Research Peptides

Jul 30, 2024

10 min read

Written by Dr. Johnathon Anderson, Ph.D., a research scientist specializing in regenerative medicine and serving as an Associate Professor at the University of California Davis School of Medicine

Overview of Research Peptides

Therapeutic peptides are a unique class of drugs made up of specific sequences of amino acids, typically weighing between 500-5000 Da. Research on these peptides began with foundational studies on natural human hormones like insulin, oxytocin, vasopressin, and gonadotropin-releasing hormone (GnRH). Since the synthesis of insulin, the first therapeutic peptide, in 1921, over 80 peptide-based drugs have gained approval worldwide, making peptide drug development a major focus in pharmaceutical research.

Early Discoveries and Insulin Breakthrough

In the early 20th century, researchers discovered bioactive peptides such as insulin and adrenocorticotrophic hormone, initially derived from natural sources. Insulin, a 51-amino acid peptide isolated by Frederick Banting in 1921, marked a monumental advance in treating diabetes. Insulin became available for patients just a year after its discovery, becoming the first commercial peptide drug in 1923. Animal-derived insulins dominated the market for decades until recombinant insulin production methods eventually took over to meet demand.

Advances from the 1950s to the 1990s

Between the 1950s and the 1990s, new peptide hormones and their receptors with therapeutic potential were identified. Advances in protein purification, synthesis, structural analysis, and sequencing led to the approval of nearly 40 peptide drugs worldwide. Synthetic peptides, such as synthetic oxytocin, synthetic vasopressin, and recombinant human insulin, emerged alongside natural peptides.

The Modern Era of Peptide Drug Development

The 21st century introduced a new phase in peptide drug development, driven by advances in structural biology, recombinant biologics, and new synthetic and analytic techniques. Today, the peptide drug development process includes discovery, design, synthesis, structural modification, and activity evaluation.

Since 2000, 33 non-insulin peptide drugs have been approved globally. These drugs now extend beyond hormone analogues to include complex, bio-inspired peptides. Notable examples include:

Enfuvirtide: A 36-amino acid peptide used to treat HIV-1.
Ziconotide: A neurotoxic peptide from the cone snail, approved in 2004 to manage severe chronic pain.
Teduglutide: A GLP-2 analogue for short bowel syndrome, produced using modified E. coli.
Liraglutide: A GLP-1 receptor agonist for type 2 diabetes, featuring a C-16 fatty acid attached to its sequence.

Peptide drugs now address a broad range of therapeutic areas, including urology, respiratory conditions, pain management, oncology, metabolic and cardiovascular health, and antimicrobial uses. Over 170 peptides are currently in clinical trials, with many more in preclinical studies, showcasing their growing role in medicine.

Therapeutic Peptides: Advantages and Limitations

Therapeutic peptides function as hormones, growth factors, neurotransmitters, ion channel ligands, or anti-infective agents. They bind with high specificity to cell surface receptors, initiating precise cellular responses. Similar to other biologics, such as proteins and antibodies, peptides have a targeted mode of action but offer lower immunogenicity and reduced production costs compared to biologics.

Comparison with Small Molecule Drugs

Small molecule drugs have a long history of therapeutic use, featuring benefits like low production costs, oral administration, and effective cell membrane penetration. These drugs, whether extracted naturally or synthesized, are generally more affordable than peptides and biologics. Oral administration provides better patient compliance, while the small molecular size allows small molecules to target intracellular molecules effectively.

However, small molecule drugs face challenges in targeting large surface interactions, such as protein-protein interactions (PPIs), due to their limited surface coverage (300–1000 Å²) compared to peptides, which can cover larger areas (1500–3000 Å²). Peptide drugs, with their flexible structures and larger size, excel in inhibiting PPIs effectively.

Small molecules also have lower specificity than peptides, which can result in unintended effects. For example, sorafenib and sunitinib are small-molecule tyrosine kinase inhibitors used to block VEGF receptors and treat cancer. However, they also affect other kinase receptors, which can lead to cytotoxicity and broader side effects. Peptide drugs, by contrast, offer a more targeted approach with reduced off-target risks.

