Human ß-defensins: The multi-functional natural peptide.

The increasing threat of antibiotic resistance among pathogenic microorganisms and the urgent demand for new antibiotics require immediate attention. Antimicrobial peptides exhibit effectiveness against microorganisms, fungi, viruses, and protozoa. The discovery of human ß-defensins represents a major milestone in biomedical research, opening new avenues for scientific investigation into the innate immune system and its resistance mechanisms against pathogenic microorganisms. Multiple defensins present a promising alternative in the context of antibiotic abuse. However, obstacles to the practical application of defensins as anti-infective therapies persist due to the unique properties of human ß-defensins themselves and serious pharmacological and technical challenges. To overcome these challenges, diverse delivery vehicles have been developed and progressively improved for the conjugation or encapsulation of human ß-defensins. This review briefly introduces the biology of human ß-defensins, focusing on their multistage structure and diverse functions. It also discusses several heterologous systems for producing human ß-defensins, various delivery systems created for these peptides, and patent applications related to their utilization, concluding with a summary of current challenges and potential solutions.

Peptide-based drug discovery: Current status and recent advances.

The progressive development of peptides from reaction vessels to life-saving drugs via rigorous preclinical and clinical assessments is fascinating. Peptide therapeutics have gained momentum with the evolution of techniques in peptide chemistry, such as microwave irradiation in solid- and solution-phase synthesis, ligation chemistry, recombinant synthesis, and amalgamation with synthetic tools, including metal catalysis. Diverse emerging technologies, such as DNA-encoded libraries (DELs) and display techniques, are changing the status quo in the discovery of peptide therapeutics. In this review, we analyzed US Food and Drug Administration (FDA)-approved peptide drugs and those in clinical trials, highlighting recent advances in peptide-based drug discovery.

Translational aspect in peptide drug discovery and development: An emerging therapeutic candidate.

In the last two decades, protein-protein interactions (PPIs) have been used as the main target for drug development. However, with larger or superficial binding sites, it has been extremely difficult to disrupt PPIs with small molecules. On the other hand, intracellular PPIs cannot be targeted by antibodies that cannot penetrate the cell membrane. Peptides that have a combination of conformational rigidity and flexibility can be used to target difficult binding interfaces with appropriate binding affinity and specificity. Since the introduction of insulin nearly a century ago, more than 80 peptide drugs have been approved to treat a variety of diseases. These include deadly diseases such as cancer and human immunodeficiency virus infection. It is also useful against diabetes, chronic pain, and osteoporosis. Today, more research is being done on these drugs as lessons learned from earlier approaches, which are still valid today, complement newer approaches such as peptide display libraries. At the same time, integrated genomics and peptide display libraries are new strategies that open new avenues for peptide drug discovery. The purpose of this review is to examine the problems in elucidating the peptide-protein recognition mechanism. This is important to develop peptide-based interventions that interfere with endogenous protein interactions. New approaches are being developed to improve the binding affinity and specificity of existing approaches and to develop peptide agents as potentially useful drugs. We also highlight the key challenges that must be overcome in peptide drug development to realize their potential and provide an overview of recent trends in peptide drug development. In addition, we take an in-depth look at early efforts in human hormone discovery, smart medicinal chemistry and design, natural peptide drugs, and breakthrough advances in molecular biology and peptide chemistry.

New Intracellular Peptide Derived from Hemoglobin Alpha Chain Induces Glucose Uptake and Reduces Blood Glycemia.

Intracellular peptides were shown to derive from proteasomal degradation of proteins from mammalian and yeast cells, being suggested to play distinctive roles both inside and outside these cells. Here, the role of intracellular peptides previously identified from skeletal muscle and adipose tissues of C57BL6/N wild type (WT) and neurolysin knockout mice were investigated. In differentiated C2C12 mouse skeletal muscle cells, some of these intracellular peptides like insulin activated the expression of several genes related to muscle contraction and gluconeogenesis. One of these peptides, LASVSTVLTSKYR (Ric4; 600 µg/kg), administrated either intraperitoneally or orally in WT mice, decreased glycemia. Neither insulin (10 nM) nor Ric4 (100 µM) induced glucose uptake in adipose tissue explants obtained from conditional knockout mice depleted of insulin receptor. Ric4 (100 µM) similarly to insulin (100 nM) induced Glut4 translocation to the plasma membrane of C2C12 differentiated cells, and increased GLUT4 mRNA levels in epididymal adipose tissue of WT mice. Ric4 (100 µM) increased both Erk and Akt phosphorylation in C2C12, as well as in epididymal adipose tissue from WT mice; Erk, but not Akt phosphorylation was activated by Ric4 in tibial skeletal muscle from WT mice. Ric4 is rapidly degraded in vitro by WT liver and kidney crude extracts, such a response that is largely reduced by structural modifications such as N-terminal acetylation, C-terminal amidation, and substitution of Leu8 for DLeu8 (Ac-LASVSTV[DLeu]TSKYR-NH2; Ric4-16). Ric4-16, among several Ric4 derivatives, efficiently induced glucose uptake in differentiated C2C12 cells. Among six Ric4-derivatives evaluated in vivo, Ac-LASVSTVLTSKYR-NH2 (Ric4-2; 600 µg/kg) and Ac-LASVSTV[DLeu]TSKYR (Ric4-15; 600 µg/kg) administrated orally efficiently reduced glycemia in a glucose tolerance test in WT mice. The potential clinical application of Ric4 and Ric4-derivatives deserves further attention.

