10.5 Peptides

Peptides are crucial building blocks in medicinal chemistry, composed of amino acids linked by peptide bonds. They play vital roles in biological processes and serve as important drug targets. Understanding peptide structure, synthesis, and interactions is key to developing effective therapies.

Peptide-based drug discovery has led to breakthroughs in treating various diseases. From peptide hormones to antimicrobial and anticancer peptides, these versatile molecules offer high specificity and low toxicity. Overcoming challenges like poor bioavailability and rapid clearance is essential for advancing peptide-based therapies.

Peptide structure and composition

Amino acid building blocks

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• Peptides are composed of amino acids, which are organic compounds containing an amino group, a carboxyl group, and a unique side chain (R group)
• There are 20 standard amino acids, each with a specific side chain that determines its chemical properties and interactions
• Amino acids are joined together by peptide bonds, formed through a condensation reaction between the carboxyl group of one amino acid and the amino group of another

Primary structure of peptides

• The primary structure of a peptide refers to the linear sequence of amino acids connected by peptide bonds
• The order of amino acids in the primary structure is determined by the genetic code and is crucial for the peptide's function and properties
• The N-terminus of a peptide has a free amino group, while the C-terminus has a free carboxyl group

Secondary structures: α-helices and β-sheets

• Secondary structures are regular, repeating patterns of local folding within a peptide chain, stabilized by hydrogen bonds between the backbone atoms
• The α-helix is a common secondary structure, characterized by a right-handed spiral conformation with 3.6 amino acids per turn, stabilized by hydrogen bonds between the carbonyl oxygen of one amino acid and the amino hydrogen of another located four residues ahead
• β-sheets are another type of secondary structure, formed by extended peptide chains that are aligned side-by-side and stabilized by hydrogen bonds between the backbone atoms of adjacent strands, creating a pleated sheet-like structure (parallel or antiparallel)

Tertiary structure and folding

• Tertiary structure refers to the three-dimensional arrangement of a peptide chain, resulting from the folding and interaction of secondary structure elements
• Folding is driven by various non-covalent interactions, such as hydrogen bonds, van der Waals forces, hydrophobic interactions, and electrostatic interactions between amino acid side chains
• The tertiary structure of a peptide is crucial for its biological function, as it determines the peptide's binding sites, catalytic activity, and interactions with other molecules
• Misfolding of peptides can lead to aggregation and the formation of insoluble fibrils, which are associated with various diseases (Alzheimer's, Parkinson's)

Peptide synthesis

Solid-phase peptide synthesis (SPPS)

• SPPS is a method for synthesizing peptides by anchoring the growing peptide chain to a solid support (resin) and adding amino acids sequentially
• The C-terminal amino acid is attached to the resin, and subsequent amino acids are coupled to the N-terminus of the growing chain using protecting groups and coupling reagents
• SPPS enables the synthesis of longer peptides with higher purity compared to solution-phase methods, as the resin-bound peptide can be easily washed and purified after each coupling step

Protecting groups and coupling reagents

• Protecting groups are chemical moieties used to temporarily block reactive functional groups (amino, carboxyl) during peptide synthesis to prevent unwanted side reactions
• Common protecting groups include Fmoc (9-fluorenylmethoxycarbonyl) for the N-terminus and tBu (tert-butyl) for side chains, which can be selectively removed under specific conditions
• Coupling reagents activate the carboxyl group of the incoming amino acid, facilitating its reaction with the N-terminus of the growing peptide chain
• Examples of coupling reagents include carbodiimides (DCC, EDC), phosphonium salts (PyBOP), and uronium salts (HBTU, HATU)

Purification and characterization techniques

• After synthesis, peptides need to be purified to remove truncated sequences, deletion products, and other impurities
• Reversed-phase high-performance liquid chromatography (RP-HPLC) is commonly used for peptide purification, separating peptides based on their hydrophobicity
• Mass spectrometry (MALDI-TOF, ESI-MS) is used to characterize the purified peptides, confirming their molecular weight and sequence
• Circular dichroism (CD) spectroscopy and nuclear magnetic resonance (NMR) spectroscopy can provide information about the secondary structure and conformation of peptides

