human breast cancer MDA-MB-435 cells. In this context, the effect of coupling technique on the uptake and cytotoxicity of p18-4 liposomal DOX in MDA-MB-435 cells was evaluated. The conventional and post conjugation methods of peptide incorporation were found to be more reliable for the preparation of p18-4 decorated liposomes for active DOX targeting to MDA-MB-435 cells. p18-4 decoration of liposomes by these methods did not have a notable effect on the size of prepared liposomes and DOX release, but increased the uptake and cytotoxicity of encapsulated DOX in MDA-MB-435 cells. The results show a potential for p18-4 decorated liposomes prepared by conventional and post conjugation method for tumor targeted delivery of DOX in breast tumor models.

Novel Peptide–Doxorubucin Conjugates for Targeting Breast Cancer Cells Including the Multidrug Resistant Cells

The efficacy of chemotherapeutic doxorubucin (Dox) in cancer treatment is limited by two main factors, nonspecific toxicity and the emergence of tumor resistance. To overcome these hurdles, in this study peptide-Dox conjugates were prepared. A decapeptide 18-4a (NH₂-WxEAAYQkFL-CONH₂) [corrected] with high specificity for breast cancer cells and improved proteolytic stability was conjugated to Dox to give peptide-Dox ester (1) and amide (2) conjugates. Cell uptake studies showed that the conjugates were 6-10 times selective for breast cancerous cells (MCF-7 and MDA-MB-435) over noncancerous cells (HUVECs and MCF-10A). Conjugate 1 displayed similar toxicity as free Dox toward the breast cancerous cells and was about 40 times less toxic toward the noncancerous cells and 4-fold more toxic toward the Dox resistant MDA-MB-435-MDR cells than the free Dox. These data suggest that conjugate 1 can be used as a potential prodrug for improving the therapeutic index of Dox and potentially many other cytotoxic drugs.

NGR peptide ligands for targeting CD13/APN identified through peptide array screening resemble fibronectin sequences.

Peptides containing the Asn-Gly-Arg (NGR) motif are known to bind CD13 isoforms expressed in tumor vessels and have been widely used for tumor targeting. Residues flanking the NGR sequence play an important role in modulating the binding affinity and specificity of NGR for the CD13 receptor. Herein, we have used a rapid, easy, and reliable peptide array-whole cell binding assay for screening a library of NGR peptides with different flanking residues. A peptide array consisting of forty-five NGR containing peptides was synthesized on a cellulose membrane, followed by screening against CD13 positive (HUVEC and HT-1080) and CD13 negative cell lines (MDA-MB-435 and MDA-MB-231). The library screening led to the identification of five cyclic and acyclic NGR peptides that display higher binding (up to 5-fold) to CD13 positive cells with negligible binding to CD13 negative cell lines when compared to the lead sequence cyclic CVLNGRMEC. Peptides with high binding affinity for the CD13 positive cells also showed improved in vitro cellular uptake and specificity using flow cytometry and fluorescence microscopy. Interestingly, the identified peptides resemble the NGR sequences present in the human fibronectin protein. These NGR peptides are promising new ligands for developing tumor vasculature targeted drugs, delivery systems and imaging agents with reduced systemic toxicity.

Synthetic peptides derived from the sequence of a lasso peptide microcin J25 show antibacterial activity

Microcin J25 (MccJ25) is a plasmid-encoded, ribosomally synthesized antibacterial peptide with a unique lasso structure. The lasso structure, produced with the aid of two processing enzymes, provides exceptional stability to MccJ25. We report the synthesis of six peptides (1-6), derived from the MccJ25 sequence, that are designed to form folded conformation by disulfide bond formation and electrostatic or hydrophobic interactions. Two peptides (1 and 6) display good activity against Salmonella newport, and are the first synthetic derivatives of MccJ25 that are bactericidal. Peptide 1 displays potent activity against several Salmonella strains including two MccJ25 resistant strains. The solution conformation and the stability studies of the active peptides suggest that they do not fold into a lasso conformation and peptide 1 displays antimicrobial activity by inhibition of target cell respiration. Like MccJ25, the synthetic MccJ25 derivatives display minimal toxicity to mammalian cells suggesting that these peptides act specifically on bacterial cells.