Key Challenges in Therapeutic Peptides

Limited Membrane Permeability

Peptides generally have low membrane permeability, which restricts their ability to enter cells and interact with intracellular targets. Factors like peptide length and amino acid composition affect this permeability. Consequently, most peptide drugs in development—over 90%, according to a 2018 study by Lau et al.—are designed to target receptors on the cell surface, such as GPCRs, GLP-1 receptors, and GnRH receptors.

Low Stability in the Body

Natural peptides are composed of amino acids linked by amide bonds, which lack the structural support of secondary or tertiary configurations. Without protective modifications, these bonds are vulnerable to breakdown by enzymes, resulting in a short half-life and rapid elimination from the body. This limited stability affects both the chemical and physical properties of peptides, making them challenging to maintain as effective treatments.

These intrinsic limitations shape both the challenges and opportunities in peptide drug design, guiding researchers toward strategies that can enhance stability, permeability, and therapeutic efficacy.

Developmental Path of Therapeutic Peptides: Discovery, Production, and Optimization

The exploration of peptide drugs began by identifying natural hormones and peptides that regulate critical physiological functions, such as blood glucose control, essential in treating hormone deficiencies. For example, managing diabetes involves either direct insulin injections or targeting receptors like GLP-1 to promote insulin production.

Early strategies in peptide drug development focused on using natural hormones or analogues from animal sources, such as insulin, GLP-1, somatostatin, GnRH, vasopressin, and oxytocin. However, limitations associated with these natural peptides spurred interest in optimizing their structures, leading to the development of hormone-mimetic peptide drugs that maintain function but offer improved stability and efficacy.

Peptides Mimicking Hormones

Glucagon-like peptide-1 (GLP-1) is a 37-amino acid hormone that regulates insulin production and secretion but has a very short half-life in the body. Researchers have modified its sequence to boost stability without sacrificing potency, which led to the creation of leading anti-diabetes drugs such as Trulicity (dulaglutide), Victoza (liraglutide), and Ozempic (semaglutide), and GnRH derived peptide drugs.

GnRH is a 10-amino acid peptide produced in the hypothalamus that regulates reproductive functions. Altering the GnRH structure enabled the development of several key drugs, including leuprolide, a GnRH receptor agonist used in prostate cancer and endometriosis, and degarelix, a GnRH antagonist designed to treat advanced prostate cancer .

Peptides from Natural Sources

Peptides are sourced from natural sources often have therapeutic properties. For instance, snake venom peptides are rich in disulfide bonds, making them highly stable and potent. These venom peptides have been modified to create therapeutic peptides that target ion channels and receptors, aiding treatments in areas like cardiovascular and nerve-related conditions.

One example, Exenatide is modeled after a peptide found in Gila monster venom, exenatide is used as a GLP-1. Another example, Ziconotide is derived from cone snail venom, this peptide is used for chronic neuropathic pain relief.

Non-ribosomal peptides (NRPs) are normally produced peptides, and non-ribosomal peptides (NRPs) are built by specialized enzymes, providing breakdown and increased stability in the body. NRPs, sourced mainly from bacteria and fungi, have yielded potent drugs like vancomycin and cyclosporin, with antibacterial and immunosuppressive properties. Other NRP classes, like cyclodepsipeptides found in plants, show increased stability for oral delivery but are challenging to synthesize and study.

These peptides from natural products showcase their promising potential for drug development, particularly when engineered for enhanced stability and targeted action.

Peptide Synthesis: An Overview

The chemical synthesis of peptides has become highly advanced, thanks largely to the development of solid-phase peptide synthesis (SPPS) by Merrifield in 1963. SPPS technology has since undergone significant improvements, both in its techniques and in the materials used, making it a cornerstone of modern peptide production. This method allows amino acid coupling and deprotection to occur in a single reactor, facilitating the use of automatic peptide synthesizers. SPPS-derived peptides are often purer than those produced by recombinant technology, as they lack biological impurities like enzymes or DNA fragments. Additionally, the impurities in SPPS products are usually limited to byproducts from synthesis reactions, making them easier to purify and analyze.