Identification of Two Novel Peptides That Inhibit α-Synuclein Toxicity and Aggregation.

Aggregation of α-synuclein (αSyn) into proteinaceous deposits is a pathological hallmark of a range of neurodegenerative diseases including Parkinson's disease (PD). Numerous lines of evidence indicate that the accumulation of toxic oligomeric and prefibrillar αSyn species may underpin the cellular toxicity and spread of pathology between cells. Therefore, aggregation of αSyn is considered a priority target for drug development, as aggregation inhibitors are expected to reduce αSyn toxicity and serve as therapeutic agents. Here, we used the budding yeast S. cerevisiae as a platform for the identification of short peptides that inhibit αSyn aggregation and toxicity. A library consisting of approximately one million peptide variants was utilized in two high-throughput screening approaches for isolation of library representatives that reduce αSyn-associated toxicity and aggregation. Seven peptides were isolated that were able to suppress specifically αSyn toxicity and aggregation in living cells. Expression of the peptides in yeast reduced the accumulation of αSyn-induced reactive oxygen species and increased cell viability. Next, the peptides were chemically synthesized and probed for their ability to modulate αSyn aggregation in vitro. Two synthetic peptides, K84s and K102s, of 25 and 19 amino acids, respectively, significantly inhibited αSyn oligomerization and aggregation at sub-stoichiometric molar ratios. Importantly, K84s reduced αSyn aggregation in human cells. These peptides represent promising αSyn aggregation antagonists for the development of future therapeutic interventions.

Solution-Phase Fmoc-Based Peptide Synthesis for DNA-Encoded Chemical Libraries: Reaction Conditions, Protecting Group Strategies, and Pitfalls.

Peptide drug discovery has shown a resurgence since 2000, bringing 28 non-insulin therapeutics to the market compared to 56 since its first peptide drug, insulin, in 1923. While the main method of discovery has been biological display-phage, mRNA, and ribosome-the synthetic limitations of biological systems has restricted the depth of exploration of peptide chemical space. In contrast, DNA-encoded chemistry offers the synergy of large numbers and ribosome-independent synthetic flexibility for the fast and deeper exploration of the same space. Hence, as a bridge to building DNA-encoded chemical libraries (DECLs) of peptides, we have developed substrate-tolerant amide coupling reaction conditions for amino acid monomers, performed a coupling screen to illustrate such tolerance, developed protecting group strategies for relevant amino acids and reported the limitations thereof, developed a strategy for the coupling of α,α-disubstituted alkenyl amino acids relevant to all-hydrocarbon stapled peptide drug discovery, developed reaction conditions for the coupling of tripeptides likely to be used in DECL builds, and synthesized a fully deprotected DNA-decamer conjugate to illustrate the potency of the developed methodology for on-DNA peptide synthesis.

Neuropeptide signalling systems - An underexplored target for venom drug discovery.

Neuropeptides are signalling molecules mainly secreted from neurons that act as neurotransmitters or peptide hormones to affect physiological processes and modulate behaviours. In humans, neuropeptides are implicated in numerous diseases and understanding their role in physiological processes and pathologies is important for therapeutic development. Teasing apart the (patho)physiology of neuropeptides remains difficult due to ligand and receptor promiscuity and the complexity of the signalling pathways. The current approach relies on a pharmacological toolbox of agonists and antagonists displaying high selectivity for independent receptor subtypes, with the caveat that only few selective ligands have been discovered or developed. Animal venoms represent an underexplored source for novel receptor subtype-selective ligands that could aid in dissecting human neuropeptide signalling systems. Multiple endogenous-like neuropeptides as well as peptides acting on neuropeptide receptors are present in venoms. In this review, we summarise current knowledge on neuropeptides and discuss venoms as a source for ligands targeting neuropeptide signalling systems.

