Next-Generation Tags for Fluorine Nuclear Magnetic Resonance: Designing Amplification of Chemical Shift Sensitivity.

Fluorine NMR is a highly sensitive technique for delineating the conformational states of biomolecules and has shown great utility in drug screening and in understanding protein function. Current fluorinated protein tags leverage the intrinsic chemical shift sensitivity of the 19F nucleus to detect subtle changes in protein conformation and topology. This chemical shift sensitivity can be amplified by embedding the fluorine or trifluoromethyl reporter within a pyridone. Due to their polarizability and rapid tautomerization, pyridones exhibit a greater range of electron delocalization and correspondingly greater 19F NMR chemical shift dispersion. To assess the chemical shift sensitivity of these tautomeric probes to the local environment, 19F NMR spectra of all possible monofluorinated and trifluoromethyl-tagged versions of 2-pyridone were recorded in methanol/water mixtures ranging from 100% methanol to 100% water. 4-Fluoro-2-pyridone and 6-(trifluoromethyl)-2-pyridone (6-TFP) displayed the greatest sensitivity of the monofluorinated and trifluoromethylated pyridones, exceeding that of known conventional CF3 reporters. To evaluate the utility of tautomeric pyridone tags for 19F NMR of biomolecules, the alpha subunit of the stimulatory G protein (Gsα) and human serum albumin (HSA) were each labeled with a thiol-reactive derivative of 6-TFP and the spectra were recorded as a function of various adjuvants and drugs. The tautomeric tag outperformed the conventional tag, 2-bromo-N-(4-(trifluoromethyl)phenyl)acetamide through the improved resolution of several functional states.

Discovery of YSR734: A Covalent HDAC Inhibitor with Cellular Activity in Acute Myeloid Leukemia and Duchenne Muscular Dystrophy.

Histone deacetylases (HDACs) have emerged as powerful epigenetic modifiers of histone/non-histone proteins via catalyzing the deacetylation of ε-N-acetyl lysines. The dysregulated activity of these Zn2+-dependent hydrolases has been broadly implicated in disease, notably cancer. Clinically, the recurring dose-limiting toxicities of first-generation HDACi sparked a paradigm shift toward safer isoform-specific molecules. With pervasive roles in aggressive diseases, there remains a need for novel approaches to target these enzymes. Herein, we report the discovery of YSR734, a first-in-class covalent HDACi, with a 2-aminobenzanilide Zn2+ chelate and a pentafluorobenzenesulfonamide electrophile. This class I selective proof of concept modified HDAC2Cys274 (catalytic domain), with nM potency against HDAC1-3, sub-µM activity in MV4-11 cells, and limited cytotoxicity in MRC-9 fibroblasts. In C2C12 myoblasts, YSR734 activated muscle-specific biomarkers myogenin/Cav3, causing potent differentiation into myotubes (applications in Duchenne Muscular Dystrophy). Current efforts are focused on improving in vivo ADME toward a preclinical covalent HDACi.

Emerging mechanisms of targeted protein degradation by molecular glues.

Targeted protein degradation has emerged as a transformative therapeutic modality for the treatment of human diseases. The clinical successes of approved protein degraders like lenalidomide and thalidomide in cancers and immune disorders, combined with the recent clinical debut of investigational heterobifunctional degraders, have demonstrated the potential of this pharmacological approach to expand the druggable proteome and improve patient outcomes. Molecular glue degraders are a class of protein degraders that operate by recruiting target proteins to cellular degradation machinery via noncanonical protein-protein interactions, inducing the destruction of the target protein. While heterobifunctional degraders consist of two distinct protein-binding moieties connected by a linker, molecular glue degraders contain a single pharmacophore and are thus more synthetically accessible, ligand-efficient, and often possess more drug-like physicochemical properties. In this chapter, we will explore the history of the field-from its conception to the recently accelerating discovery of novel glue degrader mechanisms-and contemplate its trajectory towards rational design with the emergence of new methods for protein quantification and high-throughput assays to screen for novel degraders.