ABSTRACT: Self-assembling peptides that respond to environmental stimulus, particularly pH fluctuations, are gaining significant attention for their potential use in a wide range of biomedical and biotechnological applications. These stimuli-responsive materials can adapt to varying pH conditions, which is crucial in systems such as drug delivery, More biosensing, and tissue engineering. However, the design of pH-sensitive peptide assemblies has been limited by the lack of naturally occurring amino acids that exhibit the required pH responsiveness within the relevant biological range. To address this, we have developed a novel strategy that incorporates non-natural amino acids with ionizable side chains into peptide assemblies. These amino acids, specifically designed with tertiary amine groups, undergo pH-dependent ionization, allowing for the fine-tuning of the peptide’s behavior in response to environmental pH changes. Our experimental and computational results show that these ionizable residues integrate smoothly into the peptide structure, influencing the assembly's stability and disassembly at specific pH thresholds. Additionally, the incorporation of these non-natural amino acids enhances the peptides' functionality, such as their ability to disrupt bacterial membranes at acidic pH, offering potential applications in antimicrobial therapies. This new approach to designing pH-responsive peptide materials provides enhanced control over their properties, opening the door for their use in advanced drug delivery systems, sensors, and other bio-tech applications.

ABSTRACT: Post-translational modifications (PTMs) are essential regulators of cellular processes, influencing gene expression, protein stability, and protein-protein interactions.1 Among these, lysine and arginine modifications such as acetylation, methylation, citrullination and other acylation variants are key players in epigenetic regulation.2,3 However, More the development of assay systems that can adapt to a wide range of PTMs remains a challenge. Here, we present a generalized fluorescent turn-on platform that utilizes simple peptide substrates to study the installation and removal of a diverse set of lysine and arginine PTMs, with a focus on epigenetically relevant ones. In addition to synthetic installation of native post-translationally modified residues in peptides, we employed thialysine and thiaarginine analogs to mimic modified lysine and arginine residues, enabling facile introduction of functional PTM mimetics using simple cysteine chemistry.4,5 We utilize the cleavage of peptidyl lysine and arginine bonds by trypsin, which are only removed when these residues are in their unmodified state. Conversely, in their post-translationally modified state, the peptides remain intact leading to internal fluorescent quenching, making the system adaptable to studies of both writer and eraser enzymes. Model PTMs that have been studied are removal of lysine acetylation, lactylation and β-hydroxybutyrylation by SIRT3, removal of methylated lysine variants by KDM3A and KDM4A as well as arginine citrullination by PAD4, highlighting the versatility of this approach. By integrating modularity and fluorogenic detection, this system provides an accessible, flexible, efficient, and adaptable tool for PTM studies. Its broad applicability offers significant potential for exploring enzymatic mechanisms, PTM crosstalk, and protein regulation across diverse biological contexts. References 1. B. S. Sharma, V. Prabhakaran, A. P. Desai, J. Bajpai, R. J. Verma and P. K. Swain, Oncogen, 2019, 2, 12. 2. J. Fuhrmann, K. W. Clancy and P. R. Thompson, Chem. Rev., 2015, 115, 5413-5461. 3. A. H. Shukri, V. Lukinović, F. Charih and K. K. Biggar, Biochim. Biophys. Acta Gene Regul. Mech., 2023, 194990. 4. J. C. J. Hintzen and J. Mecinović, Tetrahedron Lett., 2023, 124, 154602. 5. S. Ofori, H. S. Desai, F. Shikwana, L. M. Boatner, E. R. Dominguez Iii, J. O. Castellón and K. M. Backus, Chem. Commun., 2024, 60, 8856-8859.

ABSTRACT: mRNA display is a powerful in vitro selection and directed evolution technique that enables the screening of trillions of peptide variants for desired functions in a single experiment. Compared to other display technologies, such as phage display, mRNA display offers distinct advantages, including ultra-high-diversity libraries, in vitro selection, More and the ability to incorporate noncanonical amino acids. As a result, mRNA display has become the leading display technique for discovering novel (macrocyclic) peptide binders with antibody-like affinities and even potential oral bioavailability. PeptiFinder Biotech is the pioneering CRO specializing in mRNA display technology, providing cutting-edge services to the pharmaceutical industry. PeptiFinder mRNA display platform offers various libraries (linear, monocyclic, and bicyclic peptides) with ultra-high-diversity (up to 10^15), which can be readily screened against almost any biological target of interest (6-8 weeks) with a remarkable success rate of over >95%. Furthermore, by incorporating unnatural amino acids into macrocyclic peptide libraries, PeptiFinder mRNA display platform can generate peptide hits with enhanced physiochemical properties and optimized pharmacokinetics, streamlining the process of optimizing lead compounds into clinical candidates and accelerating drug discovery timelines for clients. PeptiFinder also offers customer library service tailored to specific client needs.

