Antimicrobial resistance (AMR) has become one of the biggest health challenges of this era, causing yearly millions of deaths worldwide. However, there has been a sharp decline in antibiotic development despite the critical need for novel therapies. In our work, we propose multiple methodologies for the discovery and design of peptidic compounds with antibacterial activities, ranging from natural products to inhibitor design. In this talk, we focus on the progress we have made in the development of nature-derived antimicrobial peptides (AMP). Lactomodulin, a 53-amino acid long microbiome-derived AMP, has both antibiotic and anti-inflammatory activity. We have analyzed the composition and structure of the parent peptide and generated shorter truncated versions analyzed based on AMP potential, helical propensity, and toxicity predictions. Out of 13 derivatives, one 15-mer LSKISGGIGPLVIPV-NH₂ and its cyclic versions have shown improved activity against Gram-positive (MIC 0.8─2.3 µM), including resistant strains, and Gram-negative bacteria (MIC 8─9.3 µM). Time killing assays further showed a fast bactericidal activity and TEM studies indicated a membrane-targeting mode of action. Interestingly, other peptides from the initial library also showed a retention of anti-inflammatory activity. These results pave the way for the development of unique peptides with Gram-positive, Gram-negative, and anti-inflammatory activities.

We have developed a novel peptide discovery technology which we called PepFusion. It is based on simultaneous identification and optimization of the sequences critical for protein-protein interactions or ligand binding. We tested it by selecting peptide antagonists of interleukin-6 (IL-6), a key mediator of several inflammatory diseases. The PepFusion library demonstrated superiority over a random library by yielding a peptide with low micromolar affinity for IL-6, whereas the random library failed. The affinity of the lead peptide was improved through additional round of mutagenesis leading to peptide variants with low nanomolar affinity toward IL-6 as well as low nanomolar IC50 in cell-based assay.

Intrinsically disordered regions (IDRs) provide structural flexibility useful for mediating diverse functions, such as cellular signaling, transcription, and regulation. Intrinsically disordered proteins exist in 40-50% of the human proteome. The structural plasticity of IDRs presents an opportunity to exploit dynamic protein-DNA binding. We used the basic region/helix-loop-helix/ leucine zipper (bHLHZ) family of transcription factors as scaffolds to construct structural analogues to the Myc/Max protein, which is associated with >70% of cancers. Our latest version, MEF, competitively binds to the E-box (enhancer box, 5’CACGTG) DNA motif, thereby inhibiting the activity of proto-oncogenic Myc/Max. To further optimize MEF, we replaced the loop in the HLH region with the longer loop of the bHLHZ transcription factor USF1. The USF1 loop can improve affinity and specificity by providing more flexibility and electrostatic contacts with DNA bases flanking the E-box. We are studying how mutations in both the length and identity of residues affect MEFU’s affinity and specificity to the DNA sequences flanking the E-box. This work can illuminate how IDRs contribute to finetuning DNA binding to optimize protein drugs against undruggable diseases.

The chemical conjugation of proteins has seen tremendous applications in the past decades, with the booming of antibody-drug conjugates and their use in oncology. While genetic engineering has permitted to produce bespoke proteins with key (un-)natural amino acid residues poised for site-selective modifications, the conjugation of native proteins is riddled with selectivity issues. Chemoselective strategies are plentiful and enable the precise modification of virtually any residue with a reactive side-chain; site-selective methods are less common and usually most effective on small and medium-sized proteins. This presentation will offer some of our lab's recent attempts at addressing these selectivity issues, with the use of multicomponent reactions, and the Ugi reaction more specifically.

Excessive mitochondrial fission contributes to a variety of pathologies, including cardiovascular diseases (CVDs), neurodegenerative disorders and cancer. Dynamin-related protein 1 (Drp1), a mitochondrial GTPase, plays a crucial role in mitochondrial homeostasis and was demonstrated to interact with Fission protein 1 (Fis1), leading to excessive mitochondrial fission, and mitochondrial impairment. Therefore, inhibition of the Drp1/Fis1 protein-protein interaction (PPI) is important for both basic research and drug discovery. Previously, we developed P110, a linear peptide that inhibits excessive mitochondrial fission and specifically targets the Drp1/Fis1 PPI. This peptide demonstrated various therapeutic potentials in a variety of disease models. Herein, based on a rational design approach and structure-activity relationship (SAR) studies, we present the development of CVP-350, a macrocyclic Drp1/Fis1 PPI inhibitor, with 'drug-like' properties. CVP-350 demonstrated: (1) Effective and specific inhibition of the Drp1/Fis1 interaction, underscoring their potential bioactivity in vitro. (2) Preservation of mitochondrial integrity and function under multiple cellular stressors in vitro, suggesting promising effects on mitigating mitochondrial-related cellular dysfunction. (3) Reduction of myocardial damage by 50-70% in a rodent infraction model, without causing any observable toxicity. Overall, our findings indicate that CVP-350 can serve as a promising lead for the treatment of diseases related to mitochondrial dysfunction.

