Development of membrane-active peptide therapeutics in oncology

Membrane-active peptides play an essential role in many living organisms and their immune systems and counter many infectious diseases. Many have dual or multiple mechanisms and can synergize with other molecules, like peptides, proteins, and small molecules. Although membrane-active peptides have been intensively studied in the past decades and more than 3500 sequences have been identified, only a few received approvals from the US Food and Drug Administration. In this review, we investigated all the peptide therapeutics that have entered the market or were subjected to preclinical and clinical studies to understand how they succeeded. With technological advancement (e.g., chemical modifications and pharmaceutical formulations) and a better understanding of the mechanism of action and the potential targets, we found at least five membrane-active peptide drugs that have entered preclinical/clinical phases and show promising results for cancer treatment. We summarized our findings in this review and provided insights into membrane-active anticancer peptide therapeutics.     

Peptides encoded by short ORFs control development and define a new eukaryotic gene family

Despite recent advances in developmental biology, and the sequencing and annotation of genomes, key questions regarding the organisation of cells into embryos remain. One possibility is that uncharacterised genes having nonstandard coding arrangements and functions could provide some of the answers. Here we present the characterisation of tarsal-less (tal), a new type of noncanonical gene that had been previously classified as a putative noncoding RNA. We show that tal controls gene expression and tissue folding in Drosophila, thus acting as a link between patterning and morphogenesis. tal function is mediated by several 33-nucleotide-long open reading frames (ORFs), which are translated into 11-amino-acid-long peptides. These are the shortest functional ORFs described to date, and therefore tal defines two novel paradigms in eukaryotic coding genes: the existence of short, unprocessed peptides with key biological functions, and their arrangement in polycistronic messengers. Our discovery of tal-related short ORFs in other species defines an ancient and noncanonical gene family in metazoans that represents a new class of eukaryotic genes. Our results open a new avenue for the annotation and functional analysis of genes and sequenced genomes, in which thousands of short ORFs are still uncharacterised.     

The noncanonical chronicles: Emerging roles of sphingolipid structural variants

Sphingolipids (SPs) are structurally diverse and represent one of the most quantitatively abundant classes of lipids in mammalian cells. In addition to their structural roles, many SP species are known to be bioactive mediators of essential cellular processes. Historically, studies have focused on SP species that contain the canonical 18‑carbon, mono-unsaturated sphingoid backbone. However, increasingly sensitive analytical technologies, driven by advances in mass spectrometry, have facilitated the identification of previously under-appreciated, molecularly distinct SP species. Many of these less abundant species contain noncanonical backbones. Interestingly, a growing number of studies have identified clinical associations between these noncanonical SPs and disease, suggesting that there is functional significance to the alteration of SP backbone structure. For example, associations have been found between SP chain length and cardiovascular disease, pain, diabetes, and dementia. This review will provide an overview of the processes that are known to regulate noncanonical SP accumulation, describe the clinical correlations reported for these molecules, and review the experimental evidence for the potential functional implications of their dysregulation. It is likely that further scrutiny of noncanonical SPs may provide new insight into pathophysiological processes, serve as useful biomarkers for disease, and lead to the design of novel therapeutic strategies.     

Translation landscape of SARS-CoV-2 noncanonical subgenomic RNAs

The ongoing COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with a positive-stranded RNA genome. Current proteomic studies of SARS-CoV-2 mainly focus on the proteins encoded by its genomic RNA (gRNA) or canonical subgenomic RNAs (sgRNAs). Here, we systematically investigated the translation landscape of SARS-CoV-2, especially its noncanonical sgRNAs. We first constructed a strict pipeline, named vipep, for identifying reliable peptides derived from RNA viruses using RNA-seq and mass spectrometry data. We applied vipep to analyze 24 sets of mass spectrometry data related to SARS-CoV-2 infection. In addition to known canonical proteins, we identified many noncanonical sgRNA-derived peptides, which stably increase after viral infection. Furthermore, we explored the potential functions of those proteins encoded by noncanonical sgRNAs and found that they can bind to viral RNAs and may have immunogenic activity. The generalized vipep pipeline is applicable to any RNA viruses and these results have expanded the SARSCoV-2 translation map, providing new insights for understanding the functions of SARS-CoV-2 sgRNAs.     

Recent advances in the structural analysis of adenylation domains in natural product biosynthesis

Adenylation (A) domains catalyze the biosynthetic incorporation of acyl building blocks into nonribosomal peptides and related natural products by selectively transferring acyl substrates onto cognate carrier proteins (CP). The use of noncanonical acyl units, such as nonproteinogenic amino acids and keto acids, by A domains expands the structural diversity of natural products. Furthermore, interrupted A domains, which have embedded auxiliary domains, are able to modify the incorporated acyl units. Structural information on A domains is important for rational protein engineering to generate unnatural compounds. In this review, we summarize recent advances in the structural analysis of A domains. First, we discuss the mechanisms by which A domains recognize noncanonical acyl units. We then focus on the interactions of A domains with CP domains and embedded auxiliary domains.     

