Jesse Jaynes

An IP-10 (CXCL10)-Derived Peptide Inhibits Angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77–98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.

LHRH-conjugated lytic peptides directly target prostate cancer cells.

Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone-releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC(50) concentrations for LNCaP cells, 4.4 and 9.1μM; for DU-145 cells, 4.8 and 5.7μM; and for PC-3 cells, 4.4 and 8.2μM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3h of exposure to either peptide; after 6h, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24h to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer.

African Americans with pancreatic ductal adenocarcinoma exhibit gender differences in Kaiso expression

Kaiso, a bi-modal transcription factor, regulates gene expression, and is elevated in breast, prostate, and colon cancers. Depletion of Kaiso in other cancer types leads to a reduction in markers for the epithelial-mesenchymal transition (EMT) (Jones et al., 2014), however its clinical implications in pancreatic ductal adenocarcinoma (PDCA) have not been widely explored. PDCA is rarely detected at an early stage but is characterized by rapid progression and invasiveness. We now report the significance of the subcellular localization of Kaiso in PDCAs from African Americans. Kaiso expression is higher in the cytoplasm of invasive and metastatic pancreatic cancers. In males, cytoplasmic expression of Kaiso correlates with cancer grade and lymph node positivity. In male and female patients, cytoplasmic Kaiso expression correlates with invasiveness. Also, nuclear expression of Kaiso increases with increased invasiveness and lymph node positivity. Further, analysis of the largest PDCA dataset available on ONCOMINE shows that as Kaiso increases, there is an overall increase in Zeb1, which is the inverse for E-cadherin. Hence, these findings suggest a role for Kaiso in the progression of PDCAs, involving the EMT markers, E-cadherin and Zeb1.

Control of Aspergillus flavus growth and aflatoxin production in transgenic maize kernels expressing a tachyplesin-derived synthetic peptide, AGM182

Aspergillus flavus is an opportunistic, saprophytic fungus that infects maize and other fatty acid-rich food and feed crops and produces toxic and carcinogenic secondary metabolites known as aflatoxins. Contamination of maize with aflatoxin poses a serious threat to human health in addition to reducing the crop value leading to a substantial economic loss. Here we report designing a tachyplesin1-derived synthetic peptide AGM182 and testing its antifungal activity both in vitro and in planta. In vitro studies showed a five-fold increase in antifungal activity of AGM182 (vs. tachyplesin1) against A. flavus. Transgenic maize plants expressing AGM182 under maize Ubiquitin-1 promoter were produced through Agrobacterium-mediated transformation. PCR products confirmed integration of the AGM182 gene, while RT-PCR of maize RNA confirmed the presence of AGM182 transcripts. Maize kernel screening assay using a highly aflatoxigenic A. flavus strain (AF70) showed up to 72% reduction in fungal growth in the transgenic AGM182 seeds compared to isogenic negative control seeds. Reduced fungal growth in the AGM182 transgenic seeds resulted in a significant reduction in aflatoxin levels (76-98%). The results presented here show the power of computational and synthetic biology to rationally design and synthesize an antimicrobial peptide against A. flavus that is effective in reducing fungal growth and aflatoxin contamination in an economically important food and feed crop such as maize.

Designed Host Defense Peptides for the Treatment of Bacterial Keratitis

Purpose To limit corneal damage and potential loss of vision, bacterial keratitis must be treated aggressively. Innovation in antimicrobials is required due to the need for empirical treatment and the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs, which provide defense against invading pathogens. This study investigates the use of novel dHDPs for the treatment of bacterial keratitis. Methods The minimum inhibitory concentrations (MICs) were determined for dHDPs on both Gram-positive and -negative bacteria. The minimum biofilm eradication concentrations (MBEC) and in vitro time-kill assays were determined. The most active dHDP, RP444, was evaluated for propensity to induce drug resistance and therapeutic benefit in a murine Pseudomonas aeruginosa keratitis model. Results Designed HDPs were bactericidal with MICs ranging from 2 to >64 μg/mL and MBEC ranging from 6 to 750 μg/mL. In time-kill assays, dHDPs were able to rapidly reduce bacterial counts upon contact with as little as 2 μg/mL. RP444 did not induce resistance after repeated exposure of P. aeruginosa to subinhibitory concentrations. RP444 demonstrated significant efficacy in a murine model of bacterial keratitis as evidenced by a significant dose-dependent decrease in ocular clinical scores, a significantly reduced bacterial load, and substantially decreased inflammatory cell infiltrates. Conclusions Innovative dHDPs demonstrated potent antimicrobial activity, possess a limited potential for development of resistance, and reduced the severity of murine P. aeruginosa keratitis. These studies demonstrate that a novel dHDP may have potential to treat patients with sight-threatening bacterial keratitis.

