Peptide-drug conjugates (PDCs) and peptide-based PROTACs represent the next generation of targeted therapeutics, combining the selectivity of peptides with the potency of small-molecule payloads or protein-degradation mechanisms. These hybrid molecules offer significant advantages over traditional systemic therapies by delivering cytotoxic agents directly to diseased cells or inducing selective degradation of disease-causing proteins. As pharmaceutical and biotech companies advance PDC and PROTAC candidates through clinical development, manufacturing these complex molecules requires specialized expertise in peptide synthesis, linker chemistry, bioconjugation methods, and analytical characterization of hybrid structures. Adesis provides integrated synthesis capabilities that eliminate the complexity of coordinating multiple vendors, enabling seamless development from initial conjugate design through clinical manufacturing under one roof.
Targeted Therapeutic Modalities: PDCs and Peptide-Based PROTACs
Peptide-drug conjugates have emerged as promising oncology therapeutics by exploiting the overexpression of specific receptors on tumor cells. Unlike antibody-drug conjugates (ADCs), which require large monoclonal antibody targeting moieties, PDCs utilize short peptide sequences that bind selectively to cancer cell surface receptors. This smaller size offers potential advantages, including better tumor penetration, faster clearance, reduced systemic exposure, and simpler manufacturing compared to biologics. The peptide component guides the cytotoxic payload to tumor cells expressing the target receptor, where internalization and linker cleavage release the active drug intracellularly, maximizing therapeutic index by concentrating drug effects at the disease site while minimizing off-target toxicity.
Several PDC candidates have demonstrated promising clinical results targeting receptors overexpressed in various cancers. Somatostatin receptor-targeting PDCs leverage the high expression of these receptors in neuroendocrine tumors, while other programs target integrins, GRP receptors, and other tumor-associated antigens. The clinical validation of this approach, including FDA approval of peptide receptor radionuclide therapies like lutetium (Lu) dotatate, has spurred increased investment in PDC development across oncology indications.
Peptide-based PROTACs employ a distinct mechanism, inducing targeted protein degradation rather than delivering cytotoxic payloads. These bifunctional molecules contain a peptide that binds the target protein, linked to an E3 ligase-recruiting element, bringing the target protein into proximity with the cellular degradation machinery. This proximity induces ubiquitination and subsequent proteasomal degradation of the target protein. The catalytic mechanism allows substoichiometric dosing, as a single PROTAC molecule can induce the degradation of multiple target protein molecules before being metabolized. This approach has shown particular promise for proteins previously considered “undruggable” by conventional small molecule inhibitors, expanding the range of therapeutic targets accessible to medicinal chemistry.
Structural Components and Design Considerations
The architecture of PDCs and peptide-based PROTACs requires careful optimization of three key components working in concert to achieve therapeutic effect. Each element contributes distinct properties that must be balanced to create effective conjugates.
The targeting peptide component determines selectivity for diseased versus healthy cells or specific protein targets. For PDCs, peptides typically range from 5 to 20 amino acids, providing sufficient length for selective receptor binding while maintaining reasonable synthetic accessibility. Receptor binding affinity must be optimized to ensure efficient cellular uptake without excessive systemic retention. Peptide sequences are often subjected to medicinal chemistry optimization to enhance stability against proteolytic degradation, improve cell penetration, or reduce immunogenicity. Common modifications include the incorporation of D-amino acids, N-methylation, cyclization, or introduction of non-natural amino acids to enhance metabolic stability.
Linker chemistry is a critical design element that determines when and where the payload or E3 ligase binder becomes active. Cleavable linkers for PDCs must remain stable in circulation to prevent premature payload release, yet cleave efficiently upon internalization into target cells. Common cleavable linker designs include valine-citrulline dipeptide sequences recognized by cathepsin enzymes in lysosomes, acid-labile hydrazone linkages that cleave in the acidic endosomal environment, or disulfide bonds reduced in the cytoplasmic reducing environment. Non-cleavable linkers rely on complete proteolytic degradation of the peptide carrier to release the payload, offering enhanced plasma stability but requiring different payload selection. For PROTACs, linker length and composition critically affect the geometry of the ternary complex formed between the target protein, the PROTAC, and the E3 ligase, making linker optimization essential for degradation efficiency.
