Discovery of a cystathionine γ-lyase (CSE) selective inhibitor targeting active-site pyridoxal 5′-phosphate (PLP) via Schiff base formation
Honami Echizen, Kenjiro Hanaoka, Kazuhito Shimamoto, Ryota Hibi, Sachiko Toma-Fukai, H. Ohno, Eita Sasaki, Toru Komatsu, Tasuku Ueno, Yukihiro Tsuchiya, Yasuo Watanabe, Takao Otsuka, Hiroaki Saito, Satoru Nagatoishi, Kouhei Tsumoto, Hirotatsu Kojima, Takayoshi Okabe, Toshiyuki Shimizu, Yasuteru Urano
Abstract
Abstract D,L-Propargylglycine (PAG) has been widely used as a selective inhibitor to investigate the biological functions of cystathionine γ -lyase (CSE), which catalyzes the formation of reactive sulfur species (RSS). However, PAG also inhibits other PLP (pyridoxal-5′-phosphate)-dependent enzymes such as methionine γ -lyase (MGL) and L-alanine transaminase (ALT), so highly selective CSE inhibitors are still required. Here, we performed high-throughput screening (HTS) of a large chemical library and identified oxamic hydrazide 1 as a potent inhibitor of CSE (IC 50 = 13 ± 1 μM (mean ± S.E.)) with high selectivity over other PLP-dependent enzymes and RSS-generating enzymes. Inhibitor 1 inhibited the enzymatic activity of human CSE in living cells, indicating that it is sufficiently membrane-permeable. X-Ray crystal structure analysis of the complex of rat CSE (rCSE) with 1 revealed that 1 forms a Schiff base linkage with the cofactor PLP in the active site of rCSE. PLP in the active site may be a promising target for development of selective inhibitors of PLP-dependent enzymes, including RSS-generating enzymes such as cystathionine β -synthase (CBS) and cysteinyl-tRNA synthetase 2 (CARS2), which have unique substrate binding pocket structures.