Rational design and biological evaluation of gemfibrozil modified Xenopus GLP-1 derivatives as long-acting hypoglycemic agents.

Novel methods for peptides structural modification and bioactivity optimization are highly needed in peptide-based drug discovery. Herein, we explored the use gemfibrozil (GFZ) as an albumin binder to enhance the stability and improve the bioactivity of peptides. Short-acting Xenopus glucagon-like peptide-1 (xGLP-1) analogues with anti-diabetic activity were selected as the starting point. Mono-GFZ conjugation, peptide sequence hybridization, and dimeric-GFZ derivatization were successively used to generate novel GFZ-xGLP-1 conjugates, biologically screened by various in vitro and in vivo models. Dimeric-GFZ modified conjugate 3b was finally identified as a promising anti-diabetic candidate with high albumin binding affinity, enhanced in vivo stability in SD rats, and long-acting hypoglycemic activity in db/db mice. Moreover, GFZ endowed 3b with strong lipid-regulating ability in DIO and db/db mice. In a twelve-week study, chronic administration of 3b in db/db mice resulted in sustained glycemic control, to a greater extent than liraglutide and semaglutide. In addition, 3b showed comparable therapeutic efficacies to liraglutide and semaglutide on HbA1c and pancreas islets protection. Our studies reveal 3b as a potential candidate for the treatment of metabolic diseases and indicate dimeric-GFZ modification as a novel method for peptide optimization.

The Place for Enzymes and Biologically Active Peptides from Marine Organisms for Application in Industrial and Pharmaceutical Biotechnology.

Since the beginning of written history, diverse texts have reported the use of enzymatic preparations in food processing and have described the medicinal properties of crude and fractionated venoms to treat various diseases and injuries. With the biochemical characterization of enzymes from distinct sources and bioactive polypeptides from animal venoms, the last sixty years have testified the advent of industrial enzymology and protein therapeutics, which are currently applicable in a wide variety of industrial processes, household products, and pharmaceuticals. Bioprospecting of novel biocatalysts and bioactive peptides is propelled by their unsurpassed properties that are applicable for current and future green industrial processes, biotechnology, and biomedicine. The demand for both novel enzymes with desired characteristics and novel peptides that lead to drug development, has experienced a steady increase in response to the expanding global market for industrial enzymes and peptidebased drugs. Moreover, although largely unexplored, oceans and marine realms, with their unique ecosystems inhabited by a large variety of species, including a considerable number of venomous animals, are recognized as untapped reservoirs of molecules and macromolecules (enzymes and bioactive venom-derived peptides) that can potentially be converted into highly valuable biopharmaceutical products. In this review, we have focused on enzymes and animal venom (poly)peptides that are presently in biotechnological use, and considering the state of prospection of marine resources, on the discovery of useful industrial biocatalysts and drug leads with novel structures exhibiting selectivity and improved performance.

PeptideNavigator: An interactive tool for exploring large and complex data sets generated during peptide-based drug design projects.

There is growing interest in peptide-based drug design and discovery. Due to their relatively large size, polymeric nature, and chemical complexity, the design of peptide-based drugs presents an interesting "big data" challenge. Here, we describe an interactive computational environment, PeptideNavigator, for naturally exploring the tremendous amount of information generated during a peptide drug design project. The purpose of PeptideNavigator is the presentation of large and complex experimental and computational data sets, particularly 3D data, so as to enable multidisciplinary scientists to make optimal decisions during a peptide drug discovery project. PeptideNavigator provides users with numerous viewing options, such as scatter plots, sequence views, and sequence frequency diagrams. These views allow for the collective visualization and exploration of many peptides and their properties, ultimately enabling the user to focus on a small number of peptides of interest. To drill down into the details of individual peptides, PeptideNavigator provides users with a Ramachandran plot viewer and a fully featured 3D visualization tool. Each view is linked, allowing the user to seamlessly navigate from collective views of large peptide data sets to the details of individual peptides with promising property profiles. Two case studies, based on MHC-1A activating peptides and MDM2 scaffold design, are presented to demonstrate the utility of PeptideNavigator in the context of disparate peptide-design projects.

Recent Advances Toward the Discovery of Drug-Like Peptides De novo.

Peptides are important natural molecules that possess functions as diverse as antibiotics, toxins, venoms and hormones, for example. However, whilst these peptides have useful properties, there are many targets and pathways that are not addressed through the activities of natural peptidic compounds. In these circumstances, directed evolution techniques, such as phage display, have been developed to sample the diverse chemical and structural repertoire of small peptides for useful means. In this review, we consider recent concepts that relate peptide structure to drug-like attributes and how these are incorporated within display technologies to deliver peptides de novo with valuable pharmaceutical properties.