The Pentelute group designs fully automated fast-flow machines to accelerate the chemical manufacture of sequence-defined biopolymers. It has built the world’s fastest and most efficient machine that can produce thousands of amide bonds an order of magnitude faster than commercially available instruments. The machine is inspired by Nature’s ribosome that can make proteins in minutes. While the Pentelute group’s fast-flow technology is not as fast as the ribosome, it can form one amide bond in 7 seconds. This technology not only facilitates rapid polypeptide generation but it has enabled the group to carry out entire D-scans of proteins to investigate folding and functions. This technology was recently used to achieve stepwise total chemical synthesis of protein chains nearing 200 amino acids in length that retained the structure and function of native variants obtained by recombinant expression. Automated flow technology may solve the manufacturing problem for on-demand personalized therapies, such as cancer vaccines.

The Pentelute group also focuses on the delivery of large biomolecules into the cell cytosol. The group has developed chemical approaches for engineering a nontoxic form of anthrax toxin, which transports proteins into cells via a protective antigen-mediated pump. This ‘protein pump’ can deliver a variety of non-native cargo molecules into cells including antibody mimics, mirror-image proteins, small molecules, enzymes, and antisense oligonucleotides. Recently, the group retargeted the anthrax protective antigen to receptors overexpressed on tumor cells and modified its lethal factor component to target cancer gene dependencies with antisense peptide nucleic acids. This discovery will significantly aid in the development of durable cell-based protein therapeutics. They also overcame the cytosolic delivery barrier with cell-penetrating peptides, chemical vectors that facilitate cellular uptake and nuclear targeting of antisense cargo. Harnessing the power of machine learning, best-in-class variants were predicted and designed de novo, outperforming all previously known peptides for antisense delivery, while being non-toxic and effective in mice.

The rapid discovery of selective affinity reagents to native proteins is a challenge in chemical biology and medicine. Leveraging expertise with high-throughput identification of peptides from ultra-large combinatorial libraries, the Pentelute group has developed an affinity selection-mass spectrometry platform for rapid de novo discovery of potent binders to proteins in solution. Using this approach, high-affinity peptidomimetic ligands were identified for the MDM2 oncogenic protein, the 12ca5 clone of anti-hemagglutinin antibody, and the signaling protein 14-3-3. This platform is currently applied for discovery of non-canonical peptide disruptors of cancer protein-protein interactions that are effective, non-toxic, long-circulating, and stable in cells and animals.

Methods for the automated high-fidelity chemical production of proteins are needed. Proteins
manufactured with mechanical machines, as opposed to bio-based systems, can bulletproof production circumventing endotoxin contamination and cell-line variability. Further, chemical synthesis enables the ability to rewire the covalent framework with non-natural amino acids, drugs, and carbohydrates.

I will discuss the fully automated single-shot chemical synthesis of protein chains up to 220 amino acids in hours. After purification and folding, the synthetic proteins functioned analogously to ribosomally produced material. In addition, rapid protein synthesis enables accelerated drug discovery when combined with our single-shot affinity-selection mass spectrometry platform. I will discuss our results toward the discovery of high-affinity peptidomimetics that target oncoproteins or SARS-CoV-2.