title: Development of novel molecular probes of the Rio1 atypical protein kinase.
abstract: The RIO kinases are essential protein factors required for the synthesis of new ribosomes in eukaryotes. Conserved in archaeal organisms as well, RIO kinases are among the most ancient of protein kinases. Their exact molecular mechanisms are under investigation and progress of this research would be significantly improved with the availability of suitable molecular probes that selectively block RIO kinases. RIO kinases contain a canonical eukaryotic protein kinase fold, but also display several unusual structural features that potentially create opportunity for the design of selective inhibitors. In an attempt to identify structural leads to target the RIO kinases, a series of pyridine caffeic acid benzyl amides (CABA) were tested for their ability to inhibit the autophosphorylation activity of Archeaoglobus fulgidus Rio1 (AfRio1). Screening of a small library of CABA molecules resulted in the identification of four compounds that measurably inhibited AfRio1 activity. Additional biochemical characterization of binding and inhibition activity of these compounds demonstrated an ATP competitive inhibition mode, and allowed identification of the functional groups that result in the highest binding affinity. In addition, docking of the compound to the structure of Rio1 and determination of the X-ray crystal structure of a model compound (WP1086) containing the desired functional groups allowed detailed analysis of the interactions between these compounds and the enzyme. Furthermore, the X-ray crystal structure demonstrated that these compounds stabilize an inactive form of the enzyme. Taken together, these results provide an important step in identification of a scaffold for the design of selective molecular probes to study molecular mechanisms of Rio1 kinases in vitro and in vivo. In addition, it provides a rationale for the future design of potent inhibitors with drug-like properties targeting an inactive form of the enzyme. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases (2012).
authors.affiliation: Polish Academy of Sciences, Institute of Biochemistry and Biophysics, Protein Biosynthesis Department, Warsaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Mielecki
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Krawiec
authors.forename: Irene
authors.initials: I
authors.lastname: Kiburu
authors.forename: Krystyna
authors.initials: K
authors.lastname: Grzelak
authors.forename: Włodzimierz
authors.initials: W
authors.lastname: Zagórski
authors.forename: Borys
authors.initials: B
authors.lastname: Kierdaszuk
authors.forename: Kamila
authors.initials: K
authors.lastname: Kowa
authors.forename: Izabela
authors.initials: I
authors.lastname: Fokt
authors.forename: Slawomir
authors.initials: S
authors.lastname: Szymanski
authors.forename: Piotr
authors.initials: P
authors.lastname: Swierk
authors.forename: Wiesław
authors.initials: W
authors.lastname: Szeja
authors.forename: Waldemar
authors.initials: W
authors.lastname: Priebe
authors.forename: Bogdan
authors.initials: B
authors.lastname: Lesyng
authors.forename: Nicole
authors.initials: N
authors.lastname: LaRonde-LeBlanc
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D001105:Archaea / Q000201:enzymology*; D019843:Archaeal Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D019843:Archaeal Proteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; C023666:pyridine