ABSTRACT: The enhancer of zeste homolog 2 (EZH2), a histone methyltransferase and a catalytic subunit of polycomb repressive complex 2 (PRC2) catalyzes trimethylation of lysine 27 of histone 3 (H3K27me3) and further alters downstream target gene levels. The genesis, progression, metastasis and invasion of many cancers have been strongly correlated with More hyperactivity of EZH2 through modulating critical gene expression. Recent studies have shown that depending on whether H3K27me3 is present or not and the various biological settings, EZH2 can also operate as a transcriptional co-activator. Here we report the use of computational tools in the development of a staple peptide that selectively binds and disrupts an intermolecular interaction within EZH2, which is crucial for PRC2 proper assembly and function. Cellular treatment with these compounds has shown a dose dependent inhibition of H3K27me3 and growth arrest through disruption of PRC2 assembly. Further, Molecular dynamic simulation, pull down and direct binding assays have validated the binding of these compounds specifically to EZH2. These compounds will help address the challenge of resistance faced by orthosteric inhibitors and provide grounds for the studies of the downstream effectors of the non-conical EZH2 function through its unique mechanism of action (MOA).

ABSTRACT: Peptides and proteins are essential biomolecules with broad applications across various industries, including pharmaceuticals, agriculture, veterinary medicine, generics, and cosmetics. However, the development of efficient production processes at an industrial scale remains challenging, as traditional methods such as chemical synthesis and More recombinant expression often fail to meet the growing demand. To address these challenges, Numaferm has introduced a novel biochemical production platform known as Numaswitch. This platform is designed to produce peptides and proteins of all lengths and functionalities with high yield and quality. The Numaswitch approach involves fusing target peptides or pepteins to Switchtag proteins, which facilitate the production of fusion proteins as inclusion bodies in Escherichia coli cells. Following extraction, Switchtags play a crucial role in promoting the correct refolding of the targets in the presence of Ca²⁺ ions, effectively overcoming the common issue of low refolding efficiencies associated with conventional IB methods. Additionally, the platform utilizes a specially engineered Numacut TEV protease, which enables precise, scarless cleavage of the Switchtag, resulting in the release of target peptides or proteins with a native N-terminus and no additional amino acids. Numaswitch is a highly reliable and universal platform for peptide and protein production aligned with the principles of green chemistry. It significantly reduces the use of hazardous raw materials, improving the safety of both the production process and the final product. Numaswitch offers a cost-effective, efficient, and sustainable alternative to traditional methods like chemical synthesis and other recombinant expression systems.

ABSTRACT: Regiostereomers and diastereomers can be separated with Phenyl or biphenyl-bonded stationary phases. The poster compares the different separation patterns achieved by Phenyl (attached via C6 chains) and biphenyl (in two different bonded ligand densities) with the conventional C18 separation. The new, different separation patterns open a dazzling More variety of ways to achieve separation in your large-scale API purification processes. Alternative separation modes may be the answer to many tough peptide purification challenges! The “PIE IN THE SKY” has been made a reality!

