David J. Olivos

Combinatorial Library Screening with Liposomes for Discovery of Membrane Active Peptides

Membrane active peptides (MAPs) represent a class of short biomolecules that have shown great promise in facilitating intracellular delivery without disrupting cellular plasma membranes. Yet their clinical application has been stalled by numerous factors: off-target delivery, a requirement for high local concentration near cells of interest, degradation en route to the target site, and in the case of cell-penetrating peptides, eventual entrapment in endolysosomal compartments. The current method of deriving MAPs from naturally occurring proteins has restricted the discovery of new peptides that may overcome these limitations. Here, we describe a new branch of assays featuring high-throughput functional screening capable of discovering new peptides with tailored cell uptake and endosomal escape capabilities. The one-bead-one-compound (OBOC) combinatorial method is used to screen libraries containing millions of potential MAPs for binding to synthetic liposomes, which can be adapted to mimic various aspects of limiting membranes. By incorporating unnatural and d-amino acids in the library, in addition to varying buffer conditions and liposome compositions, we have identified several new highly potent MAPs that improve on current standards and introduce motifs that were previously unknown or considered unsuitable. Since small variations in pH and lipid composition can be controlled during screening, peptides discovered using this methodology could aid researchers building drug delivery platforms with unique requirements, such as targeted intracellular localization.

Abstract 4545: Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Neuroblastoma is the most common extra-cranial tumor in children, with around 700 new diagnoses each year. High-risk patients have poor outcomes and only 30% survive despite the most intensive therapies available, which can also harm healthy cells. Thus, we are developing novel targeted therapies for neuroblastoma to increase treatment efficacy and limit damage to healthy cells. We utilized the One-Bead One-Compound (OBOC) combinatorial split-mix method developed by Kit Lam (Lam et al., 1991) to identify unique ligands against neuroblastoma cells for therapeutic delivery. The OBOC libraries consist of hundreds to millions of 90 μm Tentagel beads, and each bead displays 1013 copies of a peptide on its surface. Thus, each bead has one unique compound on its exterior. Twelve random OBOC libraries with varying peptide lengths (5 to 16 amino acids) were screened against three human neuroblastoma cell lines (SK-N-BE, SK-N-DZ, and IMR-32) to determine a library for more stringent screening of ligands. 37,500 beads from each library were screened against each cell line and the libraries averaged 2 positive candidates per 18,750 beads within 2 hours of incubation. The results also suggested preference for longer peptide libraries, but an intermediate length random decamer library was chosen for further screening due to both a good yield of positive beads and convenient sequencing. Each neuroblastoma line was screened against 150,000 beads from the decamer library to identify potential ligands. Positive beads were selected, retested three times, and decoded using Edman degradation. Several cancer cell lines (A549 lung cancer, and Reh and Jurkat leukemia cell lines) were used as negative controls. Ten beads per cell line displaying high binding affinity were selected. Candidate beads with positive binding to all three neuroblastoma cell lines are currently being sequenced and the ligands will be synthesized. We plan to characterize the ligands for high affinity and specificity, as well as internalization capacity, and to identify their receptors. Currently, there are very few targeted therapies for neuroblastoma. We believe our ligand discoveries can be developed for future novel targeted therapies in neuroblastoma. Citation Format: Andy Iskandar, Connie Duong, Cathy Chen, Eduardo Sanchez, David Olivos, Kit Lam, Noriko Satake. Identifying neuroblastoma-specific ligands for cancer targeted therapy using a one-bead one-compound combinatorial approach. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4545. doi:10.1158/1538-7445.AM2013-4545

Abstract A41: Dasatinib regulation of N-glycans induce anticancer activity in hematological and solid tumor malignancies.

