title: Discovery of halopyridines as quiescent affinity labels: inactivation of dimethylarginine dimethylaminohydrolase.
abstract: In an effort to develop novel covalent modifiers of dimethylarginine dimethylaminohydrolase (DDAH) that are useful for biological applications, a set of "fragment"-sized inhibitors that were identified using a high-throughput screen are tested for time-dependent inhibition. One structural class of inactivators, 4-halopyridines, show time- and concentration-dependent inactivation of DDAH, and the inactivation mechanism of one example, 4-bromo-2-methylpyridine (1), is characterized in detail. The neutral form of halopyridines is not very reactive with excess glutathione. However, 1 readily reacts, with loss of its halide, in a selective, covalent, and irreversible manner with the active-site Cys249 of DDAH. This active-site Cys is not particularly reactive (pK(a) ca. 8.8), and 1 does not inactivate papain (Cys pK(a) ca. ≤4), suggesting that, unlike many reagents, Cys nucleophilicity is not a predominating factor in selectivity. Rather, binding and stabilization of the more reactive pyridinium form of the inactivator by a second moiety, Asp66, is required for facile reaction. This constraint imparts a unique selectivity profile to these inactivators. To our knowledge, halopyridines have not previously been reported as protein modifiers, and therefore they represent a first-in-class example of a novel type of quiescent affinity label.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, Texas 78712, United States.
authors.forename: Corey M
authors.initials: CM
authors.lastname: Johnson
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Linsky
authors.forename: Dae-Wi
authors.initials: DW
authors.lastname: Yoon
authors.forename: Maria D
authors.initials: MD
authors.lastname: Person
authors.forename: Walter
authors.initials: W
authors.lastname: Fast
mesh_terms: D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001965:Bromides / Q000737:chemistry; D020134:Catalytic Domain / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D006219:Halogens / Q000737:chemistry; D057166:High-Throughput Screening Assays; D008969:Molecular Sequence Data; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011725:Pyridines / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D011726:Pyridinium Compounds / Q000737:chemistry; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D001965:Bromides; D004791:Enzyme Inhibitors; D006219:Halogens; D011725:Pyridines; D011726:Pyridinium Compounds; D000581:Amidohydrolases; C059999:dimethylargininase