ABSTRACT: The importance of peptide-based nanomaterials is rapidly expanding due to their biocompatibility, tendency to self-assemble, structural diversity and design flexibility, ease of cellular uptake, and ability to function as a drug delivery carrier. Previously, we synthesized rhodamine B-dipeptide conjugates, RhB-KK/RhB-KE (RhB: Rhodamine B, K: More Lysine, E: Glutamic acid), that form stable nanotubes at physiological pH (Imax 460 nm) but dissociate into highly fluorescent monomers (Imax 580 nm) within the acidified interior of endosomal/lysosomal cellular compartments. In this work, we have expanded the utility of our rhodamine-peptide nanotubes into a drug delivery carrier by (1) chemically conjugating 5-fluorouracil (5-FU) to RhB-KK/RhB-KE via a succinic acid linker using solid-phase peptide synthesis (SPPS) and (2) co-assembling them with CPT-KK nanotubes (CPT: Camptothecin). pH-Dependence studies have been carried out using UV-Vis, circular dichroism (CD), and fluorescence spectroscopy. RhB-KK-5-FU self-assembled into nanospheres with a diameter of ~ 16 nm, as characterized by transmission electron microscopy (TEM) and atomic force microscopy (AFM). The succinic acid linker is cleaved by intracellular enzymes through hydrolysis, releasing the free drug within the cells. Co-assembly of CPTKK and RhB-KE nanotubes resulted in helical wrapping of CPTKK around RhB-KE nanotubes. The cellular uptake would be quantified using flow cytometry, and the movement of the drug inside different cancer cell lines would be visualized in real time using confocal laser scanning microscopy (CLSM). The cellular uptake pathway(s) employed will be investigated. We are also screening the structural changes that will enhance endosomal escape and increase the bioavailability of the drug. The cytotoxicity of the system will be measured using the MTS assay. In summary, our developed system would self-report the nanotubular assembly before it gets endocytosed. Once uptaken by the cells, it would emit 580 nm (from the lysosomes), indicating the monomeric state while simultaneously releasing the free drug inside the cells.

ABSTRACT: A vast majority of peptide hydrogelators harbor a bulky, non-native aromatic moiety. Such foreign moieties raise safety concerns as far as biomedical applications of hydrogels are concerned. The hydrogel research, therefore, has branched to another dimension– to identify native or native-like short peptide stretches that could cause the gelation of More biological fluids. Using well-defined criteria to identify native peptide stretches that could form a viscous solution in water but cause gelation of phosphate-buffered saline (PBS), we identified the hexapeptide stretch from human tau, viz. tau306–311, as a promising injectable hydrogelator. The peptide causes instant gelation of PBS and the cell culture media. Such hydrogels find applications as drug delivery vehicles, scaffolds for mammalian cell culture, wound-dressing material, etc.

ABSTRACT: The Orbit Peptide Discovery platform allows for the identification of highly efficacious peptide binders. There is renewed interest in the use of peptides as targeting agents for therapeutic delivery, particularly nucleic acid (eg siRNA) or radiopharmaceuticals. These peptides bind to specific disease or tissue specific biomarkers to allow for More targeted action of a conjugated payload. Transferrin Receptor 1 (TfR1) has been a hotly pursued target, due to its ability to transcytose payloads across the Blood-Brain Barrier (BBB) for therapeutic delivery within the central nervous system. Its additional expression on the surface of muscle cells has also been exploited to deliver corrective nucleic acid in the treatment of Duchenne Muscular Dystrophy (DMD). Orbit has sought to leverage its unique affinity screening technology to identify novel peptide binders to TfR1. Subsequent validation in cells demonstrates that these peptides specifically bind TfR1 and bind in a manner non-competitive to the binding of transferrin. These hit peptides represent an ideal starting point for further development towards novel TfR1 specific treatments. ​

ABSTRACT: The identification of selective, high-affinity ligands for membrane receptors and other protein targets is a critical step in the development of novel therapeutics. For small-molecule drug discovery, high-throughput screening (HTS) enables the rapid evaluation of 10⁵–10⁶ compounds per day through automated platforms. In contrast, for biologics such More as peptides, proteins, and nucleic acids, selection technologies like phage, mRNA, ribosome, and yeast display offer significantly higher diversity, routinely screening libraries ranging from 10⁹ to 10¹³ variants. By implementing smart selection and deselection strategies, ligands with affinities in the low nanomolar range can often be identified. Previously, we developed a synthetic strategy for the generation of multicyclic peptides [2]. We have now integrated this platform with mRNA display to enable the discovery of bioactive multicyclic peptides. In this presentation, we share initial findings on potent and selective multicyclic peptide binders targeting Frizzled-5 (FZD-5) and an anti-CCR7 monoclonal antibody, with affinities in the double-digit nanomolar range. Control experiments highlight the critical role of each individual peptide loop in target engagement. Furthermore, the multicyclic peptides exhibit markedly enhanced proteolytic stability compared to their linear or monocyclic counterparts. Together, these results demonstrate the readiness of this multicyclic peptide platform for broader application in the discovery of next-generation peptide therapeutics.