Genomics-enabled discovery of phosphonate natural products and their biosynthetic pathways

Phosphonate natural products have proven to be a rich source of useful pharmaceutical, agricultural, and biotechnology products, whereas study of their biosynthetic pathways has revealed numerous intriguing enzymes that catalyze unprecedented biochemistry. Here we review the history of phosphonate natural product discovery, highlighting technological advances that have played a key role in the recent advances in their discovery. Central to these developments has been the application of genomics, which allowed discovery and development of a global phosphonate metabolic framework to guide research efforts. This framework suggests that the future of phosphonate natural products remains bright, with many new compounds and pathways yet to be discovered.     

Technological progress and challenges towards cGMP manufacturing of human pluripotent stem cells based therapeutic products for allogeneic and autologous cell therapies

Recent technological advances in the generation, characterization, and bioprocessing of human pluripotent stem cells (hPSCs) have created new hope for their use as a source for production of cell-based therapeutic products. To date, a few clinical trials that have used therapeutic cells derived from hESCs have been approved by the Food and Drug Administration (FDA), but numerous new hPSC-based cell therapy products are under various stages of development in cell therapy-specialized companies and their future market is estimated to be very promising. However, the multitude of critical challenges regarding different aspects of hPSC-based therapeutic product manufacturing and their therapies have made progress for the introduction of new products and clinical applications very slow. These challenges include scientific, technological, clinical, policy, and financial aspects. The technological aspects of manufacturing hPSC-based therapeutic products for allogeneic and autologous cell therapies according to good manufacturing practice (cGMP) quality requirements is one of the most important challenging and emerging topics in the development of new hPSCs for clinical use. In this review, we describe main critical challenges and highlight a series of technological advances in all aspects of hPSC-based therapeutic product manufacturing including clinical grade cell line development, large-scale banking, upstream processing, downstream processing, and quality assessment of final cell therapeutic products that have brought hPSCs closer to clinical application and commercial cGMP manufacturing.     

A novel peptide Phylloseptin‐PBu from Phyllomedusa burmeisteri possesses insulinotropic activity via potassium channel and GLP‐1 receptor signalling

Insulin, as one of the most important hormones regulating energy metabolism, plays an essential role in maintaining glucose and lipid homeostasis in vivo. Failure or insufficiency of insulin secretion from pancreatic beta‐cells increases glucose and free fatty acid level in circulation and subsequently contributes to the emergence of hyperglycaemia and dyslipidaemia. Therefore, stimulating the insulin release benefits the treatment of type 2 diabetes and obesity significantly. Frog skin peptides have been extensively studied for their biological functions, among which, Phylloseptin peptides discovered in Phyllomedusinae frogs have been found to exert antimicrobial, antiproliferative and insulinotropic activities, while the mechanism associated with Phylloseptin‐induced insulin secretion remains elusive. In this study, we reported a novel peptide named Phylloseptin‐PBu, isolated and identified from Phyllomedusa burmeisteri, exhibited dose‐dependent insulinotropic property in rat pancreatic beta BRIN‐BD11 cells without altering cell membrane integrity. Further mechanism investigations revealed that Phylloseptin‐PBu‐induced insulin output is predominantly modulated by K_ATP‐[K⁺] channel depolarization triggered extracellular calcium influx and GLP‐1 receptor initiated PKA signalling activation. Overall, our study highlighted that this novel Phylloseptin‐PBu peptide has clear potential to be developed as a potent antidiabetic agent with established function‐traced mechanism and low risk of cytotoxicity.     

Heat shock proteins as mediators of aggregation-induced 'danger' signals: implications of the slow evolutionary fine-tuning of sequences for the antigenicity of cancer cells

Organisms 'tune' to their environment through adaptations which confer a selective advantage. However, in complex systems, a primary change of positive adaptive value might have multiple minor secondary effects, usually of negative adaptive value, which could invoke further counter-adaptations. This 'fine-tuning', a 'debugging', mainly at the intracellular level, would appear an evolutionary burden detracting from the positive nature of the primary change. However, if the primary mutation is in a potential oncogene, secondary, short-term effects may include the recruitment, in an apparently random manner, of unmutated non-oncogene products into the antigenic repertoire of the cancer cell. This 'danger' signal, provided by the co-aggregation of oncogene and non-oncogene products, would be mediated by inducible heat-shock proteins (Hsps), and lead to display of corresponding MHC-peptide complexes. It was argued previously that T cells specific for peptides from most 'self' intracellular antigens are not eliminated during T cell 'education', and so would be available for subsequent immune activation by the corresponding peptides. These considerations might explain why cancer specific antigens have been so elusive, why cancer antigenicity is often individual specific, and why therapeutic approaches involving complexes of peptides with Hsps may be successful.     