How SPPS Works

SPPS involves attaching amino acids to a solid resin, then freeing the amino group from its protective group in a repeated cycle. Various resins, such as Wang and Merrifield resins, help introduce amide or free carboxylic groups at the peptide's C-terminus. Advances in resin technology have led to specialized resins and linkers that allow for the synthesis of long or cyclic peptides. Amino groups and side chains of amino acids are protected with different chemical groups to prevent unwanted reactions and to reduce peptide aggregation during synthesis. Two main SPPS strategies are used to remove these protection groups: Fmoc-SPPS and Boc-SPPS, which use different reagents (Fmoc for milder conditions and Boc for a more aggressive approach).

Fmoc-SPPS and Boc-SPPS

The Fmoc-SPPS method has become the preferred strategy, as Fmoc protection can be removed under milder conditions, which reduces toxicity and odor. However, Boc-SPPS is often more suitable for long peptide synthesis due to its ability to break down peptide aggregation effectively.

Challenges in Long Peptide Synthesis

While peptides under 50 residues can be routinely synthesized with Fmoc-SPPS, synthesizing longer chains remains difficult, especially on a large scale. Advanced automated synthesizers, like the CEM Liberty PRIME, enable labs to synthesize peptides quickly, even producing up to 192 different sequences simultaneously. These machines use infrared or microwave heating and UV monitoring to streamline synthesis and ensure high-quality production. However, infrared and microwave heating are challenging to scale up for larger production due to risks of uneven heating, which can create byproducts. As a result, Good Manufacturing Practice (GMP) protocols often rely on mild reaction conditions to minimize impurities, which limits the synthesis of long peptides on an industrial scale.

Impact of Peptide Synthesis on Therapeutic Development

SPPS and other chemical synthesis methods have been instrumental in advancing peptide therapeutics, allowing for precise control and modification of peptide structures. Many therapeutic peptides, including oxytocin and teriparatide, are produced via chemical synthesis, which also supports the modification and customization of peptide structures for enhanced functionality. This progress in chemical synthesis continues to accelerate the development and optimization of new peptide-based drugs.

Advancements in Peptide Drug Delivery

Peptide modifications have been critical in enhancing peptide stability and efficacy, making them more suitable as drug candidates. However, peptides are often rapidly degraded by digestive enzymes in the stomach and intestine, meaning that most peptide drugs require administration by injection. Researchers are exploring new delivery routes to address these limitations.

One promising approach is co-formulating peptides with permeation enhancers, enabling oral administration. For example, semaglutide, which is conjugated with C18 fatty acid, is administered via weekly subcutaneous injections and offers greater stability than other GLP-1 analogues. More notably, co-formulating semaglutide with sodium N-[8-(2-hydroxybenzoyl amino)] caprylate (SNAC) allows for oral administration to treat Type 2 diabetes (T2DM). SNAC protects semaglutide from stomach degradation by reducing enzyme activity and enhances its absorption through the gastric membrane, aiding its entry into circulation. This co-formulation technique, as well as other enhancers and hydrogels, has been effective in trials for other peptide drugs, like octreotide and insulin. Additionally, alternative delivery methods, such as pulmonary, transdermal, and implantable pump-based administration, are under investigation, with inhalable insulin and micro-implantable insulin pumps showing promise. These methods may broaden peptide drug application in the future.

Therapeutic Peptides for Diabetes Treatment

Type 2 diabetes mellitus (T2DM) is often treated with peptide drugs, such as GLP-1 receptor agonists (GLP-1RAs) and insulin. GLP-1 is a natural hormone produced by the intestine that interacts with receptors in various body tissues to stimulate insulin secretion, reduce glucagon release, increase satiety, and slow gastric emptying. However, GLP-1 is quickly degraded by the enzyme DPP-4, requiring synthetic GLP-1RAs to sustain its activity. Since the approval of exenatide in 2005, several GLP-1RAs, including liraglutide, lixisenatide, dulaglutide, and semaglutide, have been introduced to effectively lower blood glucose levels and improve fasting glucose levels.