The payload for PDCs typically consists of highly potent cytotoxic agents, including tubulin inhibitors, DNA-damaging agents, or topoisomerase inhibitors. These payloads must retain activity after conjugation and linker cleavage, while maintaining sufficient potency to kill cancer cells at achievable intracellular concentrations. In peptide-based PROTACs, the E3 ligase-binding component recruits the cellular protein degradation machinery. The most commonly employed E3 ligase binders target von Hippel-Lindau (VHL) protein or cereblon (CRBN), though our custom linker synthesis capabilities extend to other E3 ligase systems as programs explore alternative degradation pathways.
Conjugation Chemistry Strategies
Bioconjugation methods must achieve site-specific, high-yielding coupling between peptide and payload or E3 ligase binder while maintaining component integrity. The choice of conjugation chemistry significantly impacts manufacturing complexity, product homogeneity, and therapeutic performance.
Click chemistry approaches have gained prominence due to their exceptional selectivity and compatibility with complex molecules. Copper-catalyzed azide-alkyne cycloaddition (CuAAC) enables highly efficient coupling between azide-functionalized peptides and alkyne-functionalized payloads or vice versa. The reaction proceeds with excellent yields under mild aqueous conditions, tolerating most functional groups present in peptides and small molecules. However, the requirement for copper catalysts raises concerns about residual metal content in pharmaceutical products, necessitating thorough purification and metal testing. Strain-promoted azide-alkyne cycloaddition (SPAAC) eliminates the need for copper catalysis by employing strained alkynes like dibenzocyclooctyne (DBCO) or bicyclononyne (BCN), though these specialized reagents increase synthesis costs. The bioorthogonality of both CuAAC and SPAAC makes them particularly attractive for conjugations involving sensitive peptide sequences.
Maleimide coupling strategies exploit the selective reactivity of maleimides with cysteine thiols, providing another widely used bioconjugation approach. This chemistry offers excellent selectivity when cysteines are strategically placed in peptide sequences, enabling site-specific conjugation at defined positions. The reaction proceeds efficiently at neutral pH in aqueous buffers, making it compatible with sensitive peptides and payloads. However, maleimide conjugates can undergo retro-Michael reactions in plasma, releasing payload through thiol exchange with albumin or glutathione. This stability liability has driven the development of next-generation maleimides with improved stability, including self-hydrolyzing maleimides that form stable succinimide products after conjugation.
Alternative bioconjugation methods expand the toolbox of conjugation chemistry for specialized applications. Native chemical ligation enables conjugation via the reaction between peptide C-terminal thioesters and N-terminal cysteine residues, forming native peptide bonds in the linker region. Enzymatic conjugation using transglutaminases or sortases offers exquisite selectivity for specific recognition sequences. Oxime and hydrazone formation between ketones or aldehydes and aminooxy or hydrazine groups provides stable linkages under physiological conditions. Our process chemistry expertise enables the selection and optimization of the most appropriate conjugation strategy based on peptide sequence, payload properties, and desired linker characteristics.
Analytical Challenges for Hybrid Molecules
Characterizing PDCs and peptide-based PROTACs presents unique analytical challenges due to their hybrid nature, which combines the characterization requirements of both peptides and small molecules. Comprehensive analytical packages must confirm structure, purity, conjugation efficiency, and stability under physiological conditions.
Mass spectrometry serves as the primary tool for confirming conjugate identity and determining conjugation stoichiometry. High-resolution, accurate mass measurement verifies successful coupling by detecting the expected mass increase upon addition of the payload or E3 ligase binder. However, the large molecular weights of peptide conjugates (often exceeding 2,000 Da) can complicate ionization and detection, requiring optimization of MS conditions, including choice of ionization method, ion source parameters, and mass analyzer settings. Multiply charged species, common in peptide analysis, produce complex mass spectra that require deconvolution to determine accurate molecular weights. For conjugates with multiple potential conjugation sites, MS/MS fragmentation analysis can confirm site-specific coupling by identifying characteristic fragment ions.
