Caffeine and Nicotine with N-Substituted Diazirine Photoaffinity Labels Form Adducts at Tyrosine-39 of α-Synuclein

Aggregates of the protein α-synuclein are found in Lewy bodies in the brains of Parkinson's disease (PD) patients. Small molecules that can attenuate or halt α-synuclein aggregation have been studied as potential therapeutics for PD. However, we have a limited understanding of how these molecules bind to α-synuclein. We previously identified that caffeine, nicotine, and 1-aminoindan all bind to both the N- and C-terminus of α-synuclein, although the binding location remains unknown. In an effort to identify these binding regions on α-synuclein, we synthesized diazirine photoaffinity probes attached to caffeine (C-Dz), nicotine (N-Dz), and 1-aminoindan (I-Dz) and allowed each to react with α-synuclein <i>in vitro</i>. We then treated the incubation mixture with trypsin and employed time-of-flight mass spectrometry to analyze the resulting peptides. Our findings reveal a distinctive binding pattern among the probes: C-Dz forms covalent bonds with Tyr-39 and Glu-20, while N-Dz selectively forms a covalent bond with Tyr-39. Intriguingly, we could not detect the labeling of I-Dz to any specific amino acids. All of the diazirine-bound peptides were found near the N-terminus. Our results suggest that the N-terminal region near Tyr-39 bears further study to elucidate the binding interactions of small molecules with α-synuclein and may be a target for anti-PD agents.