GLP-1RAs are also beneficial in addressing T2DM complications. For instance, diabetic nephropathy, a kidney disease linked to T2DM, can be managed with certain GLP-1RAs. In clinical studies, lixisenatide reduced urinary excretion of specific minerals by affecting sodium transport mechanisms, while liraglutide decreased proteinuria and improved kidney function markers. GLP-1RAs also show cardiovascular benefits, with liraglutide and semaglutide reducing cardiovascular risk factors, although the underlying mechanisms are not yet fully understood. Furthermore, GLP-1RAs have helped in managing obesity in T2DM patients, with studies showing significant weight loss and fat reduction in patients using exenatide or liraglutide. There is also evidence suggesting that GLP-1RAs improve bone health by enhancing blood supply to bones, promoting strength and reducing fracture risk. Despite gastrointestinal side effects, GLP-1RAs’ additional benefits, such as renal protection and cardiovascular advantages, highlight their role in future T2DM treatment.

Therapeutic Peptides for Cardiovascular Disease

Cardiovascular disease is the leading cause of death worldwide, with hypertension as a primary risk factor. The renin-angiotensin-aldosterone system (RAAS), which regulates blood pressure, is a common target for cardiovascular therapies. ACE inhibitors, for example, reduce blood pressure by limiting angiotensin II production. Synthetic angiotensin II was approved in 2017 for boosting blood pressure in cases of septicemia, and other peptides from natural sources, like egg white-derived IRW, have shown potential as RAAS-targeting agents.

Natriuretic peptides (NPs) are another class of peptides involved in cardiovascular regulation, promoting vasodilation and reducing blood pressure. Nesiritide, a BNP analogue, was FDA-approved in 2001 for heart failure treatment, though it saw limited use due to safety concerns. More promising is cenderitide, a dual agonist targeting NPR-A and NPR-B receptors, which is undergoing clinical trials for heart and renal failure treatments. Experimental peptides, such as vasoactive intestinal peptide and cyclopeptide RD808, have also shown benefits in animal studies, reducing myocardial fibrosis and cardiac damage. The CRF-related peptide system is emerging as a potential cardiovascular target, although its role in heart health is still being studied. Additionally, DPP-4 enzyme levels have been associated with vascular function, suggesting it may be another therapeutic target for preventing cardiovascular events.

These peptide-based therapies underscore the potential of targeted peptides in managing both diabetes and cardiovascular diseases, offering new treatment avenues with multiple health benefits.

Conclusion and Future Outlook

Peptides have emerged as a unique and promising class of therapeutic agents due to their specific biochemical properties and therapeutic effectiveness. While they hold advantages over traditional small molecules and large biologics, peptides face challenges with stability and cell permeability due to limitations in amino acid composition. To address these, significant research has been dedicated to the discovery, production, and optimization of peptide drugs.

Advances in peptide discovery are driven by combining traditional techniques with modern approaches, such as rational design and phage display. This integration allows researchers to efficiently develop effective, targeted peptide leads. Additionally, single or combined methods of chemical and biological synthesis have enabled the large-scale production of peptides with consistency and reliability. By modifying peptides through chemical synthesis or genetic engineering, researchers can enhance peptide stability and optimize their therapeutic activity.

While therapeutic peptides initially focused on mimicking natural hormones, research has expanded to the intentional design of peptides with enhanced biochemical functions and targeted physiological effects. Breakthroughs in molecular biology, peptide chemistry, and delivery technologies have fueled progress across peptide drug discovery, production, and application. With over 80 therapeutic peptides already available on the market and hundreds more in clinical trials, peptides are being used to treat conditions ranging from diabetes and cardiovascular diseases to cancer, infectious diseases, and gastrointestinal issues. Given their substantial therapeutic potential, promising market prospects, and economic value, therapeutic peptides are likely to continue attracting significant investment and research, setting the stage for long-term advancements and success.

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References

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=28737935

https://doi.org/10.1111%2Fcbdd.12055