Natural products serve as important lead compounds in drug discovery attributed to their intrinsic biological activity and potential to inspire novel therapeutic agents. Peptides are known to display low toxicity, high selectivity, and high specificity allowing them to be suitable anti-cancer therapeutics. Naturally occurring cyanobactins are a notable class of small cyclic peptides, containing heterocyclics, that produce antimalarial, antitumour, and antibacterial effects. In 2016, Wewakazole B, was isolated from cyanobacterium, Moorea producens, found in the Red Sea. This cyclic dodecapeptide contains 3 heterocyclic oxazole rings and is cytotoxic against MCF7 breast cancer and H460 lung cancer cell lines, however, the mode of action is still unknown. Previously established solution phase synthetic schemes for Wewakazole B are not feasible as they require tedious and time-consuming reaction and purification steps resulting in low yields. This study details how solid phase peptide synthesis can be used to synthesize Wewakazole B and first-generation analogues with minimal purification steps, increased yield while investigating their pharmacokinetic properties. Various methods to introduce oxazoles during solid phase synthesis will be showcased. The end goal is to have a complete SAR profile of Wewakazole B and create a library of analogues with optimized cytotoxicity against drug-resistant cancer cell lines.

Natural products continue to be a source of potential drugs due to their specificity towards their targets after years of natural selection. α-Amanitin is an example of a unique octapeptide that has excellent specificity for RNA polymerase II. The design and synthesis of amanitin analogs can provide cytotoxic payloads to be used for targeted cancer therapeutics, such as antibody drug conjugates. The azaGly amanitin analog, wherein glycine-7 was replaced by an aza-glycine amino acid, resulted in increased cytotoxicity compared to the natural product. These promising results and the knowledge that aza-amino acids can enhance ß-turns in peptides, led to the exploration of functionalized aza-amino acid derivatives. Herein I report a structure-activity relationship study demonstrating the effect of replacing glycine at position 7 with aza-amino acid moieties. Three toxins, AzaVal⁷, AzaPentanyl⁷, and AzaCycloPentanyl⁷ have been synthesized, all of which display comparable cytotoxicity to the natural product, and encouragingly AzaVal⁷ was 3x more toxic on HEK293 cells.

Chemically modified genetically encoded peptide libraries encompass broader chemical space that enables rapid discovery of proteolytically resistant and potent drug leads. However, evaluating chemical modifications on phage-displayed peptide remains challenging due to their ultra-low concentrations. Despite extensive efforts to assess these modifications—such as using ESI-MS for identifying modifications of phage-displayed peptides or using MALDI-TOF-MS to detect chemical transformations on p8-displayed peptides—current methods are often time-consuming or impractical for certain applications, hindering the development of new modification strategies for generating chemically modified peptide libraries in drug discovery. Herein, we propose a strategy that enables rapid display of peptides regards of sequence and size on phage and offer convenient kinetic evaluation of chemical transformation on phage-displayed peptide. In this work, DBCO was installed on p8 proteins of M13 phage, followed by “click” chemistry attachment of chemically synthesized azido peptides. Subsequently, the kinetics of diverse chemical transformations on phage-displayed peptide were evaluated using MALDI-TOF-MS, highlighting the extreme convenience and rapidity of this method. All modification, detection, and purification (if needed) steps can be performed in parallel, with the potential for fully automation, significantly advancing the development of new chemical modification strategies of peptide libraries.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a class of immunoinhibitory cell signaling proteins with significant implications in cancer. Hypersialiation of cancer cells activates Siglecs, and this activation suppresses the immune recognition of these hyper-sialylated cells and promotes cancer-cell survival. Although Siglecs' natural substrates are gangliosides (a class primarily composed of glycoprotein with terminal sialic acid residues), Siglec proteins have a low affinity for these substrates. Hence, high-affinity inhibitors are a highly desirable focus of research.

Using genetically encoded libraries, we identified a group of bicyclic peptides with a strong affinity for Siglec-7 and Siglec-9. Specifically, we used bicyclic genetically encoded libraries modified by two-fold symmetric linkers (BiGEL2) to screen against these two targets, employing next-generation sequencing (NGS) analysis for hit nomination.

We synthesized approximately 100 peptides to explore their binding towards the Siglec targets using ELISA, BLI, and SPR. This study led to the discovery of low micromolar binders to Siglec-7 with IC50 & Ki <10 µM. Additionally, we preformed a preliminary structure-activity relationship profile using alanine scans to assist in the future development of highly potent, bioavailable, and immunosuppressive peptide binders of Siglec-7 and Siglec-9.

Peptide-based materials have many desirable qualities, such as their biocompatibility, structural tunability, and the ability to incorporate unnatural amino acids into their structure. Cyclic peptide nanotubes (cPNTs) fall into the category of peptide-based nanomaterials and are composed of cyclic peptide subunits which spontaneously form nanotubes through the formation of intermolecular hydrogen bonding. cPNTs have high functionalization potentials and exhibit enhanced cell permeation. When cPNTs insert parallel to cell membranes, this causes disruption of the membrane, leading to cell death. In this study, we prepared disulfide-stabilized tumor-targeted cPNTs to enhance cytotoxicity using peptide nanomaterials. We report the synthesis of tumour-targeted cPNTs from cyclic peptide monomers tagged with varying amounts of the tumor-homing peptide tLyp-1. cPNTs were prepared using pH-triggered cPNT self-assembly and visualized using transmission electron microscopy (TEM) confirming the formation of cPNTs of 7.6 ± 1.2 nm diameter and 74.7 ± 37.8 nm length. Additional characterization by Fourier transform infrared spectroscopy (FTIR) and dynamic light scattering (DLS) is currently underway as well as cytotoxicity studies in U87MG glioblastoma cells. Tubular nanomaterials exhibit ideal pharmacokinetic profiles and as such, tumor-targeted cPNTs hold untapped potential as a new class of therapeutic nanomaterials.