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Abstract: Cysteine is an attractive target for peptide and protein modification due to its unique reactivity and its relatively low abundance in natural proteins. Metal complexes undergoing oxidative addition have been shown to be highly effective arylation reagents; however, nucleophilic aromatic substitution as a post-translational modification in peptides and proteins has typically been limited to perfluorinated- or nitro-arenes. Moreover, selectivity can be an issue with these methods when lysine or other nucleophilic residues are present. We present a highly selective method that uses air- and water-stable organometallic reagents for aryl thioether formation in peptides and proteins. The organometallic complexes are activated to nucleophilic aromatic substitution and the modified peptides are afforded after exposure to visible light. These reagents were found to be highly selective for cysteine arylation, and no reactivity was observed with any other nucleophilic amino acid. We synthesized a series of organometallic reagents, which allowed peptide and protein modification in high yields. Notably, the low steric bulk of the complexes also gave highly selective ortho-, meta-, or para-double substitution – allowing the synthesis of peptide macrocycles and asymmetric dithioethers. This method for cysteine modification is a valuable new tool for selective and versatile functionalization of peptides and proteins.

Despite showing antiviral potential, a majority of antimicrobial peptides (AMPs) tested against Zika virus (ZIKV) have failed to advance toward clinical translation. A major limitation lies in their linear structure, which renders them highly susceptible to proteolytic degradation and limits cellular uptake—two properties crucial for effective antiviral action against an intracellular pathogen like ZIKV.
This poster proposes a shift in antiviral peptide design: from linear to cyclized AMPs. Cyclization enhances conformational rigidity, improves stability in physiological conditions, and facilitates endocytosis—all without compromising the native antiviral functionality. Drawing from established AMP sequences known to act on ZIKV and similar flaviviruses, we explore how head-to-tail or side-chain cyclization could restore therapeutic viability to sequences that failed in linear form.
The design-centered re-evaluation sets the stage for next-generation antiviral peptides tailored for real-world deployment. Cyclized AMPs offer a safer, more stable, and pharmacologically promising avenue for peptide-based therapeutics targeting emerging arboviruses like ZIKV.

Macrocyclic peptides (MCPs) hold great promise as therapeutics due to their ability to target protein-protein interactions and other challenging biological targets with high affinity and selectivity. Despite their potential, a major limitation to their broader use in drug discovery is the lack of large and diverse MCP libraries suitable for high-throughput screening (HTS). At the same time, with thousands of unique commercially available building blocks, the number of synthetically accessible macrocyclic hexapeptides is greater than 10^18 – a number far too large to comprehensively screen in functional assays. We developed BRiTeCycle to enable the sparse synthetic sampling of this 10^18 space, followed by the rapid exploration of the chemical space around any primary hits. BRiTeCycle uses a novel spatially addressed, microfluidics-based synthesis approach that has broad chemical compatibility and high stepwise efficiencies. To assess BRiTeCycle, we synthesized a 2,304-membered library of cyclic hexapeptides, in quadruplicate. Sixty amino acid building blocks were incorporated into the library. The purity of 116 samples (5% of the total) was assessed from 220 nm UPLC chromatograms. Between replicates, crude LC/MS traces are highly reproducible, suggesting reproducible robustness in our synthetic approach. In addition to the desired cyclic hexapeptide, a common side-product was the corresponding cyclic dodecapeptide that results from the cyclic dimerization of two linear hexapeptide precursors. Linear product was rarely observed. To further assess the membrane permeability of these MCPs, we applied Parallel Artificial Membrane Permeability Assay (PAMPA) to a subset of the library, which supported the membrane permeable nature of our MCPs. This work demonstrates that the BRiTeCycle platform can efficiently synthesize large libraries of diverse, cyclic hexapeptides, enabling high-throughput screening and characterization for novel drug candidates.

Macrocyclic peptides (MPs) are a class of compounds that have been shown to be particularly well suited for engaging difficult protein targets. However, their utility is limited by their generally poor cell permeability and bioavailability. Here, we report an efficient solid-phase synthesis of novel MPs by trapping a reversible intramolecular imine linkage with a 2-formyl- or 2-keto-pyridine to create an imidazopyridinium (IP+)-linked ring. This chemistry is useful for the creation of macrocycles of different sizes and geometries, including head-to-side and side-to-side chain configurations. Many of the IP+-linked MPs exhibit far better passive membrane permeability than expected for “beyond Rule of 5” molecules, in some cases exceeding that of much lower molecular weight, traditional drug molecules. We demonstrate that this chemistry is suitable for the creation of libraries of IP+-linked MPs and show that these libraries can be mined for protein ligands.

