Biology subjects

Kodadek, T.

Publications and source records attributed to Kodadek, T..

4 recordsLinked to original sources

bioRxiv2025

Macrocyclic Peptides Containing an Imidazopyridinium (IP+) Unit Display Enhanced Passive Cell Permeability

Macrocyclic peptides (MPs) have emerged as interesting therapeutic candidates due to their ability to engage difficult protein targets with high affinity and selectivity. However, their application to intracellular targets is limited by the poor passive membrane permeability of most MPs. We previously showed that incorporation of an imidazopyridinium (IP+) moiety into an MP significantly boosted passive membrane permeability, as measured by the parallel artificial membrane permeability assay (PAMPA)(Li, et al. (2024) J. Amer. Chem. Soc. 146, 14633-14644). In this study we report a detailed analysis of the entry of IP+-containing MPs into living cells. Chloroalkane penetration assay (CAPA) data show that IP+ MPs access the cytoplasm rapidly, often at rates approaching those of drug-like small molecules. Mechanistic studies, including live-cell imaging, ATP-depletion experiments, and organelle colocalization analyses, indicate that IP+ MPs traverse the plasma membrane primarily via passive diffusion, avoiding endosomal entrapment. IP+ MPs do not localize to mitochondria, as is the case for many positively charged molecules. We show that incorporation of an IP+ unit transforms a previously described membrane impermeable macrocyclic antagonist of the p53-MDM2 interaction into a bioactive inhibitor of MCF-7 proliferation. Collectively, these results establish that IP+ incorporation is an effective strategy for the development of bioactive MPs targeting intracellular proteins.

biochemistry↗

bioRxiv2025

Assessing the Suitability of Deubiquitylases As Substrates For Targeted Protein Degradation

The development of selective inhibitors of Deubiquitylase enzymes (DUBs) is difficult due to a high level of homology in the active sites of the {approx} 100 such enzymes in the human proteome. A potential way to achieve this in a more facile manner would be to develop PROTACs or molecular glues that engage the target DUB in a less conserved region outside of the catalytic domain. However, this raises the concern that auto-deubiquitylation would make DUBs poor substrates for this modality. Here we describe a chemical genetics system to evaluate this issue. We find that some DUBs are readily degradable via the Ubiquitin-proteasome pathway and some are not. Of the latter category, some resist turnover through auto-deubiquitylation and some are simply poor proteasome substrates. SignificanceDeubiquitylases (DUBs) are a family of specialized proteases that hydrolyze the isopeptide bond between a lysine and the C-terminal carboxylate of Ubiquitin. DUBs are involved in a myriad of cellular processes and many are attractive drug targets. However, it is difficult to develop selective orthosteric inhibitors due to the high degree of homology between DUB active sites. Targeted protein degradation using a proteolysis-targeting chimera (PROTAC) that recognizes the DUB in a less conserved region outside of the catalytic domain constitutes an attractive alternative strategy for selectively inhibiting a given DUB. Such ligands may not block the catalytic activity of the enzyme, raising the concern that auto-deubiquitylation will make DUBs inherently poor substrates for PROTACs of this type. Since drug-like ligands that engage DUBs outside of the active site are extremely rare, this issue is difficult to address in a straightforward fashion. In this study we establish a generally applicable chemical genetics workflow to evaluate the degradability of DUBs by a PROTAC. The data indicate that some DUBs are readily degradable and some are not. In particular, USP11, an attractive drug target in various cancers and Alzheimers disease, is shown to be rapidly degradable, while its paralogs, USP4 and USP15 resist degradation through auto-deubiquitylation.

biochemistry↗

bioRxiv2025

A Functional Assay For Mining Non-Inhibitory Enzyme Ligands From One Bead One Compound Libraries: Application to E3 Ubiquitin Ligases

Chemical dimerizers are synthetic molecules that bring into proximity two or more proteins that do not normally interact with one another. A major application of this technology is to recruit an enzyme to a target protein, resulting in its post-translational modification (PTM). In particular, chemical dimerizer-mediated poly-Ubiquitylation of proteins has garnered an enormous amount of interest as a new drug modality. A fundamental requirement for the construction of new PTM-driving dimerizers is an enzyme ligand that does not inhibit its activity. Traditional activity-based high-throughput screening platforms are not suited for this purpose. Here we describe a novel platform for screening libraries of bead-displayed compounds that links a requirement for small molecule binding to the enzyme with enzyme-mediated modification of a nearby substrate. This system ensures that the enzyme-recruiting small molecules do not interfere with the catalytic function of the enzyme. We demonstrate the utility of this system in the context of E3 Ubiquitin ligase-recruiting molecules and report the discovery of a novel, low molecular mass ligand for the Von Hippel Landau (VHL) protein.

biochemistry↗

bioRxiv2023

Chemically Induced Degradation of Native Proteins by Direct Recruitment to the 26S Proteasome

Targeted protein degradation (TPD) is a promising strategy for drug development. Most degraders function by forcing the association of the target protein (TP) with an E3 Ubiquitin ligase, which in favorable cases results in the poly-Ubiquitylation of the TP and its subsequent degradation by the 26S proteasome. Here we explore the feasibility of a different TPD strategy in which the TP is recruited directly to the proteasome without the requirement for poly-Ubiquitylation. Using an engineered cell line in which the HaloTag protein is fused to one of the Ubiquitin receptors, we show that native protein targets can be degraded in this fashion when the cells are exposed to a chemical dimerizer containing a chloroalkane and a TP ligand. The potential advantages and disadvantages of Ubiquitin-independent degraders vs. traditional proteolysis-targeting chimeras are discussed.

molecular biology↗