Challenges in peptide synthesis

• Peptide synthesis can be challenging due to the potential for side reactions, such as racemization, aspartimide formation, and peptide aggregation
• Difficult sequences, such as those containing multiple hydrophobic or β-branched amino acids, can lead to incomplete coupling and low yields
• Longer peptides (>50 amino acids) are more prone to aggregation and can be difficult to synthesize and purify
• Chemical modifications, such as cyclization, stapling, or the incorporation of non-natural amino acids, may require specialized strategies and reagents

Peptide-based drug discovery

Therapeutic targets for peptide drugs

• Peptides can be designed to target various receptors, enzymes, and other proteins involved in disease pathways
• G protein-coupled receptors (GPCRs) are common targets for peptide drugs, as they play crucial roles in signaling and are implicated in numerous diseases (diabetes, cancer, cardiovascular disorders)
• Peptides can also target ion channels, transporters, and protein-protein interactions, modulating their activity and downstream effects
• Examples of peptide drug targets include the glucagon-like peptide-1 receptor (GLP-1R) for diabetes, the μ-opioid receptor for pain management, and the vascular endothelial growth factor (VEGF) for cancer therapy

Peptide libraries and screening methods

• Peptide libraries are collections of diverse peptide sequences that can be screened for binding or activity against a specific target
• Libraries can be generated by chemical synthesis (combinatorial libraries) or biological methods (phage display, mRNA display)
• High-throughput screening (HTS) techniques, such as fluorescence polarization, surface plasmon resonance (SPR), and enzyme-linked immunosorbent assay (ELISA), are used to identify peptides with desired properties from large libraries
• Iterative rounds of screening, selection, and amplification can be employed to enrich for peptides with high affinity and specificity for the target

Peptidomimetics and peptide analogs

• Peptidomimetics are compounds that mimic the structure and function of peptides but have improved pharmacological properties, such as increased stability, bioavailability, and potency
• Peptide analogs are modified versions of natural peptides, incorporating non-natural amino acids, backbone modifications, or structural constraints to enhance their drug-like properties
• Examples of peptidomimetic strategies include the use of D-amino acids, β-amino acids, peptoids, and small molecule mimics
• Peptide analogs can be designed to improve receptor selectivity, reduce off-target effects, and increase resistance to proteolytic degradation

Peptide drug delivery systems

• Peptide drugs often face challenges in delivery due to their poor oral bioavailability, rapid clearance, and limited permeability across biological barriers
• Various drug delivery systems have been developed to overcome these limitations and improve the therapeutic efficacy of peptide drugs
• Liposomes and nanoparticles can encapsulate peptides, protecting them from degradation and facilitating their transport across membranes
• Cell-penetrating peptides (CPPs) can be conjugated to therapeutic peptides to enhance their cellular uptake and intracellular delivery
• Sustained-release formulations, such as hydrogels and microspheres, can provide controlled and prolonged release of peptide drugs, reducing the frequency of administration

Peptide-protein interactions

Peptide binding sites on proteins

• Peptides can bind to specific sites on proteins, such as active sites, allosteric sites, or protein-protein interaction interfaces
• Binding sites are typically characterized by complementary shapes, electrostatic interactions, and hydrophobic contacts between the peptide and protein surfaces
• The specificity and affinity of peptide-protein interactions are determined by the amino acid composition, sequence, and conformation of the peptide and the corresponding binding site on the protein
• Examples of peptide binding sites include the substrate-binding pocket of enzymes, the ligand-binding domain of receptors, and the groove of major histocompatibility complex (MHC) molecules