Peptide therapeutic discovery is experiencing a resurgence, particularly for challenging, historically “undruggable” targets. WuXi AppTec is leading the way in this field with innovative technologies and platforms. Traditional phage display, while cost-effective and providing substantial library diversity, is limited by its reliance on only the 20 natural amino acids, resulting in restricted chemical diversity. In response, we have developed our mRNA display capabilities, which surpasses phage display in robustness with macrocycles up to 15 amino acids long. Additionally, our peptide DNA-encoded library (DEL) service provides an alternative approach, leveraging unnatural amino acids to generate hundreds of billions of linear and cyclic peptide-like molecules. These DEL macrocycles offer broader chemical diversity and improved physicochemical properties compared to traditional peptide libraries, with smaller ring sizes (6-9 amino acids) and innovative cyclization strategies, including the ‘click’ reaction. Conversely, we can also design a focused peptide-DEL library based on an initial phage or mRNA-Display screen with up to 4 sites to include any of our 1400+ validated natural and unnatural amino acids. The DEL platform also enables the use of diverse cyclization strategies beyond disulfide and thiol-ether bonds, such as the ‘click’ reaction, which we used to produce our current libraries. In our poster we demonstrate the effectiveness of our technologies for discovering peptides including the discovery of i) a 9 nM cyclic inhibitor of the MDM2-p53 interaction, ii) potential tumor cell-specific peptide ligands that are being explored for targeted delivery of payloads via oligonucleotides or radioisotopes, iii) several peptide inhibitors of PCSK9 with nM ~ μM range binding affinity and inhibition.

Insulin therapy is vital for millions of diabetes patients but faces ongoing challenges such as high production costs, limited stability, and strict storage requirements. Our team recently discovered a minimized insulin scaffold from a venomous animal that is smaller, more stable, and highly soluble compared to human insulin. Despite its promising structure, this natural peptide exhibits minimal activity on the human insulin receptor, limiting its therapeutic potential.

To enhance receptor binding and activation, I developed an iterative AI-driven pipeline that generates and optimizes insulin variants. Beginning with ProteinMPNN for variant sequence generation, the pipeline predicts 3D structures using large language models like ESMFold and other machine learning tools such as AlphaFold3, followed by receptor docking with ZDOCK. Correct receptor engagement is verified through PyMOL and FoldSeek, while the PyRosetta DDG protocol evaluates binding affinity. Finally, molecular dynamics simulations with GROMACS assess complex stability, ensuring robust interactions. This process iterates thousands of times to refine promising candidates.

For experimental validation, designed variants are expressed via yeast display as a high-throughput method to screen for receptor binding. Successful candidates from yeast display re-enter the computational pipeline for further optimization, establishing a design–test feedback loop. Variants demonstrating strong receptor affinity proceed to chemical synthesis and comprehensive in vitro and in vivo testing, paving the way toward cost-effective, stable, and bioavailable insulin therapeutics.

Acetylation is the most dynamic protein translational modification often associated with increased DNA accessibility and transcription. These acetylated histones recruit transcription and remodeling factors, and their deregulation could result in aberrant expression of survival and growth-promoting genes. Recognition of acetylated lysine is principally mediated by bromodomains (BRDs). Recent studies have shown that BRD9 is preferentially used by cancers that harbor SMARCB1 abnormalities such as malignant rhabdoid tumors and sarcomas. BRD9 is an essential component of the SWI/SNF chromatin remodeling complex, and a critical target required in acute myeloid leukemia. As the biological function of BRD9 in tumorigenesis becomes clear, bromodomain of BRD9 has become a new hot target for effective tumor treatment method.
BRD9 has a different architecture than other bromodomains. Due to larger hydrophobic cavity of BRD9, it can recognize longer propionyl and butyryl marks on lysine. Thus, N-butyryl-lysine (BuK) can selectively bind to BRD9. Our group is specialized in the amber suppression-based noncanonical amino acid (ncAA) mutagenesis technique. We propose to extend this technique using phage-displayed ncAA-containing peptide libraries for the identification of high-affinity and highly selective BRD9 inhibitors.
Phage display is a technique for rapid screening of potential ligands. It is facilitated through the creation of a genetic fusion between a randomized peptide sequence and pIII, a phage coat protein. This direct link between genotype and phenotype allows for peptide screening. We utilized Phage-assisted, Active site Directed Ligand Evolution approach to target BRD9. To identify the binders, we choose 7mer phagemid library which generates 1.5x1010 randomized possible peptides displayed on PIII of bacteriophages. The peptides screened were tested for binding using Bio-Layer Interferometry and inhibition by Alpha Screen assay. Based on SARs second-generation focused selection was done to screen for more potent peptides. Studies resulted in identification of BRD9 binders with increased specificity and affinity. The estimated IC50 for peptide was 0.74 μM and Kd was determined to be 0.53 μM. Second generation selection peptide inhibits protein with IC50 0.54 μM and Kd value 0.104 μM. Selected peptides successfully bind and inhibit BRD9, and we aim to further optimize its cellular target engagement and on-target effects.