ABSTRACT: A.Santopretea, S.Mallartd, E. Bianchia R.Ingenitoa, P.Magottia, A.Brescianib, A.Di Marcoc, S.Espositoc, E.Monteagudoc, F.Carettic, L.Orsattic, D.Roversia, A.Santopretea, F.Tuccia, M.Venezianoc, D.Brasseurd, X.Chénédee, A.Corbiere, L.Gauzy Lazod, V.Gervatd, F.Marguetd, C.Minolettid, O.Pasquierf, B. Poiriere, A.Azame, P.Janiake, O.Duclosd, More S.Illianoeg IRBM, aPeptide Chemistry, bTranslational Biology cExperimental Pharmacology, Pomezia, Italy; Sanofi R&D, dIntegrated Drug Discovery, eCardio-Vascular and Metabolism, fDMPK, Investigative Toxicology Pre Clinical Safety Franceg Chilly Mazarin 91385 and Vitry sur Seine 94400, France Targeting the relaxin family peptide receptor 1 (RXFP1) represents a promising strategy for the treatment of cardiovascular and renal diseases. We report the development of R2R01, a novel, long-acting peptide agonist engineered for high potency, extended half-life, and improved translational potential. Initial discovery efforts identified a C18 fatty acid–modified single-chain analogue of relaxin-2 (chain B) as a potent and selective RXFP1 agonist with favourable pharmacokinetic (PK)1-2 properties. However, advanced PK profiling of this compound highlighted elevated levels of oxidative metabolism occurring in dogs and minipigs, limiting further advancement3. Through an extensive optimization program, we addressed these metabolic liabilities while maintaining sub-nanomolar RXFP1 activity. Structure-activity relationship (SAR) studies focused on modifying key molecular features—including fatty acid chain length, conjugation site, and linker composition. Notably, the introduction of α-methyl-lysine at position 30, along with additional stabilizing modifications, yielded analogs with enhanced metabolic stability, prolonged in vivo activity, and a significant reduction in mast cell–mediated pseudo-allergic reactions4. R2R01 emerged as the lead candidate from this optimized series, exhibiting a best-in-class profile in terms of receptor potency, duration of action, and preclinical safety. Its development exemplifies the power of an integrated discovery platform combining rational design, high-throughput screening, and translational in vivo models to accelerate peptide therapeutic innovation. R2R01 is currently undergoing Phase 2 clinical evaluation in patients with heart failure and hepatorenal syndrome5, supporting its potential as a transformative therapy in areas of high unmet medical need. 1S.Mallart et al. J. Med. Chem. 2021, 64(4):2139-2150, 2S.Illiano et al. Sci. Rep. 2022, 12(1):20435, 3 S.Esposito. J. Pharm. Biomed. Anal. 2023, 227, 115256, 4S.Mallart et al. J. Med. Chem. 2025, 68, 3, 3873-3885, 5B.Poirier. Br. J. Pharmacol. 2024

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: 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 More 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.

ABSTRACT: Protein modification alters structure and reactivity by adding non-native groups to specific residues. For most nucleophilic residues, multiple functionalization methods have been reported. However, there are limited methods that take advantage of the unique reactivity of methionine residues. We exploited this reactivity profile of methionine by More using a strained reactive intermediate selective for thioethers. To optimize the reaction in aqueous conditions, we altered the structure of the reactive intermediates’ precursors to increase their solubility in water, while preserving reactivity. We show that these reactive intermediates can be generated under mild conditions allowing for the preservation of peptide and protein structures. The strategy was successfully applied to a variety of natural products, peptides, and proteins. Additionally, we demonstrated the one-pot sulfonium formation then demethylation, which afforded the functionalized homocysteine product. The development of this site-selective method towards methionine will allow for valuable modifications of complex molecules in mild conditions.

ABSTRACT: Helices are particularly prevalent at PPI interfaces, and strategies to enforce helicity in peptides have been widely explored as small and more drug-like mimics of interface secondary structure. A notable approach is the hydrogen-bond surrogate (HBS) strategy, which stabilizes the H-bond in the first helical turn at the N-terminus (i to More i+4). While effective for N-terminal helicity, no robust hydrogen-bond surrogate strategy exists for stabilizing helicity from the C-terminus. This is especially important as the unfolding process of helices is thought to preferentially begin at the C-terminus and propagate toward the N-terminus. We have developed the first hydrogen-bond surrogate to nucleate the C-terminus. Through site-selective installation of an amidine in place of a native peptide bond, we effectively add another site for covalent modification. Replacing the transient hydrogen bond with a true covalent bond leads to the stabilization of the helical structure and can be tuned by varying the length of the covalent tether. This innovative work would not have been possible without the synthetic innovations our lab has recently developed. By stabilizing helices at both termini, such approaches could mitigate entropy penalties and offer new opportunities to disrupt PPIs with high specificity.