Glycans are carbohydrates covalently bound to proteins or lipids. Changes in their content and structure, particularly in asparagine-linked oligosaccharides, contribute to a variety of events during malignant transformation. These include the loss of cell-matrix adhesion, migration, invasion, metastasis, and evasion of cell death. High mannose glycans are not commonly present on the surfaces of normal mammalian cells or in serum, yet they play important roles in cancer cell biology. Therefore, modulation of glycan expression with drugs offers a promising therapeutic approach for various hematological and solid tumor cancers, several of which disproportionally afflict minority populations. Dasatinib, a dual inhibitor of Src and Abl tyrosine kinase, is used to investigate in vitro anti-cancer activity in several cancer cell lines: Jurkat lymphoblastic leukemia, Raji lymphoblastic lymphoma, SiHa cervical cancer, H1650 non small cell lung cancer, PC3N prostate cancer, SKOV3 ovarian cancer, and MCF7 breast cancer. To address how the carbohydrate moieties of the cancer cells are affected during treatment, the glycan profiles from treated cells are compared to non-treated cells and normal controls and analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). PCR arrays are used to profile glycosylation enzyme gene expression changes due to dasatinib-induced cytotoxicity. Protein expression arrays are used to investigate Src and Abl receptor tyrosine kinase signaling cascades. The results presented here demonstrate new evidence of glycosylation expression changes and the related mechanisms of their anticancer activity. Regulation of glycosylation is a promising therapeutic strategy for inducing anticancer activity through cell arrest, apoptosis, and autophagy. Citation Format: Mary R. Saunders, L. Renee Ruhaak, Cynthia Williams, David J. Olivos, Carol Stroble, Hyun Joo An, Suzanne Miyamoto, Carlito B. Lebrilla, Kit S. Lam. Dasatinib regulation of N-glycans induce anticancer activity in hematological and solid tumor malignancies. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A41.

Abstract 366: Novel self-folding tricyclic cholic acid-peptide hybrids as nanotherapeutic ligands targeting prostate and breast cancers and human embryonic stem cells

Prostate cancer is the second leading cause of death in American men accounting for 10% of all cancer-related mortalities. According to NCI statistics from 2005-2007, 1 in 6 men will be diagnosed with prostate cancer during their lifetime. Despite the use of chemotherapy for the treatment of advanced or metastatic disease, cellular resistance to anticancer drugs and adverse side effects to healthy organs limit therapeutic efficacy. A targeted pro-apoptotic drug delivery system has been developed for the selective destruction of malignant tumors and tumorigenic stem cells. A novel self-folding tricyclic branched library was synthesized. Fixed hydrophobic amino acids near the N-terminus (position 5) of twin branched peptide arms fold back onto the hydrophobic face of the planar cholic acid molecule to form a compact tricyclic molecule. The novel bracelet library was screened using a One Bead Two Compounds (OB2C) combinatorial chemistry approach. Receptors expressed on cancerous cells serve as the targets for the tricyclic molecules to induce apoptotic signaling. Receptors for LDO 18, found on prostate cancer and stem cells, serve as the carrier vector, enabling the targeted delivery of the anticancer drug to the tumor mass. This approach spares normal tissues from toxic side effects of the peptidomimetic compounds. Preliminary studies suggest that tumors of ovarian and breast origin can also be targeted using LDO18. Lead therapeutic compounds will be conjugated to LDO18 nanocarriers along with LHRH analogues to evaluate the efficacy and combined potency to prostate, ovarian, and breast cancer xenografts in nude mice and orthotropic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 366. doi:10.1158/1538-7445.AM2011-366

Abstract 2316: Therapeutic targeting of novel human stem cell and cancer associated glycans

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Host and tumor surface glycans are important in regulating pivotal pathophysiological events during the development of cancer and tumor progression. Changes in glycosylation allow tumorigenic cells to seize control over developmental processes which allow cells to proliferate, invade, promote angiogenesis, and metastasize. Malignant transformation is accompanied by the expression of oncofetal antigens which are expressed on embryonic cells, tumor cells, and a few adult cells. Glycans common to human embryonic stem cells and cancer cells can be targeted using combinatorial chemistry. Several peptidomimetic ligands have been discovered using One Bead One Compound (OBOC) and One Bead Two Compound (OB2C) methods. These cell adhering ligands possess the potential to target tumorigenic cells and mediate intracellular signaling events by binding to high mannose glycans expressed on the surface of Jurkat, TK6, Her2 expressing MCF7, chemoresistant MCF7, HeLa, and SiHa cell lines. Three lead linear peptides with alternating natural and unnatural amino acids have been investigated for their in vivo targeting potential in the lymphoid leukemia, breast, and cervical cancer mouse xenograph models. In vitro and in vivo stem and cancer cell experiments, which demonstrate genetically altered glycan function, are currently being investigated for their anticancer potential. These novel agents may be used alone or in combination with chemoradiation and anti-angiogenesis strategies for treating cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2316. doi:10.1158/1538-7445.AM2011-2316