Double-receptor-targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer

As an alternative therapeutic treatment to reduce or eliminate the current side effects associated with advanced prostate cancer (PCa) chemotherapy, a multifunctional double-receptor-targeting iron oxide nanoparticles (IONPs) (luteinizing hormone-releasing hormone receptor [LHRH-R] peptide- and urokinase-type plasminogen activator receptor [uPAR] peptide-targeted iron oxide nanoparticles, LHRH-AE105-IONPs) drug delivery system was developed. Two tumor-targeting peptides guided this double-receptor-targeting nanoscale drug delivery system. These peptides targeted the LHRH-R and the uPAR on PCa cells. Dynamic light scattering showed an increase in the hydrodynamic size of the LHRH-AE105-IONPs in comparison to the non-targeted iron oxide nanoparticles (NT-IONPs). Surface analysis showed that there was a decrease in the zeta potential values for drug-loaded LHRH-AE105-IONPs compared to the NT-IONPs. Prussian blue staining demonstrated that the LHRH-AE105-IONPs were internalized efficiently by the human PCa cell line, PC-3. In vitro, magnetic resonance imaging (MRI) results confirmed the preferential binding and accumulation of LHRH-AE105-IONPs in PC-3 cells compared to normal prostate epithelial cells (RC77N/E). The results also showed that LHRH-AE105-IONPs significantly maintained T2 MRI contrast effects and reduced T2 values upon internalization by PC-3 cells. These paclitaxel-loaded double-receptor-targeting IONPs also showed an approximately twofold reduction in PC-3 cell viability compared to NT-IONPs.

Abstract 1640: A novel peptide-suppressing M2 polarized tumor-associated macrophages enhances tumor response to chemotherapy

Introduction: Multiple reports have demonstrated that M2-polarized tumor associated macrophages (TAMs) play a significant role in promoting tumor metastasis, survival and resistance to chemotherapy. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. Objective: To determine whether targeting the Cluster of Differentiation 206 (CD206) receptor on TAMs with an engineered peptides having both CD206 specificity and apoptotic activity, will limit the protumorigenic effects of TAMs and enhance cancer cells response to chemotherapy. Method: A class of engineered peptides were designed to stimulate phagocytosis via the CD206 receptor, and to initiate apopotic cell death once endocytosed. Primary cells were isolated from C57BL/6J mice from Jackson Laboratory and were polarized to M1 and M2 phenotypes by treating with IL-4 (20ng/ml) and IFN-γ (10ng/ml) respectively from RD gemcitabine @ 40 mg/kg q4D produced a 68% reduction; however, peptide in combination with gemcitabine completely blocked tumor growth. Moreover, immunohistochemistry staining of treated tumors showed a decrease in the expression of CD206 positive M2 macrophages and CD45/CD25 positive Treg cells, and an increase in CD86 positive M1 macrophages. These results were consistent with the in vitro results obtained. Discussion and Conclusion: These results suggest that a targeting CD206 peptide engineered to promote apoptosis, enhances the tumor response to chemotherapy by limiting the pro-tumorigenic activity of TAMs. Citation Format: Anghesom A. Ghebremedhin, Clayton Yates, Jesse Jaynes, George Martin, Henry Lopez, Charles Garvin. A novel peptide-suppressing M2 polarized tumor-associated macrophages enhances tumor response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1640. doi:10.1158/1538-7445.AM2017-1640

