Introduction to Peptides: Biological Functions and Biomedical Applications

Peptides are short polymers of amino acids linked by amide bonds (peptide bonds) and play critical roles in biology and medicine. They are typically defined as molecules composed of up to ~50 amino acids, beyond which they are often considered proteins. This boundary is not strict, as some longer sequences (e.g. amyloid beta ~42 amino acids) are called peptides, whereas some small proteins (like insulin, 51 amino acids) are sometimes referred to as peptide hormones. Peptides occur naturally in all organisms and can function as hormones, neurotransmitters, growth factors, or antimicrobial agents. The field of peptide science has expanded rapidly since the first peptide hormone (insulin) was isolated in 1921, and over 80 peptide drugs have been approved for clinical use worldwide. Advances in peptide synthesis, modification, and analysis have allowed scientists to overcome many inherent drawbacks of peptides and harness them as therapeutic agents. This review provides a comprehensive overview of peptides, including their biochemical definition and classification, mechanisms of action, endogenous functions, methods of design and synthesis, research applications, current therapeutics, challenges in pharmacology, and considerations for their characterization and regulation.

Biochemical Definition and Classification of Peptides

Definition and Peptide Bonds: A peptide is a molecule consisting of two or more amino acids joined by peptide bonds (amide bonds). The peptide bond forms when the carboxyl group (C-terminus) of one amino acid reacts with the amino group (N-terminus) of another, releasing water in a condensation reaction. This covalent linkage creates a backbone of repeating units (–NH–CHR–CO–) characteristic of peptides and proteins. The term “peptide” comes from the Greek πέσσειν (“to digest”), reflecting the early discovery of peptides as protein digestion products. Peptides are distinguished from proteins mainly by size: dipeptides, tripeptides, and tetrapeptides contain 2, 3, or 4 amino acids, respectively, while oligopeptides usually have fewer than 10–15 residues and polypeptides have more than ~10–20 residues. Large polypeptides (over ~40–50 amino acids) are typically considered proteins, though the distinction is somewhat arbitrary.

Ribosomal vs. Non-Ribosomal Peptides: One way to classify peptides is by their biosynthetic origin. Ribosomal peptides are gene-encoded products synthesized via mRNA translation. They often begin as larger precursor proteins that undergo proteolytic cleavage and other post-translational modifications to yield the active peptide. Many hormones and neuropeptides fall in this class (e.g. opioid peptides, vasoactive intestinal peptide, tachykinins). Ribosomal peptides frequently require processing such as removal of signal peptides or enzymatic trimming to achieve their mature form. In contrast, non-ribosomal peptides (NRPs) are assembled by specialized enzyme complexes (non-ribosomal peptide synthetases) rather than the ribosome. NRPs often have complex cyclic or branched structures and can include unusual amino acids or modifications. They are commonly produced by fungi, bacteria, and plants and include many antibiotics and toxins (for example, the cyclic peptide antibiotic vancomycin is an NRP). Notably, non-ribosomal peptides tend to be more resistant to proteases and thus more stable in vivo than linear ribosomal peptides. A classic example of an NRP in humans is glutathione, a tripeptide (γ-Glu-Cys-Gly) that is synthesized enzymatically and serves as a key antioxidant.

Peptide Classes and Post-Translational Modifications: Beyond biosynthetic origin, peptides can be categorized by function or source. Hormonal peptides (e.g. insulin, glucagon, oxytocin) circulate as messengers regulating physiology; neuropeptides (e.g. substance P, endorphins) act as neurotransmitters or neuromodulators; cytokine and growth factor peptides (many of which are small proteins) mediate cell signaling in the immune and endocrine systems; antimicrobial peptides (e.g. defensins) provide innate immune defense. Milk peptides are generated by proteolysis of milk proteins (either during digestion or fermentation) and can have antihypertensive or immunomodulatory effects. Peptide fragments in general may result from controlled proteolysis in research (e.g. digesting a protein for analysis) or from natural protein turnover. Importantly, many bioactive peptides require specific post-translational modifications (PTMs) for full activity. For example, over half of all known peptide hormones are C-terminally amidated, a modification essential for their receptor binding and function. Amidation (conversion of a C-terminal –COOH to –CONH₂) is critical in peptides like vasopressin, oxytocin, and calcitonin gene-related peptide. Other common PTMs include N-terminal acetylation, N-terminal pyroglutamate formation (cyclization of a terminal glutamine to pyroglutamic acid, as seen in thyrotropin-releasing hormone), serine/threonine phosphorylation (important in some intracellular peptides), and tyrosine sulfation (e.g. in cholecystokinin, a gut peptide hormone). Peptides may also form disulfide bonds between cysteine residues, which cyclize the peptide and stabilize its structure (for instance, insulin’s two chains are linked by disulfides, and many small peptides like somatostatin or oxytocin are internally cyclized by disulfides). Such modifications are often crucial for biological activity, receptor recognition, or metabolic stability. In summary, peptides are chemically diverse: they vary in length, composition (including non-standard amino acids in NRPs), and can bear multiple modifications that expand their functional repertoire.

Natural vs. Synthetic Peptides: Peptides are produced naturally within organisms, but they can also be made in the laboratory. Endogenous ribosomal peptides are encoded in the genome (often as part of larger precursor proteins) and generated by the cell’s translational machinery, whereas non-ribosomal peptides are assembled by enzymatic templates. In the lab, peptides can be synthesized chemically or produced recombinantly. The first synthetic peptide (a dipeptide) was made by Emil Fischer and Ernest Fourneau in 1901, and by 1953 Vincent du Vigneaud had synthesized the hormone oxytocin (9 amino acids). Today, peptide synthesis is routine, enabling scientists to create peptides identical to natural ones or design novel sequences. Modern peptide drug development often combines both worlds: for example, semisynthesis can involve producing a peptide by recombinant DNA technology and then chemically modifying it. Whether derived from nature or synthesized de novo, peptides must be characterized carefully. Endogenous peptides often undergo PTMs as noted, and synthetic peptides can be engineered with those same modifications to mimic natural peptide structures or improve upon them for drug use.

Mechanisms of Action of Peptides

Peptides exert their biological effects primarily by binding to specific molecular targets, most commonly cell-surface receptors. Because of their size and polarity, peptides typically do not readily cross cell membranes, so the majority of peptide signals act extracellularly on receptors in the plasma membrane. Indeed, over 90% of peptides in clinical development target extracellular receptors, especially G protein-coupled receptors (GPCRs) and enzyme-linked receptors. There are, however, exceptions where peptides can access the cell interior (for example, via endocytosis or if modified for cell penetration), as discussed below.

Receptor Interactions (GPCRs and RTKs): A large number of peptide hormones and neurotransmitters are ligands for GPCRs, which are seven-transmembrane receptors coupled to intracellular G-proteins. Upon peptide binding, GPCRs activate second messenger pathways inside the cell. Examples include neuropeptides like the enkephalins and endorphins (which bind opioid GPCRs), substance P (binds neurokinin receptors), angiotensin II (binds AT₁ GPCR to regulate blood pressure), and glucagon and GLP-1 (bind class B GPCRs to regulate glucose homeostasis). Peptides can also act on receptor tyrosine kinases (RTKs) or other enzyme-linked receptors. For instance, insulin is a peptide hormone (51 amino acids, two chains) that activates the insulin RTK on cells to regulate metabolism. Another example is epidermal growth factor (EGF), a 53-amino-acid peptide growth factor that binds the EGF receptor (a tyrosine kinase) to stimulate cell proliferation. Generally, peptides display high specificity for their receptors due to a precise fit of the peptide’s 3D structure with the receptor’s binding site. Short linear peptides often adopt a particular conformation upon binding (e.g. an alpha-helix in the receptor-binding region), whereas cyclic peptides may present a rigid structure that enhances binding affinity. Importantly, some peptide ligands can trigger biased signaling at GPCRs – preferentially activating one downstream pathway over another – which is an area of intense pharmacological research (e.g. biased agonism of opioid or angiotensin receptors by certain analogs). Whether acting on GPCRs, RTKs, or ion channels (e.g. peptide toxins blocking ion channels), peptides initiate signaling cascades that modify cellular function in processes ranging from neurotransmission to gene expression.

Cellular Uptake and Membrane Transport: Unlike small molecule drugs, most peptides are not membrane-permeable. Their polar backbone and often large size prevent diffusion across the hydrophobic lipid bilayer. As a result, peptide drugs are usually administered parenterally (by injection) to reach their targets. Once in circulation, peptides generally remain extracellular, targeting receptors on cell surfaces. However, some specialized peptides can enter cells. Cell-penetrating peptides (CPPs), such as the HIV TAT peptide (a short arginine-rich peptide), can traverse cell membranes and have been used to deliver cargo molecules into cells. Certain cyclic peptides and very small peptides (di- and tri-peptides) may also be transported into cells via peptide transporters. But for the vast majority of therapeutic peptides, the targets are extracellular. This limitation is reflected by the fact that >90% of peptide drug candidates are aimed at extracellular targets. Researchers are exploring methods to improve peptide delivery, including conjugation to cell-penetrating moieties, formulation with membrane-permeabilizing excipients, or using nanoparticles to ferry peptides into cells. In physiology, some peptide hormones (like thyroid-releasing hormone, a tripeptide) can be taken up by specific transporters in the blood-brain barrier, but these cases are exceptions to the rule that peptides signal from outside the cell.

Stability and Degradation: An intrinsic challenge for peptides is their poor in vivo stability. Peptides consist of amino acid chains without the extensive tertiary structures of larger proteins; hence they are readily recognized and cleaved by peptidases. Proteolytic enzymes in blood, tissues, and the digestive tract can rapidly hydrolyze peptide bonds. As a result, many natural peptides have very short half-lives (ranging from seconds to minutes). For example, the incretin hormone GLP-1 is degraded within minutes by dipeptidyl peptidase-4, and enkephalin neuropeptides are quickly cleaved by enkephalinases. The amide bonds of peptides are susceptible to hydrolysis, and without secondary or tertiary structure for protection, linear peptides are chemically and physically unstable, leading to fast elimination. Proteases like aminopeptidases (trimming at N-termini), carboxypeptidases (trimming at C-termini), and endopeptidases (cutting internally at specific residues) all contribute to peptide degradation. This liability affects both endogenous peptide signaling (necessitating continuous production or storage of peptides in secretory granules) and the use of peptides as drugs (necessitating high or frequent dosing, or structural modifications as described later). Peptides are also generally cleared rapidly by the kidneys due to their small size and hydrophilicity; they filter into the renal tubules and, if not reabsorbed or protected by binding proteins, are excreted. To overcome these stability issues, organisms and drug designers employ several strategies. Endogenous peptide hormones often have post-translational modifications that enhance stability – for instance, C-terminal amidation can protect against carboxypeptidases, and cyclization (head-to-tail or via disulfide bonds) can make peptides less recognizable to proteases. In pharmacology, modifying peptides with non-natural features (D-amino acids, unusual linkages) or encapsulating them in delivery systems can dramatically extend their half-life (examples are given in later sections). Nonetheless, without intervention, peptides are generally short-lived molecules in circulation.

Receptor Desensitization: A pharmacodynamic consideration for peptide agonists is receptor desensitization and downregulation. Many peptide receptors (especially GPCRs) undergo regulatory feedback: upon continuous or repeated stimulation by an agonist, the receptor becomes less responsive. Mechanistically, GPCRs activated by peptide agonists can be phosphorylated by G-protein-coupled receptor kinases (GRKs) and bound by β-arrestins, leading to uncoupling from G-proteins and receptor internalization into endosomes. If agonist exposure is sustained, receptors may be targeted for degradation or recycle slowly, resulting in tolerance to the peptide. For example, chronic use of peptide analogs of GnRH initially stimulates pituitary receptors but later causes receptor downregulation and a drop in gonadotropin release – a phenomenon exploited in treating hormone-dependent diseases (using long-acting GnRH agonists to induce a chemical castration by desensitization). In general, long-term treatment with a peptide (or any agonist) can limit its effectiveness due to receptor-mediated desensitization, thereby narrowing the therapeutic window. Drug developers sometimes mitigate this by altering dosing regimens (pulsatile dosing to allow receptor recovery) or designing biased agonists that activate signaling pathways with less recruitment of desensitizing machinery. Receptor desensitization is an important aspect of peptide drug pharmacology, as it can lead to diminished response over time or after repeated doses (tachyphylaxis). It underscores the need to understand each peptide’s receptor signaling and internalization profile when developing therapeutic protocols.

Endogenous Peptides in Physiology and Systems Biology

Endogenous peptides serve as potent signaling molecules across virtually all organ systems. They coordinate complex physiological processes by acting as hormones, neurotransmitters, and paracrine signals. Below we highlight key roles of peptides in several systems biology contexts:

Neuropeptides (Neurosignaling): In the nervous system, many neurons use peptides as neurotransmitters or neuromodulators. Examples include the endorphins and enkephalins (endogenous opioid peptides that modulate pain and reward), substance P (an 11-amino-acid neuropeptide that transmits pain signals in the spinal cord and brainstem), neuropeptide Y (NPY) (a 36-residue peptide that regulates appetite, stress responses, and circadian rhythms), oxytocin and vasopressin (peptide hormones from the hypothalamus that also act in the brain to influence social bonding, stress, and water balance), and CGRP (calcitonin gene-related peptide, a vasodilatory neuropeptide involved in migraine pathology). Neuropeptides are often co-released with classical neurotransmitters and typically act on GPCRs, leading to slower but prolonged signaling effects compared to small neurotransmitters. They are critical in mediating pain, mood, feeding behavior, and autonomic regulation. For instance, substance P released from peripheral nerves triggers inflammatory responses and pain perception, while endorphins in the brainstem can inhibit pain transmission by acting on opioid receptors. Neuropeptides can also influence neuroplasticity and development. An example of systems-level neuropeptide function is neuropeptide Y’s role in metabolism and cancer: NPY not only affects feeding and energy homeostasis, but its receptors are found in some tumors, where NPY signaling can influence angiogenesis and tumor growth. This illustrates how neuropeptidergic systems intersect with other physiological processes (metabolic and vascular in this case).

Metabolic Regulation (Endocrine Peptides): Peptide hormones orchestrate many aspects of metabolism and energy balance. The pancreas produces insulin (51 aa) and glucagon (29 aa) to control blood glucose levels – insulin promotes anabolic processes (glucose uptake, glycogen synthesis) while glucagon has catabolic effects (stimulating glycogen breakdown and gluconeogenesis). The gut and adipose tissue secrete numerous metabolic peptides: GLP-1 (glucagon-like peptide-1, 37 aa) is released from intestinal L-cells in response to food and enhances insulin secretion while suppressing appetite; ghrelin (28 aa, from the stomach) stimulates hunger; leptin (167 aa protein hormone, but often discussed alongside peptides) from fat signals satiety. The coordinated actions of these peptides maintain energy homeostasis. Peptides also regulate metabolism through cross-talk between organs. For example, adipose-derived peptides (like adiponectin, a 244-aa protein hormone with modular peptide structure) influence insulin sensitivity in muscle and liver. A recent review highlighted how peptides act as fundamental regulators of metabolism and inter-organ communication, being secreted by pancreas, gut, hypothalamus, adipose tissue and other organs. Many metabolic diseases are associated with peptide dysregulation: in type 2 diabetes, GLP-1 production or action is impaired, and therapeutic analogs of GLP-1 are used to restore metabolic control. Peptide hormones such as adrenocorticotropic hormone (ACTH) (39 aa from the pituitary) regulate adrenal steroid output, linking stress to metabolism, and thyroid-stimulating hormone (TSH) (a glycoprotein composed of peptide subunits) controls basal metabolic rate via thyroid hormone release. In summary, endocrine peptides form a complex network controlling metabolism, and disturbances in this network can cause diabetes, obesity, or metabolic syndrome. Consequently, metabolic peptides like insulin and GLP-1 analogs have become cornerstone therapies for diabetes and obesity.

Immunomodulation: The immune system both produces and is regulated by peptides. Cytokines and chemokines, many of which are relatively small proteins or large peptides (typically 8–80 amino acids for chemokines), direct immune cell trafficking and activation. For example, interleukin-8 (IL-8) is a 72-amino-acid chemokine (processed to about 8 kDa) that recruits neutrophils to infection sites. Thymic peptides such as thymosin α₁ (28 aa) and thymosin β₄ (43 aa) modulate T-cell differentiation and tissue repair; thymosin β₄ has been studied for its role in promoting wound healing and new blood vessel growth (angiogenesis) in injured tissues. The nervous and immune systems intersect through neuropeptides as well: peptides like VIP (vasoactive intestinal peptide) and PACAP can suppress pro-inflammatory responses and are being explored as anti-inflammatory agents. The bradykinin peptide (9 aa) is generated during inflammation and causes vasodilation, increased vascular permeability, and pain – it exemplifies a peptide mediator of inflammation (bradykinin receptor antagonists are used to treat hereditary angioedema). Additionally, antimicrobial peptides (AMPs) are produced by immune and epithelial cells as a first-line defense against pathogens; examples include defensins and cathelicidins which can directly kill bacteria by disrupting membranes. These AMPs also have immunomodulatory roles, such as recruiting immune cells and modulating cytokine production. Thus, peptides participate in immune responses both as messengers and effectors, coordinating inflammation, cell migration, and host defense.

Angiogenesis and Cardiovascular Peptides: Vascular growth and function are influenced by peptides. The classic angiogenic growth factors like VEGF and FGF are larger proteins, but smaller peptides also play roles. For instance, angiotensin II, an octapeptide, is best known as a vasoconstrictor regulating blood pressure, but it also stimulates proliferation of vascular smooth muscle and can contribute to pathological cardiac hypertrophy. Apelin is a peptide (13–36 aa forms) secreted by adipose and other tissues that binds the APJ receptor; it regulates cardiovascular function, fluid balance, and has pro-angiogenic effects in some contexts. In the cardiovascular system, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) (28 aa and 32 aa, respectively) are cardiac-derived hormones that cause vasodilation and promote renal salt excretion, counteracting hypertension and volume overload. These peptides have diagnostic and therapeutic significance in heart failure. During tissue regeneration or cancer, various endogenous peptides modulate new vessel formation. For example, certain fragments of larger proteins act as angiogenesis inhibitors – e.g. endostatin (a 20 kDa fragment of collagen XVIII) and angiostatin (a fragment of plasminogen) – but these are larger than typical peptides. On the flip side, snake venom peptides have been found to mimic angiogenic factors; a notable example is a peptide in some snake venoms that acts as a VEGF mimic (called VEGF-F or svVEGF) and can induce angiogenesis. In summary, the cardiovascular system is regulated by an array of peptide signals that control vessel tone, cardiac remodeling, and angiogenesis, linking the endocrine, nervous, and immune systems to vascular health.

Other Systems: Nearly every physiological system utilizes peptide signals. The gastrointestinal tract produces many gut peptides (e.g. cholecystokinin (CCK), secretin, motilin, PYY) that regulate digestion, appetite, and pancreatic secretion. The renal system responds to peptides like antidiuretic hormone (ADH/vasopressin) which concentrates urine, and natriuretic peptides which encourage salt excretion. The reproductive system is controlled by peptide hormones such as GnRH (gonadotropin-releasing hormone, 10 aa) from the hypothalamus, which in turn stimulates pituitary release of LH and FSH (glycoprotein hormones) affecting gonadal function. Peptides also mediate pain and analgesia (e.g. substance P vs. endorphins), stress (corticotropin-releasing hormone, CRH, 41 aa, triggers ACTH release), and circadian rhythms (the neuropeptide orexin regulates wakefulness, and melatonin – a modified tripeptide derivative – signals darkness to the body). New peptides and their functions continue to be discovered thanks to advances in peptidomics and proteomics, which allow large-scale profiling of peptides in tissues and blood. These tools have uncovered previously unrecognized peptides and novel PTMs, expanding our understanding of the “peptidome” and its roles. In summary, endogenous peptides are integral to homeostasis, serving as key regulators in neural, endocrine, immune, and vascular networks. Their diversity and specificity make them attractive as drugs, but also pose challenges for delivery and stability, as the next sections will address.

Synthetic Peptide Design and Engineering

The ability to synthesize and modify peptides with precision has opened the door to designing peptides with improved properties for research and therapeutic use. Modern peptide design involves not only assembling the desired amino acid sequence, but also incorporating chemical modifications that enhance stability, activity, or target specificity. Key methodologies include solid-phase peptide synthesis (SPPS), strategies for cyclization, conjugation of peptides to other molecules, and systematic analog development through residue modifications.

Solid-Phase Peptide Synthesis (SPPS): Chemical peptide synthesis is most commonly done by SPPS, a technique pioneered by Robert Merrifield in 1963. In SPPS, the growing peptide chain is anchored to an insoluble resin bead while reactions are carried out, allowing easy separation of reagents and byproducts at each step. The process involves a repetitive cycle: (1) coupling of a protected amino acid to the N-terminus of the peptide on the resin, and (2) removal of the N-terminal protecting group (deprotection) to expose a free amine for the next coupling. The cycle repeats until the full sequence is assembled, after which the peptide is cleaved off the resin and any side-chain protecting groups are removed. Two main protection strategies exist: Fmoc (9-fluorenylmethyloxycarbonyl) and Boc (tert-butyloxycarbonyl). Fmoc-SPPS has become the standard in most labs because deprotection (removal of Fmoc by mild base) is gentle and compatible with a wide range of sequences. Boc-SPPS uses strong acid (trifluoroacetic acid) for deprotection and an even stronger acid (HF) to cleave the peptide, which can be harsher but sometimes yields better results for long or difficult sequences. Advances in SPPS have greatly improved its efficiency and scope – for example, automation is routine (commercial peptide synthesizers can assemble dozens or even hundreds of peptides in parallel). Innovations like microwave-assisted SPPS, low-loading resins to reduce aggregation, and pseudoproline dipeptide building blocks have enabled synthesis of peptides well over 50 amino acids in length Still, very long sequences (e.g. >100 aa) remain challenging to make purely by SPPS and may require fragment coupling or recombinant expression. SPPS has been revolutionary because it also allows incorporation of unnatural amino acids or modified residues at will – the chemist can simply use a modified amino acid building block during synthesis. This flexibility means that not only can natural peptides be synthesized in sufficient quantities, but entirely new analogs can be created by design. Once synthesized, peptides are typically purified by high-performance liquid chromatography (HPLC) and confirmed by mass spectrometry (MS) to ensure the correct mass and purity (common analytical techniques discussed later). Overall, SPPS provides a fast, reliable route to generate peptides for research or pharmaceutical development, forming the backbone of peptide engineering efforts.

Peptide Cyclization: Cyclization refers to connecting the ends or side chains of a peptide to form a cyclic structure. This can dramatically improve a peptide’s properties. Cyclization strategies include head-to-tail (joining the N-terminus to the C-terminus in a peptide chain), side chain-to-side chain (forming a bridge between two side-chain functional groups, such as via disulfide bonds or lactam links), or backbone-to-side chain linkages. Cyclization constrains the peptide’s conformation, often increasing its proteolytic resistance and sometimes its receptor affinity or selectivity. A cyclic peptide is less flexible than its linear counterpart, which can reduce the entropy loss upon binding to a target and thereby improve binding potency. Indeed, peptide cyclization is a common technique to increase proteolytic stability and even, in some cases, membrane permeability. By pre-organizing the peptide into its bioactive conformation, cyclization can also enhance biological activity. Nature employs cyclization in many bioactive peptides – for example, cyclosporine (an NRP immunosuppressant) is cyclic, and somatostatin (a 14 aa hormone) has two cysteines forming a disulfide ring. In the lab, chemists can cyclize peptides by various means: oxidizing cysteine residues to form disulfide bridges (e.g. insulin has three disulfide bonds; many venom peptides are highly crosslinked), linking amine and carboxyl groups to form lactam (amide) bonds, or using chemical linkers (such as stapling, see below). Cyclization is particularly useful for stabilizing secondary structures. For instance, an isolated short α-helix often unfurls when not part of a protein, but by cyclizing (stapling) residues i to i+4 or i+7 along the helix, one can lock the helical structure. “Stapled peptides” are a modern class of therapeutics where non-natural hydrocarbon linkers are inserted to brace an α-helix, yielding peptides that can disrupt protein–protein interactions inside cells. Cyclization can also stabilize β-turns and β-hairpins; e.g., incorporating a D-Pro–L-Pro motif or forming a head-to-tail macrocycle can nucleate a β-hairpin conformation. In summary, cyclization is a powerful design strategy to enhance peptide drug candidates, often yielding analogs with improved stability and potency. For example, the cyclic peptide octreotide (an analog of somatostatin) is far more stable and longer-acting than native somatostatin and is used clinically for acromegaly and neuroendocrine tumors.

Conjugation and Chemical Modifications: Another approach to improve peptide performance is conjugating them to other molecules. Lipidation – attaching fatty acid chains – is one such modification that has proven extremely useful. Conjugating a peptide to a fatty acid promotes binding to serum albumin and lipoproteins, effectively increasing the peptide’s molecular size and reducing renal clearance. This strategy is used in several antidiabetic GLP-1 analogs: liraglutide (a GLP-1 analog) has a C₁₆ fatty acid attached, semaglutide has a C₁₈ fatty diacid, and the insulin analog insulin degludec has a hexadecenoic acid attached these lipid modifications greatly extend their plasma half-lives by slowing renal filtration and proteolysis. Similarly, peptides can be fused or conjugated to large proteins like the Fc portion of antibodies or albumin to prolong half-life (increasing size above the kidney filtration threshold). For example, dulaglutide is a GLP-1 agonist genetically fused to an IgG Fc, allowing once-weekly dosing. Another common conjugation is PEGylation: attaching polyethylene glycol polymers to a peptide. PEG is a large, flexible hydrophilic polymer that can mask the peptide from proteases and the immune system, and also slow renal excretion by increasing effective size. Several peptide drugs have been PEGylated to enhance their pharmacokinetics; however, PEGylation can sometimes reduce activity if it sterically hinders receptor binding, so the site and size of PEG attachment must be optimized. Beyond half-life extension, conjugation can impart new functions: peptides can be linked to toxins (for targeted cancer therapy), to fluorophores or radioisotopes (for imaging and diagnostic tracers), or to other bioactive compounds (creating hybrid drugs). An example in diagnostics is Octreoscan, a radiolabeled version of the somatostatin analog octreotide, used to image neuroendocrine tumors via somatostatin receptor binding. In therapeutics, peptides have been conjugated to chemotherapeutic agents to selectively deliver the drug to cells expressing the peptide’s target receptor (analogous to antibody–drug conjugates, but using a peptide ligand). Conjugation can also improve oral stability; for instance, linking peptides to mucoadhesive or cell-penetrating sequences might help oral absorption (though oral peptide delivery remains an exceptional challenge). Overall, chemical conjugation is a versatile tool: by attaching peptides to lipids, polymers, or proteins, scientists can tune the peptide’s solubility, stability, immunogenicity, and biodistribution. The success of fatty acylation in GLP-1 analogs (liraglutide, semaglutide, etc.) highlights how conjugation has become mainstream in peptide drug design to achieve once-weekly or even oral dosing.

Analog Development and Unnatural Amino Acids: Designing peptide analogs often involves systematically exploring the structure-activity relationship (SAR) of the peptide. A classic technique is alanine scanning, where each residue in the peptide is sequentially replaced with alanine to test its importance for activity. If substituting a particular residue with Ala sharply reduces activity, that position is likely critical for receptor binding or function; conversely, if activity remains unchanged, the residue might be a candidate for modification to improve other properties. Once key residues are identified, medicinal chemists can introduce unnatural amino acids to enhance stability or affinity. One major modification is using D-amino acids in place of L-amino acids. Proteolytic enzymes are stereospecific for L-amino acids, so substituting one or more L-residues with their D-counterparts can make a peptide resistant to those enzymes. For example, the synthetic vasopressin analog desmopressin (used for diabetes insipidus) has a D-arginine, and leuprolide (a GnRH analog for prostate cancer) includes a D-leucine; these changes extend their half-lives and alter receptor interactions. Another modification is N-methylation of peptide bonds (adding a methyl group to the peptide nitrogen) – this can improve membrane permeability and protease resistance by removing a hydrogen-bond donor and increasing hydrophobicity. Incorporating β-amino acids (which have an extra methylene in the backbone) or peptoid residues (N-substituted glycine analogs) can likewise block protease action and alter conformation. A variety of non-standard amino acids are commercially available (e.g. halogenated phenylalanines, homoarginine, norleucine, etc.) and can be introduced during SPPS to modify side chain properties. For instance, replacing a serine with O-tert-butyl-serine might protect a site from phosphorylation or metabolism. The goal of analog development is often to balance affinity, selectivity, and pharmacokinetics. After identifying a “minimum active sequence”, chemists will create analogs to optimize receptor binding and reduce clearance. A case study is selepressin, an analog of vasopressin designed with modifications to improve stability and selectivity at the V₁ receptor – it has a longer plasma half-life than vasopressin while maintaining potent vasoconstrictive effects. Similarly, countless analogs of somatostatin were made by truncation and modification, leading to drugs like octreotide (8 aa, cyclic) which is more selective and stable. Through these approaches, peptide therapeutics can be refined: key binding residues are preserved or enhanced (sometimes by alpha-methylation to restrict conformations), and liabilities are eliminated (cleavage sites can be “hardened” by D-amino acids, and polar groups not needed for binding can be capped or removed to improve permeability). The introduction of non-natural features does carry a risk of immunogenicity, as the immune system might recognize an unusual structure as foreign, but small changes are generally tolerated, especially if the core sequence is close to a human peptide. Rational and combinatorial design have yielded peptide drugs with far superior properties to their natural counterparts, a theme clearly seen in the next section on therapeutic applications.

Peptides in Research and Drug Development

Peptides are invaluable tools in both basic research and clinical research. They are used to probe biological systems, map interactions, develop assays, and serve as leads for drug discovery. Several specific applications are highlighted below:

Receptor Mapping and Epitope Identification: Peptides can be used to map the binding sites of receptors or antibodies, a process often called epitope mapping. Using libraries of overlapping peptides derived from a protein sequence, researchers can determine which segment of a protein is recognized by a given antibody (linear epitope mapping). Combinatorial peptide libraries are also used to map receptor binding preferences – for example, one can determine the optimal peptide motif that binds to a receptor’s active site. A powerful technique for this is phage display, in which vast libraries (10^9 or more variants) of random peptides are expressed on the surface of bacteriophages. Phage display allows “panning” peptides that bind to a target of interest (such as a purified receptor or an antibody) by iterative selection. This approach has been used to discover peptide ligands for many receptors and to delineate their binding requirements. In fact, phage display can construct a map of ligand–receptor binding sites by analyzing which peptides are enriched against a given target. One classic outcome of phage display was the identification of the RGD motif (Arg-Gly-Asp) as a key binding sequence for integrins; a short RGD peptide can bind to α_vβ₃ integrin on angiogenic blood vessels. By selecting peptides on cells or tissues (in vivo phage display), researchers have also mapped organ-specific vascular ligands. Additionally, synthetic peptide scanning is used in receptor mapping: for instance, scanning a hormone’s sequence with alanine mutations can reveal which side chains contact the receptor. Complementarily, small peptides corresponding to fragments of a receptor can be used to map protein–protein interaction interfaces by seeing if they disrupt or mimic parts of the receptor. Overall, peptide tools have enabled fine-grained mapping of molecular interactions in a way that large proteins or small molecules alone could not, thanks to the ease of synthesis and modification of peptides.

Peptide Libraries and Combinatorial Discovery: As mentioned, libraries of peptides (whether phage-displayed, mRNA-displayed, or synthesized on solid supports) are a mainstay of drug discovery and chemical biology. Combinatorial peptide libraries allow unbiased screening for bioactive sequences. For example, one can screen a random peptide library for binding to a protein target to find lead compounds (peptide hits that could be developed into drugs). Peptide libraries have been used to find receptor agonists and antagonists, enzyme inhibitors, and cell-penetrating motifs. One technique, one-bead-one-compound (OBOC) libraries, involves synthesizing millions of peptides on tiny resin beads (each bead displaying a unique peptide); binding of a fluorescently labeled protein to some beads identifies candidate ligands, which can then be sequenced. Another use of peptide libraries is in profiling protease specificity by offering a protease a large mixture of peptide substrates (for instance, a fluorogenic library), one can deduce what sequence context the protease prefers to cut. This can yield peptide-based inhibitors tailored to that protease. In immunology, libraries of peptide–MHC complexes are screened to map T-cell epitopes, and conversely phage libraries are used to find mimotopes – peptide mimics of larger protein epitopes that can stimulate antibody responses. The versatility of peptide libraries lies in their diversity and the relative simplicity of linking genotype to phenotype (especially with phage or mRNA display, where selected peptides are directly identified by sequencing their encoding DNA/RNA). These methods have greatly accelerated the identification of novel peptides that can serve as biochemical probes or therapeutic leads. In summary, combinatorial peptide techniques supplement rational design, often revealing unexpected solutions (sequences) that bind targets in ways not predicted by theory.

Proteomics and Peptidomics: Peptides are central to the field of proteomics – the large-scale study of proteins. Proteomic analysis typically involves digesting proteins into peptides (using proteases like trypsin) and then identifying those peptides by mass spectrometry, thereby inferring the presence of specific proteins. The peptide mass fingerprint or sequence obtained via tandem MS serves as a unique signature for a protein. In this context, synthetic peptides are used as standards for quantification or to validate MS detection. On the other hand, peptidomics is the direct study of endogenous peptides in tissues or fluids, without the need for initial protease digestion. This has been facilitated by advances in high-resolution MS that can detect and sequence naturally occurring peptides, including those with PTMs. Peptidomics has led to the discovery of new bioactive peptides and provided insights into the processing and modification of known peptide hormones in vivo. For example, the orexins (hypocretins), which regulate wakefulness, were discovered by fractionating brain extracts and using orphan receptor assays to guide purification – essentially a peptidomic approach that combined biochemical extraction with cell-based assays and MS identification. Another example: Peptidomic analysis of human plasma has to contend with the fact that the majority (>95%) of detected peptides are fragments of abundant proteins (e.g. albumin) rather than bona fide hormones, making it challenging to fish out low-abundance signaling peptides. Nonetheless, databases like PeptideAtlas curate identified peptides from many experiments to help distinguish real peptides from degradation artifacts. In proteomic method development, peptides are used as standards and calibrants. For instance, sets of synthetic peptides with known retention times are used to calibrate HPLC-MS systems for more accurate peptide detection (iRT standards). In summary, peptides are both the subject and tools of proteomics: they are the units that mass spectrometrists measure to reconstruct proteomes, and synthetic peptides are indispensable for method calibration, quantitation (in targeted proteomics assays like SRM/MRM), and validation of protein identifications.

Drug Delivery and Targeting Peptides: Peptides also find use as adjuncts in drug delivery. Certain peptides can facilitate the delivery of therapeutics to specific cells or tissues. A prominent class is cell-penetrating peptides (CPPs), as noted earlier, which can ferry attached cargo (like nanoparticles, oligonucleotides, or even proteins) across cell membranes. For example, the TAT peptide from HIV can be fused to large proteins to facilitate their entry into cells. Another application targets peptides that home to particular tissues – often identified through in vivo phage display. The RGD-containing peptides targeting α_vβ₃ integrins on tumor blood vessels have been used to deliver anti-cancer drugs or imaging agents to tumors. One such peptide, iRGD, not only targets tumors but also increases vascular permeability in the tumor, improving co-delivery of other drugs. Peptides can also be used to form nanostructures, such as hydrogels or nanoparticles, that carry drugs. Some amphiphilic peptides self-assemble into micelles, which can encapsulate hydrophobic drugs. Peptide-drug conjugates represent another strategy: a cytotoxic drug linked to a peptide ligand that binds a cancer-specific receptor. Upon binding, the conjugate is taken into the cancer cell, where the drug is released – providing a targeted chemotherapy approach (several such conjugates are in development for tumors overexpressing peptide hormone receptors, for example). Radiolabeled peptides are widely used in diagnostic imaging and therapy: radiolabeled somatostatin analogs target neuroendocrine tumors (Octreoscan for imaging, or peptide receptor radionuclide therapy for treatment), and newer radiolabeled peptides target prostate-specific membrane antigen (PSMA) for imaging prostate cancer metastases. In summary, peptides contribute to drug delivery both as vehicles (delivering other payloads via conjugation or co-assembly) and beacons (guiding payloads to a target via specific binding). This area is rapidly expanding, integrating peptide chemistry with nanotechnology and biotechnology to achieve more precise therapeutic interventions.

Peptides: Current and Emerging Knowledge

Peptides have come into their own as therapeutic agents, leveraging their high specificity and potency. As of the 2020s, there are over 100 peptide-based drugs on the market, spanning indications from metabolic diseases to oncology. This section will discuss major classes of peptide therapeutics, examples of approved drugs, the importance of structure-activity relationships (SAR) in their development, pharmacokinetic considerations, and emerging frontiers.

Hormone and Metabolic Peptide Drugs: Many of the earliest peptide drugs were replacements or analogs of natural hormones. Insulin, first used in 1923, remains a lifesaving treatment for Type 1 diabetes and advanced Type 2 diabetes. Human insulin (51 aa) was originally purified from animal pancreata, but now recombinant human insulin and a multitude of analogs are available. Insulin analogs exploit SAR knowledge: for example, insulin lispro swaps two amino acids to reduce dimerization for faster onset, whereas insulin glargine has amino acid changes and additions that shift its solubility for slow release. Another early peptide drug is oxytocin (9 aa), used to induce labor; and vasopressin analogs like desmopressin (modified 9 aa) to treat diabetes insipidus and bedwetting. Glucagon-like peptide-1 (GLP-1) receptor agonists have become blockbuster drugs for Type 2 diabetes and obesity. These include exenatide (a synthetic version of a lizard peptide called exendin-4) and human analogs liraglutide, dulaglutide, semaglutide, etc., which enhance insulin secretion and promote weight loss. Native GLP-1 has a very short half-life (minutes) due to DPP-4 degradation, so analogs were engineered with improved stability: e.g. substituting D-Ala at the cleavage site (position 2) and adding fatty acid chains or large fusion partners to extend half-life. Liraglutide (C16 fatty acylated) and semaglutide (C18 acylated) can be dosed once daily or weekly, respectively. Semaglutide has even been formulated with an absorption enhancer (SNAC) to create the first oral GLP-1 pill, a notable breakthrough in oral peptide delivery. Other metabolic peptide drugs include amylin analogs (pramlintide for diabetes), PTH analogs (teriparatide, 34 aa, for osteoporosis), and leptin analogs (metreleptin for lipodystrophy).

Peptides in Cardiovascular and Renal Diseases: Peptide therapeutics also address cardiovascular and kidney conditions. Bivalirudin is a 20-residue peptide anticoagulant modeled on hirudin (a leech peptide), used in angioplasty to prevent clots. Nesiritide, a recombinant B-type natriuretic peptide (32 aa), was approved for acute heart failure (though its use is now limited) to induce vasodilation and diuresis. Carperitide (ANP analog) has seen use in Japan for heart failure. For bleeding disorders, desmopressin (mentioned above) can promote clotting factor release. Angiotensin II itself was approved as a vasoconstrictor drug (Giapreza) for septic shock, illustrating that even endogenous peptides can be harnessed as drugs in critical care. SAR plays a role in these as well – angiotensin II cannot be easily modified without losing activity, so in that case the wild-type peptide is used but careful dosing is required given its potent effects.

Neurology and Pain: A remarkable peptide drug is ziconotide, a 25-residue peptide from cone snail venom (also known as ω-conotoxin MVIIA). Ziconotide is an N-type calcium channel blocker used for refractory pain (administered intrathecally). Its development underscores the potential of natural peptides (venom peptides) as therapeutics for challenging targets like ion channels. Sumatriptan (a migraine medication) is a small molecule that mimics peptides (serotonin), but newer migraine preventives include erenumab, a monoclonal antibody against CGRP receptor, showing that peptide pathways (CGRP in this case) are key drug targets. While no endorphin or enkephalin peptides themselves are used clinically due to blood-brain barrier issues, analogs like loperamide (peripherally acting) or peptide-like drugs such as setmelanotide (a melanocortin peptide analog for obesity) show therapeutic promise in CNS or metabolic disorders. Neuropeptides are also being targeted via non-peptide drugs (e.g. substance P antagonists for nausea), but in some cases the peptide itself or a close analog is used if it can be delivered appropriately.

Anti-Cancer Peptides and Hormone Analogues: Several peptide hormones have been leveraged to treat hormone-sensitive cancers. Leuprolide, goserelin, and other GnRH analogs (10 aa) are used in prostate cancer and breast cancer to suppress sex hormone production. Interestingly, by making GnRH analogs that have higher potency and longer half-life than native GnRH, continuous administration leads to pituitary GnRH receptor desensitization and a drop in LH/FSH release – effectively a reversible chemical castration. Leuprolide’s SAR modifications include a D-Leu and a C-terminal amide, making it resistant to proteolysis and longer-acting. Another oncology example is octreotide and lanreotide, analogs of somatostatin (14 aa). Native somatostatin inhibits release of many hormones but has a half-life of ~3 minutes. Octreotide (8 aa, cyclic) is much more stable (half-life ~90 minutes IV) and selectively targets somatostatin receptors; it’s used to treat acromegaly, neuroendocrine tumors, and carcinoid syndrome. There are also peptide-based radiopharmaceuticals for cancer therapy, such as radiolabeled somatostatin analogs for peptide receptor radionuclide therapy (PRRT) to irradiate tumors from within. Emerging cancer therapeutics include peptide vaccines – short peptides from tumor antigens used to elicit an immune response – and checkpoint inhibitor peptides (small peptides that can disrupt PD-1/PD-L1 interactions, under preclinical investigation). Additionally, some antimicrobial peptides are being studied as new antibiotics, though none are yet mainline drugs except polymyxin and related lipopeptides for resistant bacteria.

Structure–Activity Relationships and Pharmacokinetics: A recurring theme in peptide drug development is optimizing the amino acid sequence for the desired activity profile. Minor changes can have large effects. For example, human insulin tends to form hexamers, which delays its action; swapping Pro^B28 and Lys^B29 in insulin (as in lispro) prevents hexamer formation, yielding a fast-acting insulin. In contrast, adding two arginines to the B-chain (as in insulin glargine) raises the isoelectric point, making it precipitate subcutaneously for slow release. Similarly, GLP-1 analogs illustrate SAR: substituting Ala⁸ with α-aminoisobutyric acid (Aib) prevents DPP-4 cleavage, and attaching a fatty acyl group at Lys²⁶ (in liraglutide) or a C18 diacid at Lys²⁶ plus a large spacer (in semaglutide) extends half-life by promoting albumin binding. These modifications maintain receptor agonism while altering pharmacokinetics (liraglutide and semaglutide have half-lives of ~13 and ~168 hours, respectively, versus 1–2 minutes for native GLP-1). Another SAR example is GnRH analogs: agonists like leuprolide preserve the His-Trp-Ser-Tyr core important for receptor activation, whereas antagonists like degarelix have multiple substitutions (including D-amino acids) that allow receptor binding but induce no activation, thereby purely blocking the receptor. Degarelix can immediately suppress testosterone without the initial surge seen with agonists, due to its antagonist action. In all these cases, rational modifications were guided by understanding which residues bind the receptor versus which can be tweaked to influence stability, solubility, or receptor kinetics.

Pharmacokinetically, peptide drugs generally have to contend with short half-lives and poor oral absorption. Strategies to improve half-life include the modifications described: lipidation, PEGylation, fusion to large proteins, cyclization, and use of D-amino acids. Oral bioavailability of peptides is usually very low due to degradation by gastrointestinal enzymes and poor permeability across the gut epithelium. However, as noted, the first oral peptide (semaglutide in a special tablet with an absorption enhancer) has been approved, and others like oral octreotide are in trials. Approaches like co-formulation with enzyme inhibitors, mucoadhesive polymer encapsulation, and even devices that inject the peptide into the gut wall (e.g. robotic pills) are being explored to further enable oral peptide delivery. Other alternative routes include inhalation (inhalable insulin was approved in the past, though with limited success), transdermal patches for small peptides, and implantable pumps for continuous infusion. The field is clearly moving toward making peptide therapies more convenient while preserving their efficacy.

Emerging Peptide Therapeutics: The horizon for peptide drugs is expanding with advances in technology. Peptide vaccines (for infectious diseases and cancer) are under development – these use synthetic peptides representing T-cell or B-cell epitopes to induce immunity. mRNA vaccine technology can also be seen as a way to deliver peptide antigens in vivo. Peptidomimetics and stapled peptides are allowing traditionally “undruggable” targets, like intracellular protein–protein interactions, to be tackled by peptide-like molecules that act inside cells. Some stapled peptides are in clinical trials for cancer (e.g. a stapled peptide antagonist of MDM2 to reactivate p53). Macrocyclic peptides identified from display libraries (including those containing non-natural amino acids) are yielding drug candidates against challenging enzymes or receptors – for example, cyclic peptides acting as allosteric inhibitors or targeting protein surfaces. Artificial amino acids that confer covalent binding abilities (e.g. electrophilic warheads) are being incorporated to create peptides that irreversibly inhibit targets – a strategy termed “covalent peptide inhibitors”. Another growth area is hybrid peptide–small-molecule conjugates, where a peptide might provide targeting while a small-molecule warhead provides potent functional effect (akin to targeted protein degradation strategies, where a targeting peptide could recruit an E3 ligase or other protein).

