pH Effects Can Dominate Chemical Shift Perturbations in 1H,15N-HSQC NMR Spectroscopy for Studies of Small Molecule/α-Synuclein Interactions.

1H,15N-Heteronuclear Single Quantum Coherence (HSQC) NMR is a powerful technique that has been employed to characterize small-molecule interactions with intrinsically disordered monomeric α-Synuclein (aSyn). We report how solution pH can impact the interpretation of aSyn HSQC NMR spectra and demonstrate that small-molecule formulations (e.g., complexation with acidic salts) can lower sample pH and confound interpretation of drug binding and concomitant protein structural changes. Through stringent pH control, we confirm that several previously identified compounds (EGCG, Baicalin, and Dopamine (DOPA)) as well as a series of potent small-molecule inhibitors of aSyn pathology (Demeclocycline, Ro90-7501, and (±)-Bay K 8644) are capable of direct target engagement of aSyn. Previously, DOPA-aSyn interactions have been shown to elicit a dramatic chemical shift perturbation (CSP) localized to aSyn's H50 at low DOPA concentrations then expanding to aSyn's acidic C-terminal residues at increasing DOPA levels. Interestingly, this CSP profile mirrors our pH titration, where a small reduction in pH affects H50 CSP, and large pH changes induce robust C-terminal CSP. In contrast, under tightly controlled pH 5.0, DOPA induces significant CSPs observed at both ionizable and nonionizable residues. These results suggest that previous interpretations of DOPA-aSyn interactions were conflated with pH-induced CSP, highlighting the need for stringent pH control to minimize potential false-positive interpretations of ligand interactions in HSQC NMR experiments. Furthermore, DOPA's preferential interaction with aSyn under acidic pH represents a novel understanding of DOPA-aSyn interactions that may provide insight into the potential gain of toxic function of aSyn misfolding in α-synucleinopathies.

Fragment-Based NMR Screening of the BPTF PHD Finger Methyl Lysine Reader Leads to the First Small-Molecule Inhibitors.

Methyl lysine readers, specifically PHD fingers, are emerging epigenetic targets in human diseases. For example, several PHD finger fusions are implicated in clinical cases of acute myeloid leukemia, highlighting the potential for PHD inhibitors in disease regulation. However, limited chemical matter targeting PHD fingers exists. Here we report the first fragment-based screen against the BPTF PHD to identify several of the first reported BPTF PHD-targeting small-molecule ligands. We used ligand-observed NMR to first screen a fragment library, followed by biophysical validation to prioritize two scaffolds, pyrrolidine- and pyridazine-containing fragments. Structural predictions show that these respective scaffolds may engage two distinct subpockets on the protein. The demonstrated ligandability of the BPTF PHD supports the future development of methyl lysine reader chemical probes to study their oncogenic functions.

19F NMR viewed through two different lenses: ligand-observed and protein-observed 19F NMR applications for fragment-based drug discovery.

19F NMR has emerged as a powerful tool in drug discovery, particularly in fragment-based screens. The favorable magnetic resonance properties of the fluorine-19 nucleus, the general absence of fluorine in biological settings, and its ready incorporation into both small molecules and biopolymers, has enabled multiple applications of 19F NMR using labeled small molecules and proteins in biophysical, biochemical, and cellular experiments. This review will cover developments in ligand-observed and protein-observed 19F NMR experiments tailored towards drug discovery with a focus on fragment screening. We also cover the key advances that have furthered the field in recent years, including quantitative, structural, and in-cell methodologies. Several case studies are described for each application to highlight areas for innovation and to further catalyze new NMR developments for using this versatile nucleus.

New Design Rules for Developing Potent Cell-Active Inhibitors of the Nucleosome Remodeling Factor (NURF) via BPTF Bromodomain Inhibition.

The nucleosome remodeling factor (NURF) alters chromatin accessibility through interactions with its largest subunit,the bromodomain PHD finger transcription factor BPTF. BPTF is overexpressed in several cancers and is an emerging anticancer target. Targeting the BPTF bromodomain presents a potential strategy for its inhibition and the evaluation of its functional significance; however, inhibitor development for BPTF has lagged behind those of other bromodomains. Here we describe the development of pyridazinone-based BPTF inhibitors. The lead compound, BZ1, possesses a high potency (Kd = 6.3 nM) and >350-fold selectivity over BET bromodomains. We identify an acidic triad in the binding pocket to guide future designs. We show that our inhibitors sensitize 4T1 breast cancer cells to doxorubicin but not BPTF knockdown cells, suggesting a specificity to BPTF. Given the high potency and good physicochemical properties of these inhibitors, we anticipate that they will be useful starting points for chemical tool development to explore the biological roles of BPTF.

4-Methyl-1,2,3-Triazoles as N-Acetyl-Lysine Mimics Afford Potent BET Bromodomain Inhibitors with Improved Selectivity.

The bromodomain and extra terminal (BET) protein family recognizes acetylated lysines within histones and transcription factors using two N-terminal bromodomains, D1 and D2. The protein-protein interactions between BET bromodomains, acetylated histones, and transcription factors are therapeutic targets for BET-related diseases, including inflammatory disease and cancer. Prior work demonstrated that methylated-1,2,3-triazoles are suitable N-acetyl lysine mimetics for BET inhibition. Here we describe a structure-activity relationship study of triazole-based inhibitors that improve affinity, D1 selectivity, and microsomal stability. These outcomes were accomplished by targeting a nonconserved residue, Asp144 and a conserved residue, Met149, on BRD4 D1. The lead inhibitors DW34 and 26 have a BRD4 D1 Kd of 12 and 6.4 nM, respectively. Cellular activity was demonstrated through suppression of c-Myc expression in MM.1S cells and downregulation of IL-8 in TNF-α-stimulated A549 cells. These data indicate that DW34 and 26 are new leads to investigate the anticancer and anti-inflammatory activity of BET proteins.

Journeying through the Field of Medicinal Chemistry: Perspectives from Graduate Researchers.

The training of new medicinal chemists is vital to the future of the field, and as graduate students at this critical stage, we are uniquely positioned to comment on our training. Herein, we discuss the perspectives from graduate researchers before, during, and after graduate school by utilizing survey data obtained from five medicinal chemistry programs in the Midwest and recent alumni of the University of Minnesota. We also reflect on the female perspective within the field of medicinal chemistry. Finally, we offer recommendations to both students and faculty in the hopes of helping future generations succeed in the field.