Design, synthesis and evaluation of antimicrobial activity of N-terminal modified Leucocin A analogues

Class IIa bacteriocins are potent antimicrobial peptides produced by lactic acid bacteria to destroy competing microorganisms. The N-terminal domain of these peptides consists of a conserved YGNGV sequence and a disulphide bond. The YGNGV motif is essential for activity, whereas, the two cysteines involved in the disulphide bond can be replaced with hydrophobic residues. The C-terminal region has variable sequences, and folds into a conserved amphipathic α-helical structure. To elucidate the structure-activity relationship in the N-terminal domain of these peptides, three analogues (1-3) of a class IIa bacteriocin, Leucocin A (LeuA), were designed and synthesized by replacing the N-terminal β-sheet residues of the native peptide with shorter β-turn motifs. Such replacement abolished the antibacterial activity in the analogues, however, analogue 1 was able to competitively inhibit the activity of native LeuA. Native LeuA (37-mer) was synthesized using native chemical ligation method in high yield. Solution conformation study using circular dichroism spectroscopy and molecular dynamics simulations suggested that the C-terminal region of analogue 1 adopts helical folding as found in LeuA, while the N-terminal region did not fold into β-sheet conformation. These structure-activity studies highlight the role of proper folding and complete sequence in the activity of class IIa bacteriocins.

Breast Cancer Targeting Peptide Binds Keratin 1: A New Molecular Marker for Targeted Drug Delivery to Breast Cancer

The biomarkers or receptors expressed on cancer cells and the targeting ligands with high binding affinity for biomarkers play a key role in early detection and treatment of breast cancer. The breast cancer targeting peptide p160 (12-mer) and its enzymatically stable analogue 18-4 (10-mer) showed marked potential for breast cancer drug delivery using cell studies and animal models. Herein, we used affinity purification, liquid chromatography-tandem mass spectrometry, and proteomics to identify keratin 1 (KRT1) as the target receptor highly expressed on breast cancer cells for p160 peptide(s). Western blot and immunocytochemistry in MCF-7 breast cancer cells confirmed the identity of KRT1. We demonstrate that the p160 or 18-4 binding to MCF-7 breast cancer cells is dependent on the expression of KRT1, and we confirm peptide-KRT1 binding specificity using SPR experiments (Kd ∼ 1.1 μM and 0.98 μM for p160 and 18-4, respectively). Furthermore, we assessed the ability of peptide 18-4 to improve the cellular uptake and anticancer activity of a pro-apoptotic antimicrobial peptide, microcin J25 (MccJ25), in breast cancer cells. A covalent conjugate of peptide 18-4 with MccJ25 showed preferential cytotoxicity toward breast cancer cells with minimal cytotoxicity against normal HUVEC cells. The conjugate inhibited the growth of MDA-MB-435 MDR multidrug-resistant cells with an IC50 comparable to that of nonresistant cells. Conjugation improved selective cellular uptake of MccJ25, and the conjugate triggered cancer cell death by apoptosis. Our findings establish KRT1 as a new marker for breast cancer targeting. Additionally, it pinpoints the potential use of antimicrobial lasso peptides as a novel class of anticancer therapeutics.

Engineered Peptides for Applications in Cancer-Targeted Drug Delivery and Tumor Detection

Cancer-targeting peptides as ligands for targeted delivery of anticancer drugs or drug carriers have the potential to significantly enhance the selectivity and the therapeutic benefit of current chemotherapeutic agents. Identification of tumor-specific biomarkers like integrins, aminopeptidase N, and epidermal growth factor receptor as well as the popularity of phage display techniques along with synthetic combinatorial methods used for peptide design and structure optimization have fueled the advancement and application of peptide ligands for targeted drug delivery and tumor detection in cancer treatment, detection and guided therapy. Although considerable preclinical data have shown remarkable success in the use of tumor targeting peptides, peptides generally suffer from poor pharmacokinetics, enzymatic instability, and weak receptor affinity, and they need further structural modification before successful translation to clinics is possible. The current review gives an overview of the different engineering strategies that have been developed for peptide structure optimization to confer selectivity and stability. We also provide an update on the methods used for peptide ligand identification, and peptide- receptor interactions. Additionally, some applications for the use of peptides in targeted delivery of chemotherapeutics and diagnostics over the past 5 years are summarized.

Cyclic AC253, A Novel Amylin Receptor Antagonist, Improves Cognitive Deficits in a Mouse Model of Alzheimer’s Disease

Amylin receptor serves as a portal for the expression of deleterious effects of amyloid β‐protein (Aβ), a key pathologic hallmark of Alzheimers disease. Previously, we showed that AC253, an amylin receptor antagonist, is neuroprotective against Aβ toxicity in vitro and abrogates Aβ‐induced impairment of hippocampal long‐term potentiation.