Challenges in Peptide Pharmacology

Despite their many advantages, peptides face several challenges that can limit their use as drugs. Key issues include:

• Poor Oral Bioavailability: Orally administered peptides are usually ineffective because they are destroyed by proteases in the gastrointestinal tract and are not easily absorbed through the intestinal lining. The stomach and pancreas secrete potent enzymes (pepsin, trypsin, chymotrypsin, etc.) that readily break peptide bonds. Moreover, the intestinal epithelium has tight junctions that prevent large polar molecules from crossing. As a result, most peptide drugs must be given by injection or alternative routes. Attempts to formulate oral peptides (e.g. enteric coatings, permeability enhancers, protease inhibitors co-formulated) have had limited success, though recent innovations like the oral semaglutide/SNAC formulation show that oral delivery is not impossible. Generally, however, achieving therapeutically meaningful bioavailability from an oral peptide is very challenging, and this remains a major hurdle for patient-friendly peptide medicines.
• Rapid Enzymatic Degradation: Even when delivered parenterally, peptides can be quickly degraded by endopeptidases and exopeptidases in blood and tissues. This leads to short circulating half-lives and the need for frequent dosing or continuous infusion. For instance, native GLP-1 and many neuropeptides have half-lives of only a few minutes in plasma. Strategies to combat this include chemical modifications such as D-amino acid substitutions, which can significantly extend peptide half-life by blocking protease action. Cyclization and capping of termini (e.g. N-acetylation, C-amidation) also protect against exopeptidases. Nonetheless, proteolysis is an ever-present elimination route for peptides. This is in contrast to small molecules, which are typically eliminated by hepatic metabolism or renal excretion – peptides are eliminated by enzymatic breakdown into amino acids (and then the amino acids are recycled or further catabolized). The rapid clearance of peptides can be advantageous in some situations (fast on/off can reduce side effects), but generally it is a limitation that requires clever design to overcome.
• Receptor Desensitization and Tachyphylaxis: As discussed, many peptide agonists can induce desensitization of their target receptors upon repeated or continuous exposure. Clinically, this can mean diminishing returns with long-term therapy or the need for drug “holidays” or pulsatile administration. For example, patients on intranasal desmopressin for bedwetting may see reduced response over time due to down-regulation of V2 receptors in the kidney. In heart failure, chronic use of β-agonist peptides (if they existed as drugs) would likely lead to β-receptor downregulation (this is seen with endogenous catecholamines and is one reason chronic heart failure is treated with β-blockers, not agonists). Opioid peptides (like endorphin analogs) cause receptor desensitization similar to morphine, leading to tolerance. Drug developers must be mindful of dosing regimens – sometimes a long-acting peptide analog that continuously stimulates a receptor may not be ideal if the biology requires pulsatile signaling. As we noted, long-term GPCR stimulation triggers internalization and degradation of receptors, limiting the therapeutic lifetime of the drug’s effect. Potential solutions include developing biased agonists that cause less recruitment of β-arrestin (hence less desensitization) or combining therapy with agents that promote receptor resensitization.
• Immunogenicity: While peptides are generally less immunogenic than larger proteins (because they may be too small to effectively cross-link B cell receptors unless attached to a carrier), they can still sometimes trigger unwanted immune responses. This risk increases if the peptide differs from any sequence in the human proteome (seen as foreign) or if it aggregates or contains impurities from synthesis. The immune system can produce anti-drug antibodies (ADAs) against peptide drugs, potentially neutralizing their effect or even causing allergic reactions. For example, ~30–40% of patients on exenatide (a lizard-derived peptide) develop low-titer antibodies, though these often do not have significant clinical impact. Factors influencing immunogenicity include the peptide’s origin (non-human sequences are more likely to be immunogenic) and modifications (certain non-natural amino acids might create novel T-cell epitopes). Additionally, impurities from synthesis, such as incomplete deprotection byproducts or aggregation, can act as adjuvants or additional antigens. Regulators like the FDA now ask for immunogenicity risk assessments for peptide products, especially “follow-on” generic versions, to ensure that differences in impurities or formulation do not heighten immunogenicity. To mitigate immunogenicity, peptide drugs are often designed to be as close to human sequences as possible (e.g. modifying exenatide to more closely resemble human GLP-1 led to analogs with potentially less immunogenicity). Also, using high purity peptides and avoiding aggregation (through proper formulation) helps. Nonetheless, the possibility of ADA formation is a challenge that needs monitoring during clinical development of peptide therapeutics. For instance, if a peptide hormone analog induces antibodies, those antibodies might cross-react with the endogenous hormone and neutralize it, potentially causing a deficiency state.

Bioactivity and Potency Assays

In addition to chemical analysis, peptide drugs (especially if complex or large) often require bioactivity assays to confirm that the manufacturing process yields a biologically active conformation. For instance, for peptide hormones, a cell-based assay measuring receptor activation (cAMP production for a GPCR agonist, for example) might be used to define potency in units. However, many peptide drugs can be well-characterized enough chemically that bioassays serve mainly as a secondary measure. Still, ELISA (enzyme-linked immunosorbent assay) or RIA (radioimmunoassay) techniques are sometimes used to measure peptide levels or potency in samples. ELISAs are commonly employed in pharmacokinetic studies to quantify peptide drug concentrations in blood – antibodies specific to the peptide can capture and detect it. For example, a clinical trial of a GLP-1 analog might use an ELISA to measure plasma drug levels over time. ELISAs can also detect anti-drug antibodies in patients, which is a regulatory requirement for biologics and increasingly for peptides if immunogenicity is a concern. In research, ELISAs often use peptides as standards – a synthetic peptide of known concentration is used to generate a standard curve in an assay measuring, say, an endogenous peptide hormone in patient samples. The high specificity of antibodies in ELISA allows discrimination between similar peptides (though cross-reactivity must be evaluated). Along with ELISA, techniques like surface plasmon resonance (SPR) are used in development to characterize binding kinetics of peptides to their targets. SPR is a label-free method that measures real-time binding and dissociation on a sensor chip. It can provide the association rate (k_on) and dissociation rate (k_off) and equilibrium affinity (K_D) for a peptide–receptor or peptide–antibody interaction. For instance, SPR might be used to compare how tightly a peptide analog binds to a receptor versus the native peptide, guiding lead optimization with kinetic insight (a slower k_off often correlates with longer duration of action in vivo). Other biophysical assays like isothermal titration calorimetry (ITC) can directly measure the binding affinity and thermodynamics of peptide–protein interactions, although ITC typically requires larger quantities of material.