Shusuke Tomoshige

To expand the range of available E3 ligase resources, we developed novel proteolysis targeting chimeras (PROTACs) utilizing the carboxyl terminus of the Hsc70-interacting protein (CHIP) E3 ligase, which was previously unutilized.

Targeted protein degradation (TPD), a technology that induces the degradation of a protein of interest (POI) by a chemical degrader, is a major trend in drug discovery research. Proteolysis targeting chimeras (PROTACs) are representative degraders that induce the ubiquitination of a POI, leading to its proteasomal degradation. Following the report of PROTAC efficacy in animals approximately a decade ago, TPD research continues to flourish. Unlike the occupancy-driven mechanism of traditional inhibitors, TPD employs an event-driven mechanism. Consequently, TPD is a highly anticipated technology capable of addressing therapeutic targets previously considered "undruggable." Against this backdrop, our group is actively pursuing research focused on the targeted degradation of undruggable proteins. This symposium review summarizes our studies. It focuses on two distinct areas: the targeted degradation of aggregation-prone proteins for the development of neurodegenerative disorder treatments, and the targeted degradation of mitochondrial matrix-localized proteins for mitochondria-related diseases.

Proteolysis-targeting chimeras (PROTACs) represent a promising therapeutic modality, but their clinical translation is often hindered by poor pharmacokinetic properties associated with their location in the "beyond Rule of 5" chemical space. Using the BRD4 degrader dBET1 as a model, this study explored a dual approach to improve the cellular permeability of PROTACs by combining amide-to-ester substitution with the strategic linker methylation to induce stereochemistry-driven conformational modulation. Substitution with ester enhanced both permeability and degradation potency, while methylation afforded two diastereomers with different permeability profiles. Steered molecular dynamics and enhanced conformational sampling in polar and nonpolar environments revealed distinct chameleonic behaviors, with the more permeable diastereomer 2b adopting folded conformations with a lower solvent-accessible 3D polar surface area in the nonpolar environment. These findings were supported by 2D NMR and hydrogen-bond acidity analyses (A_NMR). Notably, low-energy "congruent conformation" accessible in both environments was identified for 2b. This work establishes a viable strategy for the design of membrane-permeable PROTACs.

The de novo total synthesis of position-selectively glycosylated aggregation-prone proteins has established the importance of posttranslational glycosylation in suppressing aggregation and reducing cytotoxicity. Here, building on our strategy of disrupting intermolecular interactions, we focused on tyrosine carbon as a candidate target for glycosylation to suppress aggregation. We report a laccase-catalyzed, tyrosine residue-selective biorthogonal reaction with sugar-conjugated 1-methylurazoles, enabling tyrosine-C-selective pseudo-glycosylation at Tyr10 of amyloid β1-40 (Aβ40) and at TyrA14 of human insulin. Pseudo-glycosylated Aβ40s exhibited enhanced aggregation resistance and reduced cytotoxicity. Notably, in situ pseudo-glycosylation of aggregated Aβ induced disaggregation, i.e., reversal of fibrillation, which has been considered to be irreversible under physiological conditions. Pseudo-glycosylated insulin displayed increased aggregation resistance without loss of bioactivity. Our findings underscore that protein aggregability can be controlled by targeting residues involved in strong intermolecular interactions as well as increasing hydration without strictly mimicking natural posttranslational modification. This single-step pseudo-glycosylation strategy could contribute to the development of novel treatments for neurodegenerative disorders and improving the stability of water-soluble protein therapeutics.

A key challenge in drug discovery is to endow candidate bioactive molecules with druglike properties. However, the optimization of critical, often-conflicting physicochemical properties through chemical modifications is particularly difficult, and the term Aufheben has been adopted to describe the simultaneous preservation and modification of apparent opposites to achieve improvement. This perspective examines strategies to reconcile conflicting parameters, such as lipophilicity/hydrophilicity, druglikeness/flatness, and druglikeness/molecular weight, in order to improve the aqueous solubility and membrane permeability of drug candidates. We review and categorize numerous molecular design strategies that have been investigated to address these challenges and highlight some recent successful examples.

Lipids, including fatty acids and phospholipids, play crucial roles in biological systems and are widely utilized in pharmaceutical and biomedical applications. However, their inherent hydrophobicity poses significant challenges for formulation and administration. In this study, we aimed to enhance the aqueous solubility of lipidic compounds by leveraging light-responsive molecular design. We synthesized azo-lipids by incorporating azobenzene units into a fatty acid and phosphatidylcholine, hypothesizing that light-induced trans–cis isomerization would improve solubility. The synthesized compounds exhibited reversible photoisomerization upon alternating UV (365 nm) and visible light irradiation, as confirmed by UV-vis spectroscopy and reverse-phase HPLC. The solubilization of these azo-lipids was quantified under UV-unirradiated and irradiated conditions. Azobenzene-incorporated phosphatidylcholine 2 exhibited a drastic increase in solubilization from 2.030 to 1008 µM (496-fold) after UV irradiation. This significant improvement was attributed to efficient photoisomerization and molecular bending in the cis, cis conformation, reducing intermolecular interactions. Our findings suggest that this on-demand aqueous solubilization strategy offers a novel approach for improving the handling, storage, and potential therapeutic administration of lipid-based compounds.

Our study shows efficient tyrosine labeling using 1-methyl-4-arylurazole (MAUra) with laccase under mild conditions. This method achieves a high efficiency (k_cat/K_m = 7.88 × 10⁴ M⁻¹ s⁻¹), selectively targeting exposed tyrosine sites on proteins.

Mitochondrial-targeted protein degradation (mitoTPD) is explored using small-molecule degraders that leverage a mitochondria-localized protease. This approach restored mitochondrial morphology and shows potential for drug discovery.