ABSTRACT: Recent advancements have optimized the incorporation of thioimidates into peptide backbones, enhancing their utility in solid-phase peptide synthesis (SPPS). This study investigates the stability of thioimidate-modified peptides on solid supports and evaluates the efficiency of hydrolyzing these intermediates directly from the resin to yield More C-terminal thioesters. The approach aims to streamline the synthesis process while preserving the stereochemical integrity of the peptides. Such methodologies are particularly valuable for applications like native chemical ligation, where access to a c-terminal thioesters will be accessible from commercially available resins, amino acids, and require no post-synthetic modifications.

ABSTRACT: IL-17 is a key mediator of psoriasis, psoriatic arthritis, hidradenitis suppurativa, and spondylarthritis. There are currently multiple approved injectable IL-17 antagonists but no approved agents for orally delivered antagonists. Clinical trials of these agents have shown that inhibition of IL-17 three dimeric forms (AA, AF, and FF) yield greater More efficacy in psoriasis than inhibition of IL-17A alone. Here we report for the first time the preclinical characterization of PN-881, an orally delivered macrocyclic peptide that potently and selectively binds IL-17A and F, thus blocking all three dimeric forms (AA, FF, and AF). PN-881 is also resistant to the proteolytic and reducing environment of the gastrointestinal tract and stable in serum after absorption, making it a suitable candidate for oral delivery. Orally dosed PN-881 demonstrated target engagement in PD-based mouse model, as well as in 5-day efficacy study in a dose-dependent manner.

ABSTRACT: The insulin/IGF-1 signaling axis regulates cell metabolism and growth, and its dysregulation is implicated in several diseases, including cancer. Elevated IGF-1 levels and IGF1R overexpression drive tumor proliferation, survival, and therapy resistance. While IGF1R is a promising oncology target, selective inhibition remains challenging due to More structural homology with the insulin receptor (IR). Existing IGF1R inhibitors lack specificity and cause hyperglycemia by inhibiting IR signaling. Our lab recently identified viral insulin/IGF1-like peptides (VILPs), a novel protein family encoded by Iridoviridae viruses, sharing ~30–50% identity with human insulin and IGF-1. Chemically synthesized VILPs bind both IR and IGF1R, with most acting as agonists. Remarkably, VILPs from Mandarin fish ranavirus (MFRV) and Lymphocystis disease virus-1 (LCDV1) function as natural IGF1R-selective antagonists, sparing the IR. Preliminary studies using chimeric VILPs demonstrate that the LCDV1-VILP C-domain and a Gly8>Ser substitution in the B-domain convert IGF-1 into an IGF1R antagonist; reversing these changes abolishes antagonism. To further define structural features driving antagonism, we are engineering IGF-1/MFRV-VILP chimeras and mutating five conserved residues shared among antagonistic VILPs. Candidate peptides will be screened in IGF1R-overexpressing murine embryonic fibroblasts to assess effects on receptor autophosphorylation and Akt/Erk signaling. Promising hits will advance to recombinant production and detailed analysis of IGF1R versus IR selectivity. This platform provides a rational path to designing next-generation IGF1R antagonists with minimal IR cross-reactivity, addressing a critical limitation of current therapies and offering a novel therapeutic avenue for IGF1R-driven diseases.