Amidines are an under-explored isostere of the amide bond that are emerging as a promising candidate for peptide bond surrogates because they more closely approximate the properties of the native amide bond. Amidines have been reported in natural as well as artificially synthesized peptide backbones and have shown to possess potent antibiotic properties. Amidines modulate hydrogen bonding interactions and stabilize helical structure of peptides much like amides, reinforcing their significance as an ideal amide bond isostere. Despite the significance of amidines, their incorporation into linear peptides remains synthetically challenging. To date, the sole reported strategy for installing amidines on linear peptides—via the attack of an amine on an electrophilic thioimidate—suffers from slow kinetics, undesired side reactions and difficult to monitor solid-phase steps, thereby limiting its broad applicability. We have developed strategies for rapid and direct installation of amidines into linear peptides which is compatible with the standard Fmoc solid phase peptide synthesis conditions. This work aims to overcome the critical roadblock of laborious amidine installation on linear peptides and enable their facile incorporation into peptide backbones.

Through continuous innovation in the field of solid-phase peptide synthesis (SPPS), a novel bromine-functionalized polystyrene/ DVB resin was designed to enhance the synthesis of C-terminal acid peptides, achieving high yields and low impurities.
Currently, the industry relies on two widely accepted resins for synthesizing acid peptides: the Wang resin and the 2-CTC resin. Each of these resins has its own well-documented advantages and limitations, making the selection process crucial for specific applications.
The case studies presented herein demonstrate the effectiveness of the MBH-Br (4-Methylbenzhydryl bromide) resin in addressing the challenges associated with the synthesis of acid peptides showing low DKP and high stability to peptide elongation. These studies highlight the resin’s ability to facilitate the production of both protected and unprotected acid peptides, showcasing its potential to improve efficiency and purity in peptide synthesis and consequently supporting the peptide manufacturers in achieving better output.

Sponsored Talk by Asymchem
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Susan Li
Peptide Chief Scientist, CMC development and manufacturing, Asymchem Group

Forced Degradation Study of Peptides

Abstract

Peptides are a-amino acid polymers composed of 40 or fewer amino acids. Therapeutic peptides are less immunogenic and offering greater safety, selectivity and specificity. By understanding the degradation behavior of peptides, safer and more effective peptide drugs can be designed. Degradation studies are conducted including a selection of stress conditions, degradation products formed, and different peptide degradation mechanisms. A case study on the degradation profile of existing peptide drugs is presented as well.

Bio

Susan Li holds a PhD in Polymer Chemistry and Physics from Nankai University in China. She completed a postdoctoral fellowship at the University of Texas Southwestern Medical Center (UTSW) in the United States. Before joining Asymchem, she previously worked at PolyPeptide Group where she focused on CMC development for peptide APIs. She also served as a Senior Scientist in the Department of Macromolecular Life Sciences and Cancer Biology at the Stanford Research Institute (SRI), leading efforts to establish a peptide-targeted drug platform for cancer treatment and diagnosis. In 2019, Susan became the Director of Peptide R&D at Sinopep Biopharmaceutical Co., Ltd. in China, where she led the development of peptide APIs, including Semaglutide and Desmopressin. Under her leadership, the DMFs for these compounds were successfully completed and found to meet FDA standards. Currently, Susan works as the Peptide Chief Scientist at Asymchem Group, overseeing peptide CMC development and manufacturing.