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 discovery of active peptides, such as antimicrobial, antiviral, catalytic, or self-assembling is challenging, due to the vast search space and limited understanding of how peptide sequences correlate with desired activities and/or functions [1]. The exponential growth of the peptide permutation space with increasing sequence length makes it More difficult to explore all possibilities efficiently. To avoid expensive and time-consuming guesswork and experimental failure, we combine machine learning (ML)-based predictions with genetic algorithm-based optimizations to accelerate peptide discovery. ML can find patterns or regularities in data, build mathematical models based on the theory of statistics and make up for the lack of knowledge. We curated a dataset of experimentally validated self-assembling peptides and combined aggregation propensity data from molecular dynamics (MD) simulations with a sequential properties representation scheme to train a neural network classifier able to identify peptides with self-assembly propensity. The ML classifier achieved 81.9% accuracy, outperforming the current state-of-the-art models. Furthermore, we developed a flexible and adaptive generative model based on the ML-driven genetic algorithm that allows for a directed search of the sequence space by promoting self-assembly propensity. This enabled the discovery of sequences in unexplored regions of the peptide space, with low similarity to the dataset, which were validated using MD simulations and experiments [2]. This approach not only improves the efficiency of the search but also contributes new knowledge to expand existing peptide datasets, driving future advancements in the field. [1] Dražić, E., Jelušić, D., Janković Bevandić, P., Mauša, G., Kalafatovic, D. (2025) ACS Nano, 10.1021/acsnano.5c00670 [2] Njirjak, M., Žužić, L., Babić, M., Janković, P., Otović, E., Kalafatovic, D., Mauša, G. (2024) Nat. Mach. Intell., 6, 1487–1500.

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. ​