ABSTRACT: Infectious diseases remain a major public health threat, with limited therapeutic options available for several pathogens, including multidrug-resistant Pseudomonas aeruginosa. To address this challenge, we have developed a novel approach using bifunctional molecules (chimeras) that recruit complement protein C3 and activate the immune system to More eliminate infections. C3 is a highly abundant plasma protein (5–13 µM) and a central component of all three major pathways of the innate immune system: the classical pathway, alternative pathway, and lectin pathway. Deposition of C3 on the bacterial surface initiates a complement activation cascade, leading to bacterial clearance via multiple mechanisms, including membrane lysis by the membrane attack complex and immune cell recruitment through opsonization. To identify C3 binders, we screened several DNA-encoded libraries and discovered hits with low micromolar affinity for C3. The hits were validated using surface plasmon resonance (SPR) and STD-NMR. Incorporating proprietary lysine-targeting handles into the hit structures enabled chemoproteomic analysis, which localized the binding site near Lys678 of C3. Subsequent medicinal chemistry optimization yielded binders with varying affinities. Conjugating these C3 binders to bacterial-targeting peptides produced bifunctional molecules (chimeras) capable of inducing C3 deposition on the P. aeruginosa membrane and inhibiting bacterial growth at sub-micromolar concentrations. This effect was shown to be dependent on both complement-active serum and the C3-binding moiety. Our lead chimera demonstrates favorable pharmacological properties, including good aqueous solubility, plasma stability, moderate microsomal stability, no hemolysis at 2 µM, and no cytotoxicity in mammalian cells at 10 µM. Pharmacokinetic studies in mice reveal an in vivo half-life of approximately 5 hours and efficient accumulation in lung tissue. In murine lung infection models using both carbapenem-sensitive (ATCC27853) and -resistant (AR #0246) P. aeruginosa strains, treatment with our lead compound at 29 µmol/kg (BID) resulted in a robust 2-log reduction in bacterial load. The complement-recruiting chimera platform is now being expanded to target other pathogens and holds promise in the fight against antimicrobial resistance.

ABSTRACT: Glucagon Like Peptide (GLP-1) can be used in treatments of diabetes type 2, where carbohydrates are not metabolized due to insulin resistance or lack of insulin, resulting in high level of glucose in the blood. The approval of the GLP-1 receptor agonist semaglutide for weight regulation in January 2023 ushered in a new era of obesity therapy. More According to Frost & Sullivan survey, the GLP-1 drug market is expected to reach US$28.3 billion in 2025, with an annual growth rate of 16.6% from 2020 to 2025, and will exceed US$40 billion in 2030. Silica reversed phase chromatography (RPC) is a popular purification tool for peptide-based therapeutics such as insulins and GLP-1 compounds. As GLP-1 drug market grows, there is a big demand for bulk media with high performance and much longer lifetime for purification of peptide-based drugs. NanoMicro Technology is the only company to commercialize monodispersed silica bulk media for separation and purification process. Our innovative technology allows us to manufacture monodispersed silica particles in large quantity (several hundred kilograms a batch with almost 100% yield), thus dramatically reduces cost and meets high demands of purification process market needs. In this report, we would like to introduce our UniSil Revo 10-100 particles, discuss their physical properties and chemical stability, and show their applications in purification of insulin and GLP-1. With an inert surface coating, we have achieved lifetime of UniSil Revo 10-100 at least 2-3x better than silica particles at 0.1M NaOH flash condition. With pore size of 100Å, UniSil Revo 10-100 particles have very similar selectivity and retention time to Kromasil 100 particles, making direct replacement of Kromasil 100 very feasible, without changing of purification methods, or sacrificing yields and purity of GLP-1 drugs.

ABSTRACT: We have developed streaMLine, an innovative platform for peptide drug discovery that greatly shortens the time from initial hit to clinical drug candidate. The platform allows for high-throughput synthesis and screening. Thousands of peptides are systematically screened in in vitro assays and on physicochemical parameters, whereby the streaMLine More platform enables complete sequence exploration and simultaneous optimization of key parameters. We employ a fully digitalized laboratory system where detailed information on all aspects of sample lifetime is tracked. Using a machine learning approach, this enables accurate distinction of key chemical peptide modifications from artefact background effects. This unique strategy for peptide screening integrates with state-of-the-art in vivo pharmacology facilities, including advanced animal models and rapid determination of PK/PD relationships. Using the streaMLine platform, we developed novel GLP-1R agonists, to demonstrate how high-throughput screening peptide libraries and machine learning guided drug design can be applied to accelerate drug discovery. We systematically synthesized and screened a total of 2,688 peptides in a parallelized optimization workflow. Using this approach, we identified a vast chemical solution space for generating novel GLP-1R agonists based on an alternative peptide starting point, i.e. the secretin backbone. To validate the pipeline, we conducted an in-depth profiling of a GLP-1R agonist that showed high receptor selectivity, attractive physicochemical properties, a potent weight-lowering in vivo efficacy, and a pharmacokinetic profile compatible with a once-weekly human dosing regimen.