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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: 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.

Panel Discussion: Peptides as Radiopharmaceuticals
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Murray Wan
Principal Scientist, Mariana Oncology

Peptide Radiopharmaceutical Panel

Abstract

Mariana Oncology is designing, developing, and manufacturing a new generation of precision radioligand therapies that represent a revolutionary approach to cancer treatment.

Bio

Murray Wan is a Principal Scientist in the Medicinal Chemistry group at Mariana Oncology. He earned his Master's degree in Organic Chemistry from Baylor University in 2017, following undergraduate studies in Organic Chemistry at the University of Rochester. After completing his studies, he joined Merck's Discovery Chemistry group in Boston. Initially focused on small molecule drug discovery, he later transitioned to peptide chemistry. Now at Mariana Oncology, Murray contributes to discovery chemistry efforts on various Radio Ligand Therapy (RLT) programs.

Chemistry of Complex Peptides
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Akshay Shah
Director of Chemistry, Pinnacle Medicines

Enablement of Late-Stage Functionalization and High-Throughput Parallel Chemistry for Efficient Peptide Optimization

Abstract

Late-stage-enabling, high-throughput chemistries have been gaining momentum in the peptide drug discovery campaigns as chemists strive for fuller, shorter and fewer design-test-make cycles to optimize chemicals leads into clinical candidates. However, one must strike a balance between the high throughput and the late-stage of diversity introduction as they often run counter to one another. It is up to the ultimate objective one hopes to achieve that may tilt the balance in the favor of one over the other. The presentation will highlight: 1) photoredox-catalyzed decarboxylative Giese additions to dehydroalanine derivatives; and 2) Suzuki cross-couplings with 4-Br phenylalanine derivatives and draw focus on chemistry entablements that maximize the late-stage versus high throughput diversity introduction.

Bio

Dr. Akshay Shah is an experienced medicinal chemists with 10 years of combined drug discovery experience across modalities that include small molecules and peptides. He has authored 15+ peer-reviewed publications and hold inventorship on 10+ patents while frequently providing peer-reviews to top journals. Upon completion of PhD at indiana University, Bloomington in 2014 focusing on natural product synthesis and subsequent postdoctoral stint with renowned chemist Prof. K. C. Nicolaou at Rice University, Dr. Shah joined medicinal chemistry department at Pfizer as a senior Scientist and focused on development of novel, high-throughput parallel chemistries for SAR expansions. In 2017, Dr. Shah moved to Merck Research Labs in West Point, PA where he made key contributions to identification of small molecule clinical candidate. His interest in peptide discovery led him to Johnson & johnson in 2021 as a principal Scientist where Dr. Shah led medicinal chemistry efforts on multiple programs delivering on key discovery milestones. Dr. Shah is currently the Director of Chemistry at Pinnacle Medicines where his efforts build and advance a deep discovery chemistry portfolio focused on cyclic peptides.

Chemistry of Complex Peptides
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Cesar de la Fuente
Presidential Associate Professor, University of Pennsylvania

AI for Antibiotic Discovery

Abstract

Traditional antibiotic discovery has long relied on physically exploring nature, collecting soil and water samples to isolate active compounds through a painstaking trial-and-error process. This approach has become increasingly unsustainable in the face of rising antibiotic resistance and the urgent need for new therapeutics. In this talk, I will discuss how our lab has moved beyond this paradigm over the past decade by leveraging artificial intelligence (AI) to digitally mine the world’s biological information—genomes, proteomes, and metagenomes. Computers excel at pattern recognition in images and text, but their application in biology and medicine is still nascent. In this talk, I will discuss our decade-long advances accelerating antibiotic discovery. We pioneered designing antibiotics using artificial intelligence (AI), achieving efficacy in preclinical animal models and demonstrating that machines could create therapeutic molecules. For the first time, we mined the human proteome to identify antibiotic candidates. Building on this work, we hypothesized that similar compounds exist throughout evolution. We expanded our efforts to extinct species, where our AI-driven approach led to the discovery of the first therapeutic molecules from organisms like Neanderthals and woolly mammoths. This work launched the field of molecular de-extinction and yielded preclinical candidates such as neanderthalin, mammuthusin, and elephasin. Furthermore, we expanded our antibiotic discovery efforts to explore other branches of the tree of life beyond eukaryotes. By computationally analyzing microbial dark matter, we identified nearly one million new antibiotic molecules, which have been made freely available to encourage global researchers to synthesize and develop them. Additionally, through computational exploration of thousands of human microbiomes, we discovered new antimicrobial agents, including prevotellin-2 from the gut microbe Prevotella copri. Collectively, our efforts have dramatically accelerated antibiotic discovery, reducing the time to identify preclinical candidates from years to just a few hours. We are on the cusp of a new era where AI advances will help control antibiotic resistance, infectious disease outbreaks, and pandemics.