Peptide-mediated signaling pathways

• Peptides can act as ligands for cell surface receptors, triggering intracellular signaling cascades that regulate various cellular processes, such as proliferation, differentiation, and apoptosis
• Peptide hormones and neuropeptides bind to specific GPCRs, leading to the activation of G proteins and downstream effector molecules (adenylyl cyclase, phospholipase C)
• Peptide growth factors, such as insulin-like growth factor (IGF) and epidermal growth factor (EGF), bind to receptor tyrosine kinases (RTKs), initiating phosphorylation cascades that control gene expression and cell fate
• Peptides can also modulate signaling pathways by interfering with protein-protein interactions, such as the binding of adaptor proteins or the formation of signaling complexes

Peptide inhibitors and agonists

• Peptides can be designed to act as inhibitors or agonists of specific protein targets, modulating their activity and downstream effects
• Peptide inhibitors can block the binding of natural ligands, substrates, or protein partners, preventing the activation of the target protein
• Examples of peptide inhibitors include the HIV-1 fusion inhibitor enfuvirtide, which blocks the interaction between the viral envelope protein and the host cell membrane, and the cancer therapeutic cilengitide, which inhibits the αvβ3 and αvβ5 integrins involved in angiogenesis
• Peptide agonists can mimic the effect of natural ligands, activating the target protein and inducing a biological response
• Examples of peptide agonists include the GLP-1 receptor agonist exenatide for the treatment of type 2 diabetes and the parathyroid hormone (PTH) analog teriparatide for the treatment of osteoporosis

Peptide-based protein-protein interaction modulators

• Protein-protein interactions (PPIs) play crucial roles in many biological processes and are attractive targets for therapeutic intervention
• Peptides can be designed to modulate PPIs by mimicking the binding interface of one protein partner, competing with the natural interaction
• Peptide-based PPI inhibitors can disrupt the formation of protein complexes, preventing their functional activity and downstream effects
• Examples of peptide-based PPI modulators include the p53-MDM2 interaction inhibitor PMI, which reactivates the tumor suppressor function of p53, and the BCL-2 family inhibitor ABT-737, which induces apoptosis in cancer cells
• Peptide-based PPI stabilizers can also be developed to promote the formation of specific protein complexes, enhancing their activity and biological effects

Peptide stability and metabolism

Enzymatic degradation of peptides

• Peptides are susceptible to degradation by various proteolytic enzymes, such as peptidases and proteases, which cleave peptide bonds at specific sites
• Endopeptidases, such as trypsin and chymotrypsin, cleave peptide bonds within the peptide chain, while exopeptidases, such as aminopeptidases and carboxypeptidases, remove amino acids from the N-terminus or C-terminus, respectively
• The stability of peptides towards enzymatic degradation depends on their amino acid composition, sequence, and structure
• Peptides containing certain amino acids, such as proline, glycine, and D-amino acids, are more resistant to proteolysis due to their conformational constraints or lack of recognition by enzymes

Strategies for improving peptide stability

• Several strategies can be employed to enhance the stability of peptides and protect them from enzymatic degradation
• Incorporation of non-natural amino acids, such as D-amino acids, β-amino acids, or unnatural side chain modifications, can reduce the susceptibility of peptides to proteolysis
• Cyclization of peptides, either head-to-tail or through disulfide bridges, can improve their stability by constraining their conformation and reducing the accessibility of cleavage sites
• Peptide stapling, which involves the introduction of covalent cross-links between amino acid side chains, can stabilize α-helical conformations and increase resistance to proteolysis
• PEGylation, the attachment of polyethylene glycol (PEG) chains to peptides, can increase their size, hydrophilicity, and steric hindrance, reducing their clearance and enzymatic degradation

Peptide half-life and clearance

• The half-life of a peptide refers to the time required for its concentration to decrease by 50% in the body
• Peptides often have short half-lives due to rapid clearance by the kidneys, liver, and other organs, as well as enzymatic degradation in the blood and tissues
• The clearance of peptides is influenced by their size, charge, hydrophobicity, and binding to plasma proteins
• Strategies to extend the half-life of peptides include increasing their size (PEGylation, fusion to albumin or Fc domains), reducing their renal filtration (charge modification, glycosylation), and improving their binding to plasma proteins (fatty acid conjugation)

