Foundational Genetically-encoded libraries (GEL) platforms like phage-, yeast-, mRNA-display construct libraries using 20 natural amino acids (20AA). GEL can be expanded by unnatural amino acids (UAA) and chemical post-translational modification (cPTM). The standard procedure involves incorporating UAA or cPTM into a "naïve" library, followed by multiple rounds selection. However, such approach uses zero knowledge of binding interactions which might have been discovered from 20AA libraries. There is currently no consensus whether libraries containing pre-existing knowledge can offer an effective path for discovery of molecular interactions. To explore this, we evaluated the feasibility of discovering macrocyclic peptide ligands from "non-zero knowledge" libraries by chemically reshaping pre-selected phage-displayed 20AA binders against the NS3aH1 protease. The re-shaping is performed using a novel C2-symmetric linchpin, 3,5-bis(bromomethyl)benzaldehyde (termed KYL). KYL diversified peptide libraries into bicyclic architecture and delineated 2 distinct sequence populations: (i) peptides with HXDMT motif retained binding upon bicyclization (ii) peptides without HXDMT motif lost binding once chemically modified. The same HXDMT family can be found in selections starting from naïve KYL-modified library. Our report provides a case study for discovering bicyclic ligands using pre-selected 20AA libraries, suggesting that other 20AA-based selections potentially could be used for discovery advanced peptide ligands.

Subunit vaccines are safer than live-attenuated or inactivated vaccines. However, their limited immunogenicity and susceptibility to metabolic degradation require strong adjuvants and/or conjugation to delivery systems to induce a robust, antigen-specific immune response. Interestingly, synthetic peptides can be useful and polyvalent building blocks for development of self-adjuvanted nanoparticles. Recent studies showed that amphiphilic peptides can increase antigen density, promote cellular uptake by APCs and activate T and B lymphocytes. Additionally, short peptide monomers forming cross-β-sheet fibrils such as I₁₀ have shown potential in inducing strong immunity against the grafted epitope. However, direct comparison of the intrinsic immunogenicity of peptide amphiphilic cylindrical micelles and cross-β peptide fibrils has never been performed. In this work, the amphiphilic peptide C₁₆-V₃A₃K₃(PA) and the I₁₀ β-peptide were each linked to two epitope models, OVA_253-266 and OVA_323-339, able to polarize the resulting adaptive immune response differently. Biophysical analysis showed that both nanoplatforms formed β-sheet-rich nanofilament structures with epitopes exposed on their surfaces. Intramuscular administration in mice led to a strong, antigen-specific humoral response without additional adjuvants. This study illuminates the potential of synthetic self-assembling nanoplatforms as universal antigen carriers, enabling the rapid development of vaccines to combat infectious diseases.

Naturally derived biomaterials offer biocompatibility but may lack batch-to-batch consistency and precise chemical tunability, limiting their use in advanced applications like drug delivery and tissue repair (1). Synthetic biomimetic peptides can address these limitations by producing consistent, peptide-only hydrogels that are both biocompatible and tunable in their properties at the atomic level (2).

Using optimized solid-phase peptide synthesis, we produced high-purity biomimetic peptides engineered with light responsive functional groups for rapid, light-triggered crosslinking. The peptides were purified by RP-HPLC, with identify verified by mass spectrometry, and analyzed for stable conformation and supramolecular assembly through circular dichroism. To leverage the spatial and temporal control offered by light activation, we introduced alkene and thiol moieties, enabling rapid thiol-ene reactions that allow for controlled material assembly (3). By systematically varying the peptide structure, we investigated how functional group configuration impacts the physical properties of hydrogels.

Our custom-designed biomimetic peptides formed strong hydrogels with finely tunable mechanical properties. Rheometry demonstrated that increasing reactive group density enhanced gel elastic modulus (G’) up to the solubility limit. Allowing time for peptide folding of 1 hour before activation further strengthened the hydrogels, especially at lower concentrations.