Bio

César de la Fuente is a Presidential Associate Professor at the University of Pennsylvania, where he leads the Machine Biology Group. He completed postdoctoral research at the Massachusetts Institute of Technology (MIT) and earned a PhD from the University of British Columbia (UBC). He is best known for pioneering computational and artificial intelligence approaches to antibiotic discovery, which have drastically accelerated the time needed to identify preclinical candidates, from years to hours. These candidates show promise for therapeutic intervention against currently untreatable infections. Moreover, he spearheaded the discovery of therapeutic molecules from extinct organisms, and his lab has uncovered a myriad of novel peptide molecules across the tree of life, revealing a previously unrecognized branch of host immunity. Professor de la Fuente has received numerous awards for his contributions, including the Princess of Girona Prize and the Fleming Prize. He is a Fellow of the American Institute for Medical and Biological Engineering, a Sloan Fellow, and a National Academy of Medicine Emerging Leader. De la Fuente has authored more than 170 publications and holds multiple patents.

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 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!

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 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).

Drug Delivery
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Guizhi Zhu
Associate Professor, University of Michigan

Engineer and deliver peptide-based proteolysis-targeting vaccines (PROTAX) for cancer immunotherapy

Abstract

Synthetic long peptide (SLP) vaccines hold great potential to improve the tumor therapeutic efficacy of immune checkpoint blockade (ICB). However, current SLP vaccines elicit limited T cell responses pivotal for tumor immunotherapy. Here, we present SLP-based proteolysis-targeting vaccines (PROTAX) that facilitate antigen proteolytic processing and cross-presentation to potentiate T cell responses for robust ICB combination immunotherapy of tumors. PROTAX are modular conjugates of SLP antigens, E3 ligase-binding ligands, and linkers. In antigen-presenting cells (APCs), PROTAVs bind to E3 ligases to rapidly ubiquitinate PROTAV antigens, thereby facilitating antigen proteolytic processing by proteasome and promoting antigen cross-presentation to T cells. In mice, when co-delivered with bi-adjuvants activating cyclic-GMP-AMP synthase (cGAS) and Toll-like receptor 9 (TLR9) using lipid nanoparticles (LNPs), PROTAVs promoted the quantity and quality of CD8+ T cells against monovalent or multivalent SLP antigens. Combining PROTAX and ICB remodels tumor immune microenvironment. As a result, the combination of PROTAX with ICB promotes the complete regression rates of murine melanoma and human papillomavirus (HPV)-associated tumors, and eradicates 50%-100% ICB-resistant large tumors (300 mm3) in HPV-associated tumors, B16F10 melanoma, and SM1 Braf(V600E) allograft melanoma, and significantly promoted the tumor therapeutic efficacy of chemically-induced autochthonous Braf(V600E) melanoma in a genetically engineered mouse model. Overall, PROTAX represent a simple and broadly applicable platform for robust tumor combination immunotherapy.

Bio

Dr. Guizhi Zhu is currently Ara Garo Paul Associate Professor at University of Michigan – Ann Arbor. He received BS degree in Biotechnology from Nankai University, PhD degree in Biomedical Sciences from the University of Florida, followed by a postdoc training on drug delivery and bioimaging at the National Institute of Biomedical Imaging and Bioengineering (NIBIB) of NIH. His multidisciplinary research group (https://www.guizhizhu.org/) studies the engineering and delivery of nucleic acid and peptide therapeutics and vaccines for the treatment and prophylaxis of cancer, infectious diseases, autoimmune diseases, and genetic diseases. He has published over 100 peer-reviewed papers. He has received prestigious awards, including 2022 Oligonucleotide Pharmaceutical Society (OTS) Young Investigator Award and 2022 American Association of Pharmaceutical Scientists (AAPS) Emerging Leader Award.

