<i>N</i>‑Myristoyltransferase Inhibition Induces ER-Stress, Cell Cycle Arrest, and Apoptosis in Cancer Cells

<i>N</i>-Myristoyltransferase (NMT) covalently attaches a C14 fatty acid to the N-terminal glycine of proteins and has been proposed as a therapeutic target in cancer. We have recently shown that selective NMT inhibition leads to dose-responsive loss of <i>N</i>-myristoylation on more than 100 protein targets in cells, and cytotoxicity in cancer cells. <i>N</i>-myristoylation lies upstream of multiple pro-proliferative and oncogenic pathways, but to date the complex substrate specificity of NMT has limited determination of which diseases are most likely to respond to a selective NMT inhibitor. We describe here the phenotype of NMT inhibition in HeLa cells and show that cells die through apoptosis following or concurrent with accumulation in the G1 phase. We used quantitative proteomics to map protein expression changes for more than 2700 proteins in response to treatment with an NMT inhibitor in HeLa cells and observed down-regulation of proteins involved in cell cycle regulation and up-regulation of proteins involved in the endoplasmic reticulum stress and unfolded protein response, with similar results in breast (MCF-7, MDA-MB-231) and colon (HCT116) cancer cell lines. This study describes the cellular response to NMT inhibition at the proteome level and provides a starting point for selective targeting of specific diseases with NMT inhibitors, potentially in combination with other targeted agents

Bicyclization and Tethering to Albumin Yields Long-Acting Peptide Antagonists

Proteolytically stable peptide architectures are required for the development of long-acting peptide therapeutics. In this work, we found that a phage-selected bicyclic peptide antagonist exhibits an unusually high stability in vivo and subsequently deciphered the underlying mechanisms of peptide stabilization. We found that the bicyclic peptide was significantly more stable than its constituent rings synthesized as two individual macrocycles. The two rings protect each other from proteolysis when linked together, conceivably by constraining the conformation and/or by mutually shielding regions prone to proteolysis. A second stabilization mechanism was found when the bicyclic peptide was linked to an albumin-binding peptide to prevent its rapid renal clearance. The bicyclic peptide conjugate not only circulated 50-fold longer (<i>t</i>_1/2 = 24 h) but also became entirely resistant to proteolysis when tethered to the long-lived serum protein. The bicyclic peptide format overcomes a limitation faced by many peptide leads and appears to be suitable for the generation of long-acting peptide therapeutics

Chemical Macrocyclization of Peptides Fused to Antibody Fc Fragments

To extend the plasma half-life of a bicyclic peptide antagonist, we chose to link it to the Fc fragment of the long-lived serum protein IgG1. Instead of chemically conjugating the entire bicyclic peptide, we recombinantly expressed its peptide moiety as a fusion protein to an Fc fragment and subsequently cyclized the peptide by chemically reacting its three cysteine residues with tris-(bromomethyl)­benzene. This reaction was efficient and selective, yielding completely modified peptide fusion protein and no side products. After optimization of the linker and the Fc fragment format, the bicyclic peptide was fully functional as an inhibitor (<i>K</i>_i = 76 nM) and showed an extended terminal half-life of 1.5 days in mice. The unexpectedly clean reaction makes chemical macrocyclization of peptide-Fc fusion proteins an attractive synthetic approach. Its good compatibility with the Fc fragment may lend the bromomethylbenzene-based chemistry also for the generation of antibody–drug conjugates

Bicyclic Peptide Ligands Pulled out of Cysteine-Rich Peptide Libraries

Bicyclic peptide ligands were found to have good binding affinity and target specificity. However, the method applied to generate bicyclic ligands based on phage-peptide alkylation is technically complex and limits its application to specialized laboratories. Herein, we report a method that involves a simpler and more robust procedure that additionally allows screening of structurally more diverse bicyclic peptide libraries. In brief, phage-encoded combinatorial peptide libraries of the format X_<i>m</i>CX_<i>n</i>CX_<i>o</i>CX_<i>p</i> are oxidized to connect two pairs of cysteines (C). This allows the generation of 3 × (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) different peptide topologies because the fourth cysteine can appear in any of the (<i>m</i> + <i>n</i> + <i>o</i> + <i>p</i>) randomized amino acid positions (X). Panning of such libraries enriched strongly peptides with four cysteines and yielded tight binders to protein targets. X-ray structure analysis revealed an important structural role of the disulfide bridges. In summary, the presented approach offers facile access to bicyclic peptide ligands with good binding affinities

Development of a Photo-Cross-Linkable Diaminoquinazoline Inhibitor for Target Identification in <i>Plasmodium falciparum</i>

Di­amino­quina­zolines represent a privileged scaffold for antimalarial discovery, including use as putative <i>Plasmodium</i> histone lysine methyltransferase inhibitors. Despite this, robust evidence for their molecular targets is lacking. Here we report the design and development of a small-molecule photo-cross-linkable probe to investigate the targets of our di­amino­quina­zoline series. We demonstrate the effectiveness of our designed probe for photoaffinity labeling of <i>Plasmodium</i> lysates and identify similarities between the target profiles of the probe and the representative di­amino­quina­zoline BIX-01294. Initial pull-down proteomics experiments identified 104 proteins from different classes, many of which are essential, highlighting the suitability of the developed probe as a valuable tool for target identification in <i>Plasmodium falciparum</i>