Sulfilimines, the mono aza-analogues of sulfoxides have sparked acute interest in the fields of chemistry and biology with the discovery of a S=N crosslink in collagen IV. They serve as potential bioconjugation handles, bioisosteric pharmacophores, and are precursors to medicinally relevant S(VI) scaffolds. S-imination strategies to access sulfilimines have been employed for methionine (Met) functionalization in peptides and proteins for conjugation and also for chemoproteomic profiling of Met. Herein, we report the use of diphenylphosphinylhydroxylamine (DPPH) for S-imination to access sulfiliminium salts and sulfoximines under safe, mild, metal-free, and bio-molecule-compatible conditions with excellent chemoselectivity, broad functional group tolerance, and applicability in late-stage derivatization. These S-imination methods afforded successful chemoselective installation of sulfiliminium and sulfoximine scaffolds on clinically relevant methionine, buthionine, and also on complex peptides such as cholecystokinin and bombesin. α-amanitin is a sulfoxide bearing bicyclic octapeptide which is a potent inhibitor of RNA polymerase II, explored as a payload in targeted cancer therapy. DPPH mediated S-imination renders late-stage synthetic access to sulfilimine, sulfoximine, and sulfondiimine amatoxins; and cytotoxicity assays were employed to address their potential bioisosterism. Collectively, these results attest to the robustness of this S-imination strategy for chemoselective installation of versatile sulfilimine and sulfoximine scaffolds on peptides.

The Glucose Regulated Protein of 78 kilodalton (GRP78) is a main chaperone assisting in protein folding in the endoplasmic reticulum (ER) during cell stress conditions. Furthermore, GRP78 is overexpressed and translocated to the cell surface of cancer but is absent (or minimally expressed) on normal tissues, acting as a clinical biological marker (biomarker) for cancer-targeted therapy. This research is based on the discovery of cell surface (cs)GRP78 peptide binding ligands for cancer-targeted gene (short interfering RNA, siRNA, and plasmid DNA, pDNA) delivery and therapy applications. Synthetic fluorescein-labeled amphiphilic peptides composed of csGRP78 targeting and penetrating domains were investigated for anti-cancer utility. These peptides folded into unusual helical-coiled structures that enabled self-assembly into nanofibers. The peptides also displayed GRP78-dependent cell uptake in a representative GRP78 overexpressing prostate cancer (DU145) cell line. The detected cytosolic and nuclear accumulation of fluorescein-labeled peptides underscored their utility in gene delivery applications. Transfections (siRNA and pDNA) in the DU145 cells indicated the potential to silence (e.g., GRP78 siRNA) and activate (e.g., p53 pDNA) key biomarkers implicated in the cancer cell death response. This presentation will thus serve to highlight the importance of targeted gene delivery approaches for precision oncology applications.

Protease-activated receptor 2 (PAR2) is constitutively expressed on the endothelial cells of blood vessels, playing a role in numerous physiological processes such as cell migration, vasodilation, and inflammation. Upon PAR2 activation, several second messenger signaling cascades are activated to regulate these cellular functions. Studies have found that certain ligands can promote selective activation of one or more signalling pathways, a concept referred to as biased agonism. However, it is unclear which PAR2-mediated functions are coupled to each signalling pathway in endothelial cells. This work aims to design PAR2 activating peptides that produce endothelium-dependent vasodilation with little to no inflammation.

A library of 25 PAR2-targeted seven-mer peptides was synthesized by automated Fmoc solid-phase peptide synthesis, purified by preparative HPLC, and characterized by high-resolution mass spectrometry. Modifications to the C-terminal region explored the importance of different structural features on receptor activation. Initial results revealed peptides that were biased towards specific G protein pathways when containing a positively charged C-terminus, and other pathways when containing short aliphatic or polar uncharged amino acids at position 6. These structural features will also be evaluated for functional selectivity, providing insight into how the different PAR2 driven functional responses are coupled to each signalling pathway.