Peptides in the Clinic
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Kathy Miller
Ballve-Lantero Professor, Indiana University

Peptide vaccines for prevention and treatment of cancer

Abstract

Vaccines targeting infectious diseases have had a profound impact on human health but early clinical efforts to develop cancer vaccines were disappointing. Peptide vaccines can be broadly characterized into two groups: cellular (T cell) and humoral (B cell) vaccines. Hundreds of tumor antigens, both shared and personalized neo-antigens, have been identified. Most vaccines in clinical development are designed to activate Th-1 cytotoxic T- cells (CTL) which play a major role in tumor rejection. However, humoral arm also plays a critical role in the generation of an antitumor response. The successful clinical usage of passively infused monoclonal antibodies points to the effectiveness of the humoral arm of the immune system. B cell epitope vaccines are designed to induce a protective humoral response. This response includes creation of antibody-producing plasma cells as well as immunologic memory. Several factors need to be considered in formulating an effective peptide vaccine; these factors include inclusion of a universal T helper epitope and the necessity to mimic the structure of the parent antigen to generate high-affinity Abs. We’ll review data from early clinical trials with both cellular and humoral vaccines, then highlight ongoing trials and opportunities for development.

Bio

Kathy D. Miller received her MD in 1991 from the Johns Hopkins School of Medicine in Baltimore, MD. Dr. Miller completed internal medicine training at Hopkins, then returned to her native Midwest for fellowship training at Indiana University, serving as Chief Fellow in 1997. She returned to Indiana University in 1999, attaining the rank of Professor and Ballvé-Lantero Scholar in 2014. She served as the Associate Director for Clinical Research for the IU Melvin and Bren Simon Comprehensive Cancer Center from 12/17-3/25. Dr. Miller’s career has combined both laboratory and clinical research in breast cancer. She was chair of the ECOG-ACRIN Breast Core Committee in January 2014 through Dec 2017 when she was elected co-chair of NCI’s Breast Cancer Steering Committee, completing two terms in 3/25. In those roles she worked with academic scientists and community oncologists to develop trials that combine clinical and biologic endpoints yet remain feasible in non-academic settings.

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 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.

Chemistry of Complex Peptides
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Soren Ostergaard
Senior Principal Scientist, Novo Nordisk A/S

Half-life extension of peptides using fatty acid derivatization; past, present and future opportunities

Abstract

Semaglutide and tirzepatide approved for type 2 diabetes and obesity are redefining peptide therapeutics. A molecular engineering technology, fatty acid derivatization, initially applied to insulin, then semaglutide, has now enabled otherwise short-lived peptides to be dosed once weekly and moreover, this technology has unlocked unprecedented efficacy in terms of biological activity. Central to this advancement is the derivatization with a fatty acid, which facilitates the binding of the peptide or protein of interest to albumin (1). Due to the well-defined chemical nature of fatty acids, inherent safety, ease of synthesis and upscale, this technology offers a unique versatile approach to peptide and protein drug discovery.
In this presentation, the focus will be on early and current inventions, illustrating how this technology has been applied to address the challenges in designing long-acting peptide-based therapeutics. Additionally, how these endeavors might pave the way for future advancements in peptide drug design will be discussed.
(1) P. Kurtzhals, S. Østergaard, E. Nishimura, T. Kjeldsen, 2022, Nature Rev. Drug Disc, 22, 59-80, doi.org/10.1038/s41573-022-00529

Bio

Soren Ostergaard is a senior principal scientist at Novo Nordisk A/S where he has been working in various peptide projects including peptide synthesis technologies for the last +30 years. He has a long track record of successful peptide drug projects that have resulted in marketed products or compounds in clinical trials. He has been optimizing novel half-life extension methodologies of peptides in order to be able to dose once-weekly or beyond of peptide drugs. Another focus area has been improving the potency/selectivity profile e.g., endogenous peptides or de novo design of dual acting peptides for subcutaneous or oral dosing. In addition, he has also worked with challenges in the bRo5 space, immunogenicity of peptides and proteins (B- and T- cell epitope mapping) and integrating these technologies into the drug discovery process
More than 20 years ago he introduced high throughput array and plate synthesis and have been an early key driver in the implementation of these robotic technologies to successfully speed and improve the peptide drug discovery process at Novo Nordisk.
He is collaborating with academia and has through the years served as supervisor for PhD students. He attends career events representing Novo Nordisk as talent ambassador, giving educational lectures at various courses at Universities in Denmark and Sweden and from time to time invited as PhD assessor. He is the author or co-author of +90 publications and patents.