ABSTRACT
The prevalence of autoimmune diseases is on the rise globally. Currently, autoimmunity presents in over 100 different forms and affects around 9% of the world's population. Current treatments available for autoimmune diseases are inadequate, expensive, and tend to focus on symptom management rather than cure. Clinical trials have shown that live helminthic therapy can decrease chronic inflammation associated with inflammatory bowel disease and other gastrointestinal autoimmune inflammatory conditions. As an alternative and better controlled approach to live infection, we have identified and characterized two peptides, Acan1 and Nak1, from the excretory/secretory component of parasitic hookworms for their therapeutic activity on experimental colitis. We synthesized Acan1 and Nak1 peptides from the Ancylostoma caninum and Necator americanus hookworms and assessed their structures and protective properties in human cell-based assays and in a mouse model of acute colitis. Acan1 and Nak1 displayed anticolitic properties via significantly reducing weight loss and colon atrophy, edema, ulceration, and necrosis in 2,4,6-trinitrobenzene sulfonic acid-exposed mice. These hookworm peptides prevented mucosal loss of goblet cells and preserved intestinal architecture. Acan1 upregulated genes responsible for the repair and restitution of ulcerated epithelium, whereas Nak1 downregulated genes responsible for epithelial cell migration and apoptotic cell signaling within the colon. These peptides were nontoxic and displayed key immunomodulatory functions in human peripheral blood mononuclear cells by suppressing CD4+ T cell proliferation and inhibiting IL-2 and TNF production. We conclude that Acan1 and Nak1 warrant further development as therapeutics for the treatment of autoimmunity, particularly gastrointestinal inflammatory conditions.
Subject(s)
Ancylostomatoidea/chemistry , Colitis/drug therapy , Colitis/prevention & control , Leukocytes/immunology , Peptides/therapeutic use , Amino Acid Sequence , Ancylostoma , Animals , Cell Proliferation/drug effects , Cytokines/metabolism , Disease Models, Animal , Gene Expression Regulation/drug effects , Humans , Inflammation Mediators/metabolism , Intestines/pathology , Kv1.3 Potassium Channel/antagonists & inhibitors , Kv1.3 Potassium Channel/metabolism , Leukocytes/drug effects , Magnetic Resonance Spectroscopy , Male , Mice, Inbred C57BL , Necator americanus , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacology , Principal Component Analysis , Protein Domains , Protein Folding , T-Lymphocytes/cytology , Trinitrobenzenesulfonic Acid , Xenopus laevisABSTRACT
The peptide recifin A is the inaugural member of the structurally intriguing new fold referred to as a tyrosine-lock. Its central four stranded ß-sheet is stabilized by a unique arrangement in which three disulfide bonds and their interconnecting backbone form a ring that wraps around one of the strands, resulting in a Tyr side chain being buried in the molecular core. Here we aimed to establish a synthetic route to this complex class of natural products. Full length recifin A was successfully generated through native chemical ligation chemistry joining two 21 amino acid residue fragments. Surprisingly, reduced linear recifin A readily adopts the correct, topologically-complex fold via random oxidation of the cysteines, suggesting it is highly energetically favored. Utilizing our synthetic strategy, we generated five recifin A analogues to investigate the structural role of the central Tyr residue and provide the first insights into the structure activity relationship of recifin A towards its cancer target tyrosyl-DNA phosphodiesterase I.
ABSTRACT
BACKGROUND AND PURPOSE: Over past decades, targeted therapies and immunotherapy have improved survival and reduced the morbidity of patients with BRAF-mutated melanoma. However, drug resistance and relapse hinder overall success. Therefore, there is an urgent need for novel compounds with therapeutic efficacy against BRAF-melanoma. This prompted us to investigate the antiproliferative profile of a tachykinin-peptide from the Octopus kaurna, Octpep-1 in melanoma. EXPERIMENTAL APPROACH: We evaluated the cytotoxicity of Octpep-1 by MTT assay. Mechanistic insights on viability and cellular damage caused by Octpep-1 were gained via flow cytometry and bioenergetics. Structural and pharmacological characterization was conducted by molecular modelling, molecular biology, CRISPR/Cas9 technology, high-throughput mRNA and calcium flux analysis. In vivo efficacy was validated in two independent xerograph animal models (mice and zebrafish). KEY RESULTS: Octpep-1 selectively reduced the proliferative capacity of human melanoma BRAFV600E -mutated cells with minimal effects on fibroblasts. In melanoma-treated cells, Octpep-1 increased ROS with unaltered mitochondrial membrane potential and promoted non-mitochondrial and mitochondrial respiration with inefficient ATP coupling. Molecular modelling revealed that the cytotoxicity of Octpep-1 depends upon the α-helix and polyproline conformation in the C-terminal region of the peptide. A truncated form of the C-terminal end of Octpep-1 displayed enhanced potency and efficacy against melanoma. Octpep-1 reduced the progression of tumours in xenograft melanoma mice and zebrafish. CONCLUSION AND IMPLICATIONS: We unravel the intrinsic anti-tumoural properties of a tachykinin peptide. This peptide mediates the selective cytotoxicity in BRAF-mutated melanoma in vitro and prevents tumour progression in vivo, providing a foundation for a therapy against melanoma.