Anticancer peptide NK-2 targets cell surface sulphated glycans rather than sialic acids

Abstract Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.     

Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Single-cell analysis of tumors: Creating new value for molecular biomarker discovery of cancer stem cells and tumor-infiltrating immune cells

Biomarker-driven individualized treatment in oncology has made tremendous progress through technological developments, new therapeutic modalities and a deeper understanding of the molecular biology for tumors, cancer stem cells and tumor-infiltrating immune cells. Recent technical developments have led to the establishment of a variety of cancer-related diagnostic, prognostic and predictive biomarkers. In this regard, different modern OMICs approaches were assessed in order to categorize and classify prognostically different forms of neoplasia. Despite those technical advancements, the extent of molecular heterogeneity at the individual cell level in human tumors remains largely uncharacterized. Each tumor consists of a mixture of heterogeneous cell types. Therefore, it is important to quantify the dynamic cellular variations in order to predict clinical parameters, such as a response to treatment and or potential for disease recurrence. Recently, single-cell based methods have been developed to characterize the heterogeneity in seemingly homogenous cancer cell populations prior to and during treatment. In this review, we highlight the recent advances for single-cell analysis and discuss the challenges and prospects for molecular characterization of cancer cells, cancer stem cells and tumor-infiltrating immune cells.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

Role of antimicrobial peptides in host defense against mycobacterial infections

Worldwide, tuberculosis remains the most important infectious disease causing morbidity and death. Currently, at least one-third of the world's population is infected with Mycobacterium tuberculosis. In addition, the World Health Organization estimates that about 8-10 million new tuberculosis cases occur annually worldwide and this incidence is currently increasing. Moreover, multidrug-resistant tuberculosis has been increasing in incidence in many areas during the past decade. These situations underscore the importance of the development of new therapeutic agents against mycobacterial infectious diseases. In this article, it is review current progress in the understanding of antimicrobial peptides as potential candidates to develop an alternative/adjunct therapeutic strategy against tuberculosis. This immunoadjunctive therapy might be evaluated in the context of possible drug resistance. This review also summarizes the knowledge about the functions of antimicrobial peptides in the pulmonary innate host defense system and their role in mycobacterial infection, and at the same time outlines recent advances in our understanding of the combined effect of antimicrobial peptides and anti-tuberculosis drugs against intracellular mycobacteria. A concerted effort should now focus on the clinical application of antimicrobial peptides for their practical use.     

Proteomic analysis of mitochondria from Caenorhabditis elegans

Mitochondria play essential roles in cell physiological processes including energy production, metabolism, ion homeostasis, cell growth, aging and apoptosis. Proteomic strategies have been applied to the study of mitochondria since 1998; these studies have yielded decisive information about the diverse physiological functions of the organelle. As an ideal model biological system, the nematode Caenorhabditis elegans has been widely used in the study of several diseases, such as metabolic diseases and cancer. However, the mitochondrial proteome of C. elegans remains elusive. In this study, we purified mitochondria from C. elegans and performed a comprehensive proteomic analysis using the shotgun proteomic approach. A total of 1117 proteins have been identified with at least two unique peptides. Their physicochemical and functional characteristics, subcellular locations, related biological processes, and associations with human diseases, especially Parkinson's disease, are discussed. An orthology comparison was also performed between C. elegans and four other model organisms for a general depiction of the conservation of mitochondrial proteins during evolution. This study will provide new clues for understanding the role of mitochondria in the physiological and pathological processes of C. elegans.     

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as “thymic hormones,” are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting.     

Biotechnological implications of the salivary proteome

Although very attractive for noninvasive specimen collection, saliva has not yet been considered a relevant bodily fluid for the diagnosis and prognosis of diseases. The functional roles of specific salivary peptides and proteins have also not yet been studied in detail. Recent proteomic analysis of human whole saliva has shown that salivary biomarkers could contribute to the detection of local and systemic diseases, provided the standardization of proper sampling procedures exists. Recently, interesting and novel functions for different families of specific secretory peptides and proteins have been demonstrated, which could be a basis for the design of peptidomimetics with relevant biotechnological applications. In this review, we focus on the most recent advances in analysing salivary proteins and their potential application in biotechnology.