Abstract 2170: Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer

Prostate Cancer (PCa) is the second leading cause of cancer-related deaths among men in the United States. Due to the harsh side effects of conventional chemo- and radiotherapies, targeted drug delivery is now gaining the focus of cancer researchers. The key for success of any newly developed targeted drug delivery systems is the novel design of targeting peptides which have high binding affinities to differentially over-expressed receptors on the surface of PCa cells. Two over-expressed such receptors, which can be targeted to the cell surface of PCa cells are: Gonadotropin Releasing Hormone Receptor (GnRH-R), and Prostate Specific Membrane Antigen (PSMA). The analysis done from molecular dynamics simulation data gave an important information about each interaction; such as RMSD, The number of contacts between receptor and peptide within a cutoff distance of 5 A and the binding free energies, ΔGGBSA of the interactions. In all systems, the back bone RMSD deviation of the receptor was calculated using the last 10 ns of the sampling process and averaged over all trajectories. The RMSD data deviated between 1.22 - 2.51 A. The free binding energies or ΔGGBSA was calculated from the last 10 ns of the sampling process. The QH1 system has the more negative ΔGGBSA -36.15 kcal mol-1 and the QH4 system has the least negative ΔGGBSA of -18.4 kcal mol-1 among the eight systems of GnRH-R. QH2 system exhibits a highly interactive hydrogen bond (bond distance is 2.65 A) between 134th position of the receptor amino acid sequence GLU and 5th position of the qh2 peptide9s TYR. The ΔGGBSA is for the qh2 system is – 34.92 kcal mol-1. In the PSMA and its targeting peptide interactions, T2IA has the least negative ΔGGBSA of -47.37 kcal mol-1. A salt bridge was observed between the 10th position of the T2IA peptide sequence ARG and the 328 position of the 3RBU receptor9s GLU. Based on our molecular modeling studies, we have determined the best targeting peptides among all the newly designed peptides. The binding efficiency of the targeting peptides with the lowest binding energies will be further investigated surface plasmon resonance (SPR) in near future. Citation Format: Ahmad Salam, Vincent Hembrick, Jesse Jaynes, Timothy Turner, Mohamed Abdalla. Molecular modeling of novel peptide-receptor interaction for targeted drug delivery of prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2170.

Abstract PR03: Synthetic peptides suppress M2 macrophages and synergize with chemotherapy in prostate and breast cancer models

Multiple lines of evidence suggest that macrophages, in particular M2-polarized tumor associated macrophages (TAMs), promote tumor aggressiveness and chemoresistance. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. To assess this potential, we synthesized artificial peptides (10-12mers) with a striapathic arrangement of hydrophobic and hydrophilic amino acids, designed to specifically target TAMs within the tumor microenvironment. These peptides, including drug candidate RP-182, are basic in design and bind to human albumin prior to binding to their eventual target, which enhances peptide half-life to around 2 hours. In vitro experiments demonstrated that exposure to 50uM peptide significantly reduced M2 macrophage viability and increased apoptosis after 48 hours of exposure, but had no effect on M1 macrophages. Interestingly, we did not observe a significant effect of RP-182 in vitro on various cancer cell lines, including MDA-MB-231, C42B or cells isolated from KPC960 mice. To determine whether RP-182 had in vivo efficacy we used multiple animal models, beginning with inflammatory indications where macrophage activity is also implicated, and progressing to solid organ cancer models: (1) In bleomycin animal models of lung and skin fibrosis, mice developed very high levels of inflammatory markers including IL6, which remained at control levels in mice treated with 10 mg/kg RP-182 qD subcu. At increased pulmonary bleomycin levels, all control animals died while 65% of treated animals survived. (2) In xenograft models of triple negative breast cancer (MDA-MB-231) mice treated with peptide (10 mg/kg qD subcu) demonstrated 37% reduction in MD-MB-231 tumor growth; gemcitabine @ 40 mg/kg q4D demonstrated 70% reduction; however, peptide plus gemcitabine completely blocked tumor growth. (3) In a xenograft model of castration resistant prostate cancer (CRPC) (C42B) mice treated with 2.5 mg/kg Docetaxel q7D demonstrated 45% reduction in tumor growth relative to control; mice treated with 10 mg/kg RP-182 qD demonstrated 52% reduction in tumor growth; but Docetaxel plus RP-182 reduced tumor growth 65%. (4) In the KPC960 model of pancreatic cancer, RP-182 treatment caused a 30% reduction in tumor growth and reduced the expression of a number of inflammatory genes in the tumors. (5) Finally, in the KRAS/p53 transgenic model of pancreatic cancer, treatment with peptide for one week reduced tumor growth, and was associated with 5.8 fold reduction in PDL1 and a 5.5 fold reduction in PDL2 expression in tumors. Toxicity testing on animals given much higher doses of RP-182 showed no changes in histology, blood counts, body or tissue weight. These results suggest that these novel designed peptides suppress macrophages in the tumors, enhancing the tumor response to chemotherapy and downregulating certain checkpoint proteins. This abstract is also presented as Poster B23. Citation Format: Henry Lopez, George R. Martin, Jesse M. Jaynes, Clayton Yates. Synthetic peptides suppress M2 macrophages and synergize with chemotherapy in prostate and breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR03.