Subject(s)
Antineoplastic Agents , Melanoma , Adenosine Triphosphate , Animals , Antineoplastic Agents/pharmacology , Calcium , Cell Line, Tumor , Humans , Melanoma/drug therapy , Melanoma/pathology , Mice , Mutation , Octopodiformes/chemistry , Peptides/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/therapeutic use , RNA, Messenger , Reactive Oxygen Species , Tachykinins/genetics , Tachykinins/therapeutic use , Zebrafish/geneticsABSTRACT
Introduction: Animal venoms are a complex mixture of bioactive molecules that have evolved over millions of years for prey capture and defense from predators. Venom consists of many different types of molecules, with disulfide-rich peptides being a major component in most venoms. The study of these potent and highly selective molecules has led to the development of venom-derived drugs for diseases such as type 2 diabetes mellitus and chronic pain. As technologies have improved, more bioactive peptides have been discovered from venomous animals. Many of these molecules may have applications as tools for understanding normal and disease physiology, therapeutics, cosmetics or in agriculture.Areas covered: This article reviews venom-derived drugs approved by the FDA and venom-derived peptides currently in development. It discusses the challenges faced by venom-derived peptide drugs during drug development and the future for venom-derived peptides.Expert opinion: New techniques such as toxin driven discovery are expanding the pipeline of venom-derived peptides. There are many venom-derived peptides currently in preclinical and clinical trials that would have remained undiscovered using traditional approaches. A renewed focus on venoms, with advances in technology, will broaden the diversity of venom-derived peptide therapeutics and expand our knowledge of their molecular targets.
Subject(s)
Diabetes Mellitus, Type 2 , Venoms , Animals , Diabetes Mellitus, Type 2/drug therapy , Drug Development , Drug Discovery/methods , Peptides/chemistry , Peptides/pharmacology , Venoms/chemistry , Venoms/pharmacology , Venoms/therapeutic useABSTRACT
Melanoma is the main cause of skin cancer deaths, with special emphasis in those cases carrying BRAF mutations that trigger the mitogen-activated protein kinases (MAPK) signaling and unrestrained cell proliferation in the absence of mitogens. Current therapies targeting MAPK are hindered by drug resistance and relapse that rely on metabolic rewiring and Akt activation. To identify new drug candidates against melanoma, we investigated the molecular mechanism of action of the Octopus Kaurna-derived peptide, Octpep-1, in human BRAF(V600E) melanoma cells using proteomics and RNAseq coupled with metabolic analysis. Fluorescence microscopy verified that Octpep-1 tagged with fluorescein enters MM96L and NFF cells and distributes preferentially in the perinuclear area of MM96L cells. Proteomics and RNAseq revealed that Octpep-1 targets PI3K/AKT/mTOR signaling in MM96L cells. In addition, Octpep-1 combined with rapamycin (mTORC1 inhibitor) or LY3214996 (ERK1/2 inhibitor) augmented the cytotoxicity against BRAF(V600E) melanoma cells in comparison with the inhibitors or Octpep-1 alone. Octpep-1-treated MM96L cells displayed reduced glycolysis and mitochondrial respiration when combined with LY3214996. Altogether these data support Octpep-1 as an optimal candidate in combination therapies for melanoma BRAF(V600E) mutations.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Melanoma/drug therapy , Mutation , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Sirolimus/pharmacology , Skin Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation/drug effects , Energy Metabolism/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Melanoma/enzymology , Melanoma/genetics , Melanoma/pathology , Signal Transduction , Skin Neoplasms/enzymology , Skin Neoplasms/genetics , Skin Neoplasms/pathologyABSTRACT
Venoms act with remarkable specificity upon a broad diversity of physiological targets. Venoms are composed of proteins, peptides, and small molecules, providing the foundation for the development of novel therapeutics. This study assessed the effect of venom from the red-bellied black snake (Pseudechis porphyriacus) on human primary leukocytes using bead-based flow cytometry, mixed lymphocyte reaction, and cell viability assays. We show that venom treatment had a significant immunosuppressive effect, inhibiting the secretion of interleukin (IL)-2 and tumor necrosis factor (TNF) from purified human T cells by 90% or greater following stimulation with mitogen (phorbol 12-myristate 13-acetate and ionomycin) or via cluster of differentiation (CD) receptors, CD3/CD28. In contrast, venom treatment did not inhibit TNF or IL-6 release from antigen-presenting cells stimulated with lipopolysaccharide. The reduced cytokine release from T cells was not associated with inhibition of T cell proliferation or reduction of cell viability, consistent with an anti-inflammatory mechanism unrelated to the cell cycle. Deconvolution of the venom using reverse-phase HPLC identified four fractions responsible for the observed immunosuppressive activity. These data suggest that compounds from P. porphyriacus venom may be potential drug leads for T cell-associated conditions such as graft versus host disease, rheumatoid arthritis, and inflammatory bowel disease.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Elapid Venoms/pharmacology , Immunosuppressive Agents/pharmacology , Animals , Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , Cytokines/immunology , Elapidae , Humans , Lipopolysaccharides/pharmacologyABSTRACT
Helminths have evolved to become experts at subverting immune surveillance. Through potent and persistent immune tempering, helminths can remain undetected in human tissues for decades. Redirecting the immunomodulating "talents" of helminths to treat inflammatory human diseases is receiving intensive interest. Here, we review therapies using live parasitic worms, worm secretions, and worm-derived synthetic molecules to treat autoimmune disease. We review helminth therapy in both mouse models and clinical trials and discuss what is known on mechanisms of action. We also highlight current progress in characterizing promising new immunomodulatory molecules found in excretory/secretory products of helminths and their potential use as immunotherapies for acute and chronic inflammatory diseases.