Regulation of the Escherichia coli AtoSC two component system by synthetic biologically active 5;7;8-trimethyl-1;4-benzoxazine analogues Panagiota S. Filippou a,  , Eftychia N. Koini b,  , Theodora Calogeropoulou b , Panagiota Kalliakmani a , Christos A. Panagiotidis c , Dimitrios A. Kyriakidis a,⇑ a Laboratory of Biochemistry, Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece b National Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, 48 Vas. Constantinou Ave., 11635 Athens, Greece c Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece article info Article history: Received 21 December 2010 Revised 1 June 2011 Accepted 8 June 2011 Available online 21 June 2011 Keywords: AtoSC two component system atoDAEB operon Kinase activity 5,7,8-Trimethyl-1,4-benzoxazine derivatives Escherichia coli abstract The Escherichia coli AtoSC two component system;upon acetoacetate induction;regulates the expression of the atoDAEB operon;through His ? Asp phopshotransfer;thus modulating important cellular pro- cesses. In this report the effect of seven 5,7,8-trimethyl-1,4-benzoxazine derivatives on the regulation of the E. coli AtoSC system was studied. The new compounds were tested for their effectiveness on the expression of the atoC and the regulated atoDAEB operon. The non-substituted 5,7,8-trimethyl-1,4-ben- zoxazine (4a), was the most potent inducer on atoC transcription;resulting in accumulation of AtoC pro- tein. The induction of atoC by 4a was specific;since no effect was observed on the other genes of the system (atoS and atoDAEB). Moreover;compound 4a was shown to significantly up-regulate the in vitro kinase activity of the histidine kinase AtoS without altering the protein levels in the cell. Inter- estingly;this compound appeared to modulate the acetoacetate-mediated induction of the atoDAEB pro- moter by the AtoSC system. These data provide the first evidence for a differential modulator role of 5,7,8- trimethyl-1,4-benzoxazine;on the AtoSC two component system mediated signaling. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction AtoSC is a well characterized two component system (TCS) in Escherichia coli, which relies on transmembrane sensor AtoS histi- dine kinase (HK) autophosphorylation and subsequent AtoC re- sponse regulator (RR) phosphorylation. AtoS was reported to be in trans-phosphorylated between the monomers of a homodimer in a conserved H-box containing the phosphorylation site His- 398 residue. 1 His-398 is lying in proximity to the AtoC Asp-55 res- idue upon AtoSC interaction for the subsequent phosphotransfer and AtoC phosphorylation necessary for AtoSC regulatory actions, 2 the transcriptional regulation of the atoDAEB operon that encode for proteins involved in short-chain fatty acid metabolism. 2–5 By governing the atoDAEB operon;this system regulates;amongst oth- ers;the biosynthesis and the intracellular distribution of cPHB, 6,7 the flagella synthesis; 8 the chemotactic behaviour and the involve- ment of extracellular Ca 2+ on cPHB synthesis regulated by the AtoSC system. 9 We have recently reported the regulation of the AtoSC system by polyamines and histamine, as cellular components that enable path- ogenic bacteria to survive and overcome host defence mechanisms. 10–12 Putrescine, the non-natural diamine 1,3-diami- nopropane and their synthetic analogues elicited a pronounced atoC transcriptional activation, without activating neither the atoDAEB operon promoter nor atoS. 11 Histamine, a biogenic amine, elicited a polyamine-mediated inductive phenotype and induced atoC expression, without activating the atoDAEB operon promoter. 12 Fur- thermore, the involvement of TCSs including AtoSC, in specialized functions including the bacterial pathogenicity, virulence and host-microbe interactions 3,4 warrant the investigation of a number of synthetic amine-containing compounds, including 1,4-benzoxa- zines, on their possible involvement on the signaling cascade. The 2H-1,4-benzoxazine-3-(4H)-one and 3,4-dihydro-2H-1,4- benzoxazine systems have been studied extensively for building natural and designed biologically active compounds, which span from herbicides, fungicides, cardiovascular agents, K ATP channel openers, compounds against diabetes, neuroprotectants and agents against anxiety and depression. 13–17 Thus, the above mentioned heterocycles can be considered as privileged scaffolds for the development of potential new drugs. We recently reported, the synthesis of 5,7,8-trimethyl-1,4-benzoxazine derivatives encom- passing the pharmacophore aminoamide functionality of lidocaine and their biological evaluation as novel antiarrhythmics against ischemia-reperfusion injury. 18 Furthermore, the 5,7,8-trimethyl- 1,4-benzoxazine moiety can be considered as a bioisostere of the 5,7,8-trimethyl-1,4-benzopyran nucleus of the well known antiox- idant vitamin E. 0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2011.06.029 ⇑ Corresponding author. Fax: +30 2310997689. E-mail address: kyr@chem.auth.gr (D.A. Kyriakidis).   PSF and ENK contributed equaly in this work. Bioorganic & Medicinal Chemistry 19 (2011) 5061–5070 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc