Throughout evolution, Nature has developed molecular machines to rapidly manufacture, tailor, and deliver large functional biopolymers such as proteins into specific cells. Inspired by these mechanisms of Nature, the Pentelute Lab aims to invent new chemistry for the efficient and selective modification of proteins, to ‘hijack’ these biological machines for efficient drug delivery into cells and to create new machines to rapidly and efficiently manufacture peptides and proteins.
A main goal of the Pentelute Lab is to invent new chemistry to modify Nature’s proteins to enhance their therapeutic properties for medicine. This goal has posed challenges because proteins contain 20 amino acids that present different reactive functional groups and have a flexible structure in solution. Chemical modifications of proteins need to be biocompatible, site-selective, quantitative, and carried out in water at reasonable temperatures to maintain structural integrity and function. The Pentelute Lab has developed a series of highly efficient and selective chemistries that can selectively modify the amino acids cysteine and lysine within unprotected peptides and proteins. These newly developed chemistries can be catalyzed by enzymes, promoted by short reactive motifs, such as the ‘pi-clamp’ and ‘DBCO-tag’ discovered by the group, or mediated by transition metals such as palladium. This extensive protein modification toolkit has enabled the production of potent bioconjugate molecules including peptide macrocycles that cross cell membranes to disrupt cancer or antibody-drug conjugates targeting several disease indications.
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.