Peptide prodrugs and bioconjugation

• Peptide prodrugs are inactive precursors that are converted to the active peptide drug by enzymatic or chemical transformations in the body
• Prodrug strategies can be used to improve the stability, bioavailability, and targeting of peptide drugs
• Examples of peptide prodrugs include esterification of carboxyl groups to increase lipophilicity and oral absorption, and conjugation to targeting moieties (antibodies, aptamers) for site-specific delivery
• Bioconjugation involves the covalent attachment of peptides to other molecules, such as polymers, lipids, or small molecules, to modify their pharmacokinetic and pharmacodynamic properties
• Examples of peptide bioconjugates include peptide-drug conjugates (PDCs) for targeted delivery of cytotoxic agents, and peptide-oligonucleotide conjugates (POCs) for improved cellular uptake and gene silencing

Peptide-based therapies

Peptide hormones and neurotransmitters

• Peptide hormones are endogenous signaling molecules that are produced by endocrine glands and released into the bloodstream to regulate various physiological processes
• Examples of peptide hormones include insulin (glucose metabolism), glucagon (blood sugar regulation), oxytocin (social bonding, lactation), and vasopressin (water balance, blood pressure)
• Peptide neurotransmitters are signaling molecules that are released by neurons and bind to receptors on target cells to modulate synaptic transmission and neuronal activity
• Examples of peptide neurotransmitters include endorphins (pain modulation, reward), substance P (pain sensation, inflammation), and neuropeptide Y (appetite, stress response)
• Synthetic analogs of peptide hormones and neurotransmitters can be developed as therapeutic agents to treat disorders associated with their dysregulation, such as diabetes, obesity, and neurological diseases

Antimicrobial and anticancer peptides

• Antimicrobial peptides (AMPs) are a diverse group of peptides that exhibit broad-spectrum activity against bacteria, fungi, and viruses
• AMPs typically have a cationic charge and amphipathic structure, which allows them to interact with and disrupt the negatively charged cell membranes of microorganisms
• Examples of AMPs include defensins, cathelicidins, and magainins, which are found in various organisms as part of their innate immune defense
• Anticancer peptides (ACPs) are peptides that selectively target and kill cancer cells while sparing healthy cells
• ACPs can act through various mechanisms, such as membrane disruption, apoptosis induction, and inhibition of angiogenesis and metastasis
• Examples of ACPs include the proapoptotic peptide (KLAKLAK)2, the antiangiogenic peptide anginex, and the cell-penetrating peptide Tat-KLA

Peptide vaccines and immunotherapies

• Peptide vaccines are designed to elicit an immune response against specific antigens, such as viral proteins or tumor-associated antigens, by presenting peptide epitopes to the immune system
• Peptide vaccines can be used for the prevention or treatment of infectious diseases, cancer, and autoimmune disorders
• Examples of peptide vaccines include the HPV vaccine (cervical cancer), the influenza vaccine (seasonal flu), and the gp100 vaccine (melanoma)
• Peptide-based immunotherapies leverage the ability of peptides to modulate the immune system, either by stimulating an immune response or suppressing an overactive immune reaction
• Examples of peptide immunotherapies include the T cell receptor (TCR) mimic peptides for cancer treatment, the tolerogenic peptides for autoimmune diseases, and the checkpoint inhibitor peptides for immune activation

Clinical applications and challenges

• Peptide-based therapies have been successfully applied to the treatment of various diseases, such as diabetes (insulin, GLP-1 receptor agonists), cancer (goserelin, octreotide), and HIV (enfuvirtide)
• Peptides offer several advantages as therapeutic agents, including high specificity, low toxicity, and the ability to target protein-protein interactions and other challenging targets
• However, peptide-based therapies also face challenges, such as poor oral bioavailability, rapid clearance, and immunogenicity
• Strategies to overcome these challenges include the use of peptide anal