HPLC method development for PDCs and PROTACs requires balancing the separation requirements of both peptide and small molecule components. Reverse-phase HPLC methods must accommodate the amphipathic nature of these conjugates, which often exhibit retention characteristics different from those of either the unconjugated peptide or the payload alone. Gradient optimization, column selection, and mobile phase additives require empirical optimization for each conjugate structure. Establishing methods to separate conjugated product from unconjugated peptide, free payload, and potential side products (multiply conjugated species, partially degraded conjugates) ensures accurate purity assessment. Our analytical characterization capabilities enable the development of robust, validated methods specific to each conjugate’s unique properties.
Linker stability assessment under physiological conditions provides critical information for predicting in vivo behavior. Stability studies in human plasma, buffer solutions at various pH values, and in the presence of glutathione or other biological nucleophiles reveal potential liabilities. For cleavable linkers, confirming selective cleavage under intended conditions (lysosomal cathepsins, endosomal pH, cytoplasmic reducing environment) while remaining stable in plasma validates the conjugate design. These studies guide formulation development and inform dosing strategies based on expected stability profiles.
Chemical Modification Expertise
Strategic chemical modifications to the peptide component can significantly enhance PDC and PROTAC performance by improving metabolic stability, pharmacokinetics, or membrane permeability. Our expertise in peptide modifications enables optimization of conjugate properties beyond what standard amino acid sequences achieve.
PEGylation involves the attachment of polyethylene glycol chains to peptide side chains or termini, increasing the hydrodynamic radius and reducing renal clearance. This modification extends the circulation half-life, potentially reducing dosing frequency and improving tumor accumulation through enhanced permeability and retention. PEGylation also reduces immunogenicity and proteolytic degradation by shielding peptide epitopes from immune recognition and enzyme active sites. The length and placement of PEG chains require optimization, as excessive PEGylation can reduce receptor binding affinity or impair cellular uptake.
Cyclization strategies impose conformational constraints that enhance metabolic stability and can improve target-binding affinity by preorganizing bioactive conformations. Disulfide bond formation between cysteine residues creates reversible cyclic structures, while lactam bridges between lysine and aspartate/glutamate side chains form irreversible constraints. Peptide stapling using hydrocarbon crosslinks or other non-natural linkages enhances alpha-helical content and protease resistance. These modifications typically improve plasma stability while maintaining or enhancing receptor binding properties.
N-Methylation of peptide backbone amides eliminates hydrogen-bond donors, thereby enhancing cell permeability and reducing proteolytic susceptibility. This modification proves particularly valuable for peptide-based PROTACs, where cell permeability is essential for accessing intracellular protein targets. Strategic N-methylation at one or two positions can substantially improve permeability without completely disrupting peptide secondary structure or receptor binding.
Integrated Synthesis Advantages
Manufacturing PDCs and peptide-based PROTACs through integrated capabilities under one roof eliminates the coordination challenges inherent in multi-vendor approaches. When peptide synthesis, linker production, and conjugation chemistry are handled by separate suppliers, project timelines extend through multiple technology transfers, quality agreement negotiations, and intellectual property disclosures. Each handoff introduces opportunities for miscommunication, scheduling delays due to conflicts, and potential quality issues arising from inconsistent manufacturing practices.
Our integrated approach allows the same scientific team to oversee all synthesis components [CZ1] , maintaining institutional knowledge from initial route design through manufacturing. Process optimization occurs seamlessly across all components, enabling rapid troubleshooting when conjugation yields prove suboptimal or analytical characterization reveals unexpected impurities. Quality systems remain consistent throughout development, simplifying regulatory documentation and ensuring coherent control strategies. Most importantly, intellectual property remains protected within a single organization rather than being disclosed to multiple contract manufacturers.
Contact our peptide-drug conjugate team to discuss how our integrated synthesis capabilities can accelerate your PDC or PROTAC program from discovery through clinical development.
[CZ1]We do not work with cytotoxic payloads for PDC chemistry.