Many cancers are difficult to treat due to challenges in targeting proteins that drive cancer development. The Myc/Max transcription factor is such a target that it is associated with >70% of cancers. One promising avenue is to design Myc/Max mimics that inhibit the Myc/Max transcription factor from binding its DNA target, which is the E-box. The Myc/Max protein structure, the basic/helix-loop-helix/zipper (bHLHZ), inspired us as our design scaffold. Building upon our successful Myc/Max protein mimics, particularly MEF, we aimed to enhance MEF’s E-box binding specificity and affinity by introducing intrinsically disordered regions (IDRs). We hypothesized that incorporating IDRs into the loop of the helix-loop-helix region would optimize MEF’s selective targeting of the E-box, as we found in earlier designed proteins. This project involves two key phases: evaluating the IDR loop as an independent module that enhances E-box binding and exploring the impact of loop length and sequence on MEF’s selectivity for the E-box. By employing bacterial one-hybrid assay and electrophoretic mobility shift assays, we aim to create next-generation protein drugs exclusively targeting the Myc/Max/E-box network, thereby offering a unique strategy against undruggable cancers.

Most small molecule drug candidates being developed focus on their ability to bind to a protein’s pockets to inhibit or block the binding of the natural substrates. However, they fail to inhibit protein-protein interactions, which have garnered significant attention in the pharmaceutical industry in recent years. Peptides’ high structural compatibility with the targeted proteins have the ability to disrupt such protein-protein interfaces.

Efficient in silico design of high-affinity peptide ligands is an ever-growing field that still demands the synthesis to confirm the desired activity.¹Batch-mode solid-phase peptide synthesis has been the standard for drug discovery; however, synthesizing a library of candidates is time- and resource-consuming.²

In this work, we present our efforts for the rational design of two libraries of peptides targeting HLA-DR and Hsp90; as well as the use of CF-SPPS for synthesizing them to evaluate both the design model and their biological activity.

(1) Vanhee, P.; Rousseau, F.; Schymkowitz, J. Computational Design of Peptide Ligands. Trends in Biotechnology. May 2011, pp 231–239.
(2) Ruhl, K. E.; Schultz, D. M.; Lévesque, F.; Mansoor, U. F. Continuous-Flow Solid-Phase Peptide Synthesis to Enable Rapid, Multigram Deliveries of Peptides. Org Process Res Dev. 2024

Since Merrifield’s development of solid-phase peptide synthesis, we have seen explosive growth in the number of synthetic building-blocks that can be incorporated into peptides. This has created a problem: the number of possible molecules that could be synthesized is many orders of magnitude greater than the largest conceivable combinatorial libraries. Computational design, based on combinatorial optimization algorithms, addresses this problem by proposing sequences likely to have desired folds and functions. These computational methods complement experiments by reducing astronomically large numbers of combinatorial possibilities to experimentally tractable shortlists. This presentation describes our robust, versatile methods, made available to peptide scientists in the Rosetta and Masala software suites, for designing peptides that fold into rigid conformations. Our physics-based methods generalize to exotic chemical building blocks poorly amenable to machine learning-based methods for want of training data. Our pipeline has produced experimentally-validated mixed-chirality peptides that bind to targets of therapeutic interest, and peptides that diffuse across cell membranes. Ongoing research is mapping the sequence optimization problem (which grows intractable even for supercomputers as the number of candidate chemical building blocks grows very large) to current and near-future quantum computers, allowing use of quantum algorithms in the context of the existing, widely-used design protocols.

Antimicrobial resistance (AMR) has become a major challenge in the prevention and treatment of bacterial infections. Faced with this critical situation, the development of new antimicrobials with new modes of action has become a global priority. Antimicrobial peptides have been recognized as an interesting tool to fight AMR as they often show broad spectrum of activity and act via original modes of action. The cationic lipopeptide brevibacillin is particularly interesting because of its significant inhibitory activity against several clinically relevant bacteria, including multidrug-resistant strains. To better understand its mode of action and optimize its pharmacological properties, our objective was to develop a straightforward chemical synthesis and perform a structure-activity study.

In this study, brevibacillin and a series of analogues were produced by solid-phase peptide synthesis and their antimicrobial activity and cytotoxicity evaluated. Some analogues showed antimicrobial activity comparable to native brevibacillin against the tested bacteria. This structure-activity study identified key features of brevibacillin that allow modifications without affecting the inhibitory activity, while significantly reducing toxicity. The study highlights the great potential of brevibacillin, as well as opportunities for modifications to increase production yields, enhance stability, optimize activity, and reduce cytotoxicity for applications in the food, veterinary, and medical sectors.