Thomas Kodadek

A 30 residue FOLDON peptide effectively trimerizes monomeric proteins. Multivalent interactions allow trimeric FOLDON fusions to be retained on beads displaying low molecular weight fragments.

Chemically induced proximity is a powerful modality for manipulating protein function. Most of the effort in this field has focused on targeted protein degradation, but recruitment of other types of post-translational modification enzymes to a target protein is also of interest. To construct such reagents, one would ideally like to have ligands that engage the enzyme without inhibiting its activity. In this study, we describe a screening platform for the discovery of noninhibitory macrocyclic ligands for a protein tyrosine phosphatase, using PTP1B as an exemplary model target. This workflow involves sequential screens of small libraries of bead-displayed macrocycles in which only one position of the macrocycle is varied in each round of screening while the others are held as invariant placeholders. The beads co-display a high KM substrate for the phosphatase, allowing ligand-dependent recruitment of the enzyme to the bead surface to be coupled to dephosphorylation of the co-displayed substrate. This is detected by staining with a labeled anti-phosphotyrosine antibody. Finally, we demonstrate that the same general approach can be applied to proteins lacking enzymatic activity by screening against biotin ligase-target protein fusions and employing a proximity labeling-like assay to register screening hits.

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 boosted passive membrane permeability significantly, as measured by the parallel artificial membrane permeability assay (PAMPA) (Li et al. (2024) J. Am. 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 the mitochondria, as is the case for many positively charged molecules. We show that the 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. Enhanced permeability is observed when the IP⁺ unit is positioned either within the macrocyclic backbone or on a side chain. Collectively, these results establish that IP⁺ incorporation is an effective strategy for the development of bioactive MPs targeting intracellular proteins.

Many important solid-phase synthesis reactions proceed slowly and inefficiently, for example, the coupling of an activated ester to a sterically hindered, bead-displayed amine, a critical step in the synthesis of many peptides. Forcing conditions are often required to achieve acceptable yields. We show here that such reactions can be accelerated by tethering to the bead a nucleophilic pyridine catalyst, which reacts with the activated ester, creating a high local concentration of a reactive acylpyridinium intermediate that couples efficiently to the bead-displayed amine. In many cases, the “cleanliness” of reactions carried out on resins equipped with an immobilized catalyst is substantially better than the analogous reactions in which the catalyst is added to the solution.

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.

Fragment-based drug discovery (FBDD) is a powerful approach to the development of pharmaceuticals and probe molecules and there is broad interest in the development of new platforms for their discovery. Here, we introduce a workflow in which low molecular weight organic molecules displayed on the surface of TentaGel beads are exposed to a multimeric, fluorescently labeled target protein. Using tetrameric or dimeric protein targets, we show that beads that display even weak ligands (KDs in the high μM to low mM range) stably capture the protein due to avidity effects, thus allowing a simple “pull-down” protocol to be employed for fragment discovery. We also demonstrate that the platform is capable of supporting a “fragment growth” screen, which is a typical strategy to advance a fragment to a higher-affinity lead molecule. This platform is inexpensive and requires no specialized infrastructure.

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 polyubiquitylation 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-Lindau (VHL) protein.