Abstract 3675: Double receptor targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer

Purpose: As an alternative to the drawbacks of current advanced prostate cancer chemotherapy, we propose a multifunctional double targeting drug delivery system that utilizes the combination of two cancer-targeting peptides: a modified luteinizing hormone releasing hormone (LHRH), the ligand for luteinizing hormone releasing hormone receptor (LHRH-R), and AE105, the ligand for urokinase type plasminogen activation receptor (uPAR). Both ligands are fused to amphiphilic polymer coated iron oxide nanoparticles (IONPs) and loaded with the anticancer drug Paclitaxel (PTX) as the payload. Materials and Methods: Targeted IONPs were characterized by both gel electrophoresis and dynamic light scattering (DLS). PTX was absorbed on the IONPs. Drug loading and release characteristics were conducted by HPLC. The binding specificity and the internalization efficiency of the targeted IONPs were both examined by Prussian blue staining and magnetic resonance imaging (MRI). Cytotoxicity of the targeted IONPs was evaluated by MTT assay. Results: Characterization of targeted IONPs by gel electrophoresis confirmed the successful attachment of the peptides to IONPs. Conjugation of peptides to carboxylic groups of polymer coating on IONPs resulted in an increase in the average hydrodynamic size of targeted IONPs (16.34 nm) as compared to non-targeted IONPs (12.51 nm), as well as a decrease of zeta potential from -70.43 mV to -58.06 mV, respectively. Prussian blue staining demonstrated that both, LHRH and AE105 conjugated IONPs were internalized efficiently by cells of the human prostate cancer cell line, PC3. In vitro MRI results showed that double-targeted IONPs significantly maintained T2 MRI contrast effect and reduction of T2 values upon internalization by PC3 cells. MRI imaging confirmed the preferential binding and accumulation of double-targeted IONPs in PC3 cells when compared to normal prostate epithelial cells (RC77N/E). PTX loaded double-targeted IONPs were stable at physiological pH and efficiently released around pH 4, the optimum pH inside the tumor cells. PTX loaded double-targeted IONPs showed an approximately 2-fold reduction in PC3 cell viability when compared to non-targeted IONPs. In addition, the percentage of cell death resulting from the PTX loaded, double-targeted IONPs was very similar to the percentage of cell death attributed to 100 ng/ml of free PTX, thereby demonstrating that their capability of reducing drug concentration. Conclusions: Our results indicate that we have developed a LHRH-R and uPAR targeted IONPs drug delivery system that potentially provides a MRI tractable delivery of cancer therapeutics such as PTX to prostate cancer cells. Therefore, our optimized double-targeted IONPs drug delivery system has the potential to significantly improve the health outcomes and quality of life for cancer patients as a novel type of targeted nanomedicine therapy. Citation Format: Md Shakir U. Ahmed, Mohamed O. Abdalla, Clayton Yates, Jesse Jaynes, Timothy Turner. Double receptor targeting multifunctional iron oxide nanoparticles drug delivery system for the treatment and imaging of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3675. doi:10.1158/1538-7445.AM2015-3675