Abstract A49: Multifunctional nanocarriers against cervical cancer

Cervical cancer accounts for 500,000 new cases and 250,000 deaths in women worldwide. In low-income countries, cervical cancer is the most common cause of cancer-related deaths among women. Novel approaches for the early detection and aggressive treatment of cervical cancer are urgently needed to increase the survival rates. To this end, a promising strategy that might improve the specificity of current diagnostic and therapeutic agents is the development of drugs or contrast agents targeting glycans, polysaccarides decorating normal and cancerous cells that provide a unique fingerprint on the cell surface. Tumor glycans are important in regulating pivotal events during the development and progression of cancer. Changes in their sequence and structure are known to be responsible for cell proliferation, invasion, angiogenesis, and metastasis. Malignant transformation of cervical cells is accompanied by the expression of specific glycans that are absent on normal uterine cervix cells. Peptide-based ligands specific for these cervical cancer glycans have been discovered, and their use as tumor-homing agents for diagnostics and therapeutic purposes has been explored in xenograph models. We have currently developed an innovative drug delivery vehicle for the simultaneous and highly specific imaging and treatment of cervical cancer. To take full advantage of the enhanced permeability and retention effect (EPR) of the nanocarriers and to have deep penetration inside the tumor mass, we have developed a telodendrimer system, comprised of clusters of oligomeric cholic acids (CA) linked to a polyethylene glycol (PEG) chain which self-assemble within an aqueous environment with tunable sizes and efficient drug loading capacity. This vehicle consists of a nanocarrier loaded with the powerful anticancer drug, doxorubicin, and decorated with cervical cancer specific ligands targeting cell surface glycans. Thanks to the presence of the targeting ligands, the nanocarriers carry the otherwise non-selective drug specifically into the targeted cancer cells leading to release of anticancer drug only where needed. Because of the lower doses and the relatively low cost of the nanoparticle employed as drug carriers, we envision that this technology will be amendable for clinical translation and will be a powerful therapeutic tool to address the needs and disparities typical of less developed countries. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A49.

Abstract A50: Targeting nanotherapeutics for breast, ovarian, and prostate cancer

African Americans have the highest incidence and mortality rates from cancer despite representing a minority of the population of the United States. In particular, breast and prostate cancer significantly contribute to these disproportionate rates in African American women and men respectively compared to other ethnic groups. We have developed a series of novel self-folding combinatorial libraries composed of cholic acid or cholesterol functional groups with single and branched hexamer peptide arms. Fixed hydrophobic amino acids near the N-terminus (position 5) of the peptide arms interact with the hydrophobic face of the planar cholic acid or cholesterol molecule to form 3-Dimensional compact molecules offering a variety of unique peptide sequence presentations. Receptors overexpressed on cancerous cells serve as the targets for these synthetic molecules. Screening of One-Bead One-Compound (OBOC) combinatorial libraries under stringent conditions provides a powerful approach to test hundreds of thousands to millions of compounds in one batch. Novel cholic-peptide hybrid molecules containing RGD motifs binding to vβ3 integrin were discovered against MDA-MB-231 breast cancer. An NGR motif known to target receptors up regulated in the tumor vasculature and perivascular cells were found in cholesterol-peptide protein conformation mimetics binding to A2780 chemo-resistant ovarian cancer. Targeting ligands to PC3N and LnCap prostate cancers from both cholesterol and cholic acid library sets were discovered. Based on these findings, focused libraries were constructed and screened for enhanced affinity and specificity. Candidate peptidomimetics are equipped on our novel cholic acid nanocarriers enhancing nanotherapeutic targeting, efficacy, and limiting toxicity. The reduced toxicity stemming from tumor targeting and increased efficacy from the combination of nanocarrier EPR effects and targeting ligands result in longer, more tolerable therapeutic cycles that might significantly extend the life expectancy and quality of life of breast and prostate cancer in patients, for which current treatment options are ineffective. Developing novel treatment strategies which include the selective delivery of cytotoxic agents for the treatment of cancers disproportionately afflicting minority populations may be translated for clinical development as a promising diagnostic and therapeutic tools. Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A50.