Biology subjects

Kougentakis, C.

Publications and source records attributed to Kougentakis, C..

3 recordsLinked to original sources

bioRxiv2025

The pKa values of buried ionizable amino acids can be determined by the thermodynamic stability of the protein

Ionizable groups in hydrophobic environments in proteins usually titrate with anomalous pKa values. The ionization of these buried residues is often coupled to transitions between conformational states. Here we test the hypothesis that because the thermodynamic stability of a protein ({Delta}G{degrees}H2O) determines the probability of conformational transitions, the apparent pKa values of buried residues can be governed by {Delta}G{degrees}H2O. Variants of staphylococcal nuclease (SNase) with either Lys-66 or Lys-92 buried in its hydrophobic interior were engineered along with surface mutations that alter {Delta}G{degrees}H2O without affecting the electrostatic properties of the internal microenvironments of the buried Lys residues. The measured pKa values of these Lys residues largely correlates with {Delta}G{degrees}H2O. NMR spectroscopy was used to demonstrate that the structural changes of the protein backbone coupled to the ionization of Lys-66 or Lys-92 are comparable regardless of the {Delta}G{degrees}H2O of the protein. NMR spectroscopy confirmed that global unfolding of the Lys-92 variant coincides with the apparent pKa of the Lys side chain. The data presented show that the anomalous pKa values measured for internal residues in proteins do not necessarily report on local dielectric or electrostatic properties of the microenvironments around the ionizable group; rather, they can report on the energetics of pH-driven conformational transitions. These data suggest that accurate structure-based calculation of pKa values will require de novo prediction of partially unfolded conformations, and accurate calculation of free energy differences between conformational states, both of which remain formidable challenges.

biophysics↗

bioRxiv2025

Catalytic degradation of circulating targets with FcRn-mediated cycling LYTACs

Circulating proteins are common targets for the discovery of occupancy-based inhibitors including monoclonal antibodies. Effective inhibition of target pathogenicity with blocking approaches, however, is often challenged by target parameters that lead to insufficient occupancy and/or incomplete pharmacology limited by only single site binding. Extracellular targeted protein degradation approaches, such as lysosomal targeting chimeras (LYTACs), offer an opportunity to minimize these challenges by an event-driven mechanism that selectively, thoroughly and irreversibly eliminates drivers of disease. First generation LYTACs, designed to traffic to the lysosome, show limited durability since the therapeutic is degraded along with the target protein of interest. Here we describe cataLYTACs, which overcome this limitation by combining stabilized asialoglycoprotein (ASGPR) ligands, pH-sensitive target binding and recycling via the neonatal Fc receptor (FcRn). These cataLYTACs degraded superstoichiometric levels of a target protein, IgE, in vitro and demonstrated deep and sustained clearance of human IgE in mouse models. In non-human primates, cataLYTACs resulted in >98% clearance of circulating endogenous IgE for 2 weeks and outperformed the standard of care blocking antibody, omalizumab (Xolair(R)), in both free IgE elimination and duration of action. CataLYTACs represent a new therapeutic modality for a wide range of disease states driven by circulating factors, with the potential for superior efficacy and duration of action compared to traditional inhibitors.

cell biology↗

bioRxiv2024

Monitoring lysosomal catabolism: a sensitive probe for assessing targeted lysosomal degradation of extracellular proteins

Extracellular targeted protein degradation (eTPD) is an emerging therapeutic field. The Lysosome targeting chimera (LYTAC) is a therapeutic modality that promotes degradation of extracellular drivers of disease in the lysosome. While widely available pH-sensitive probes may report on lysosome delivery, these probes do not necessarily report on the enzymatically active functional state of the lysosome. We report the development and application of a sensitive fluorescent probe, LysoLight Deep Red, to monitor catabolism of internalized proteins in the lysosome based on cleavage by cathepsin proteases. We demonstrate the application of Lysolight Deep Red to monitor the catabolic fate of therapeutic monoclonal antibodies, ASGPR-targeted LYTAC therapeutics and LYTAC targets in immortalized cell lines and in primary human hepatocytes.

cell biology↗