Kinetics of Ligand Interaction, Substrate Turnover and Oxime Reactivation in Cholinesterases
Skaggs School of Pharmacy and Pharmaceutical Sciences
University of Zagreb, Zagreb, Croatia
Dr. Radić is a graduate of the University of Zagreb in Croatia where he started his research in acetylcholinesterase (AChE) reaction kinetics under mentorship of late Dr. Elsa Reiner, one of founding contributors in the field of cholinesterases. His interests in the AChE molecular structure brought him to the laboratory of Dr. Palmer Taylor at UCSD where he contributed to functional mapping of the AChE molecule using site directed mutagenesis and defined structural domains and amino acid residues critical for catalytic activity and ligand interaction. He proposed a common, simplified kinetic scheme and equation to describe acetylcholine (ACh) turnover by both AChE and its structurally and functionally close relative butyrylcholinesterase (BChE) that included both substrate inhibition of AChE and substrate activation of BChE, two prominent forms of deviation from Michaelis- Menten kinfkovarik etcs. Later this scheme and mechanism proved useful for the description of oxime reactivation kinetics of nerve agent organophosphate (OP) and OP pesticide conjugated AChE and BChE including positive allosteric modulation in reactivation of OP-BChE and negative allosteric modulation of OP-AChE conjugates. Dr. Radić developed intrinsic tryptophane fluorescence based assays as a valuable tool for in vitro monitoring of time resolved and equilibrium interactions of cholinergic ligands with native nicotinic acetylcholine receptor ligand binding domain surrogates, ACh binding proteins (AChBPs), as well as with AChE and BChE. This assay proved essential in functional characterization of one of tightest known, high affinity binding small molecule femtomolar AChE inhibitors developed in collaboration with Dr. Barry Sharpless of The Scripps Research Institute in La Jolla (TSRI). Dr. Radić leads, as a PI, NIH funded collaborative projects with researchers from Oak Ridge National Laboratory (Dr. Andrey Kovalevsky) University of Utah (Dr. Donald Blumenthal) and The Ohio State University (Dr. Xiaolin Cheng) towards development of novel, improved oxime reactivators of OP-AChE and OP-BChE conjugates, both in vitro and in vivo. Also, ongoing are international collaborations with University of Hradec Kralovy, Czech Republic (Dr. Kamil Kuca, Rector) and University Hospital Hradec Kralovy (Dr. Ondrej Soukup and Dr. Jan Korabecny), Institute for Medical Research and Occupational Health in Zagreb, Croatia (Dr. Zrinka Kovarik) and Dr. Jonah Cheung of the New York Structural Biology Center.
Education: B.Sc., M.Sc. and Ph.D. in Chemistry and in Biochemistry from University of Zagreb, Croatia.
Awards and Honors:2016 Spiridion Brusina Medal; New York Academy of Science Professional Member, IREX Graduate Student Fellow and Fulbright and Fogarty Postdoctoral Research Fellow at UCSD, La Jolla.
Leadership Experience: Treasurer, Croatian Society of Biochemistry and Molecular Biology (1990).
Teaching:Advanced Visualization of Drug-Macromolecule Structural Interactions (SSPPS elective), Visualization of macromolecules and their interactions with drugs in 3D (WARR 87); Enzymes: Kinetics and Reaction Mechanisms, (graduate course at University of Zagreb)
- Outlined joint simplified kinetic scheme and model for substrate turnover by AChE and BChE.
- Developed intrinsic tryptophan based assay for in vitro characterization of ligand interaction with AChBP, AChE and BChE.
- Described allosteric modulation of oxime reactivation of OP-BChE conjugates as a basis for development of a new class of small molecule measures to counteract OP poisoning and improve associated therapy.
- Kovalevsky A et al. (2016). Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning. Ann N Y Acad Sci. 78: 41-49.
- Sit RK et al. (2018) Pharmacology, Pharmacokinetics, and Tissue Disposition of Zwitterionic Hydroxyiminoacetamido Alkylamines as Reactivating Antidotes for Organophosphate Exposure. J. Pharmacol. Exp. Ther. 367: 363 – 372.
- Rosenberg Y et al. (2017) Post-exposure treatment with the oxime RS194B rapidly reverses early and advanced symptoms in macaques exposed to sarin vapor. Chem. Biol. Interact. 274: 50-57.
Selected Recent Publications (from >100 articles)
Drug discovery for therapy of OP intoxication.