Biosci. Biotechnol. Biochem., 8 (10), 2306-2310, 2001 Note 3a Antibacterial Agents that Inhibit Histidine Protein Kinase YycG of Bacillus subtilis Kaneyoshi Yamamoro,! Takashi Kirayama,! Shu Minacawa,! Takafumi Waranase,! Seiji Sawapa,? Tadashi Oxamoro,! and Ryutaro Ursunt!t \Department of Agricultural Chemistry, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan *Kyoto University of Education, Kyoto612-0863, Japan Received March 30, 2001; Accepted June 6, 2001 ‘We demonstrated in vitro that YycG-YyeF of Bacillus subtilis constitutes two-component system and shows a specificity of the sensor protein for the cognate phos- phorylation partner. Based on inhibition of such an au- tophosphorylation of YycG, we searched imidazole and zerumbone derivatives to identify the antibacterial a- gents such as NH125, NH126, NH127, and NHO891. Key words: two-component system; signal transduc- tion; histidine Kinase inhibitors; an- timicrobial agents, Histidine protein kinases (HPKs), one of the major components in the two-component regulatory sys- tem, play a key role in prokaryotic signal transduc- tion, which is required for response and adaptation to various environmental stresses. Two-component signal transduction systems consist of a histidine kinase and its cognate response regulator. In response to an environmental signal, the ki tophosphorylates a highly conserved His residue, The high-energy phosphate group on the His residue is then transferred to an Asp residue of the response regulator, therby altering its activity.” Recently, several essential two-component systems have been found: HP166, HP1043, and HP1021 in Helicobac- ter pylori; CekA-CirA in Caulobacter erescentus and YyeG-YyeF in Bacillus subiiis*®, Staphylococ- cus aureus’, and Streptococcus pneumoniae.’” YyoG-ycF is also conserved among other Gram- positive pathogen bacteria and is considered @ novel target for antibacterial agents." Natural products and synthetic inhibitors of bac- terial HPKs, which include hydrophobic tyramines and substituted salicylanilides, have been described. These compounds have MICs in a range of 1-10 ug/ml and their 50% HPK inhibitory concen- trations (IC50s) are in the low micromolar range." However, despite the correlation between bacteric- dal activity and inhibition of HPKs, whether HPK inhibition accounts for the bactericidal activity has kinase au- + Towhom correspondence should be addressed. Tel: 81-742-43-7273 (xt. 3309); Fax: 81 not been established. ‘The deduced sequences of the putative YycG and YycF proteins have structural similarity to histidine Kinases and response regulators, respectively, but their biochemical properties have not been clarified. In this study, we constructed a YycG-YycF signal transduction system in vitro. Based on inhibition of the autophosphorylation reaction of YycG, we tested newly synthesized imidazole and zerumbone deriva- tives for their antibacterial activities. To obtain the cytoplasmic signaling domain of YyeG, the yycG (151746 to 153000 in GenBank BSUB0021) gene containing the coding region (204 to 611 aa of YycG) was amplified by PCR with pBY33, (Table 1) as the template, and (5*GCAGGATCCAT- TACCCACCC3’) and (5 ‘TGCAGACGAG- TACCGT3’) as the primers. After that, it was digested with BamHI and PstI and ligated into the corresponding sites of pQE30 to obtain pYycG. To make a mutation at the putative autophosphorylated site of YycG (His386), we used a QuickChange Site-Directed Mutagenesis Kit (Stratagene). pYycGH386R and pYycGH386A were derived from pYycG, carrying yycG with changes of His386 (CAT) to Arg (CGA) and Ala (GCO), respectively. Primers used for the site-directed mutagenesis were as follows: primerl, (5’-CGITGCGAATGTATCG- (CGAGAGCTGCGGACGC-3") and (5’-GCG TCC- GCAGCTCTCGCGATACATTCGCAACG-3'); primer2, (5’-CGTTGCGAATGTTCGGCCGAGCT- GCGGACGC-3") and (5'-GCGTCCGCAGCTC GGCCGATACATTCGCAACG-3"). Their mutation sites were confirmed by digestion with Nru I and Eco52 I, respectively, and DNA sequencing, YycG, YycGH386R, YycGH386A, and YycF (see Table!) were purified as N-terminal His6-tag proteins in E. coli M15 (pREP4) containing pYycG, pYycGH386R, pYycGH386A, and pYycF,, respectively, as previous ly described." We incubated YycG, YycGH386R, or YycGH386A (1 ys) with 37 KBq of [y-"P] ATP in the 2-43-1485; E-mail: wisumi@nara kinda. cepInhibitors of Histidine Protein Kinase ¥yeG 2307 Table 1, Bacterial Steans and Plasmids Sond oe ee or ence Bacterial strains acs svbails 168 mpc H. Yoshikawa cherish colt MCH00 —— F° araD139A(argFac) U169 rpL150 psiF25 i0B3301 rbsR deoCl rlAl 0) MIS ipREPA] F>reca* wr" fac” ara” gal mil acl. Kn’ Nef Str rit iawn Plasmids DlluesriptlSK” cloning vector, Ap” Swratagene POE ‘expression vector, Ap’ Qiagen mova ‘ter the 6.0K fragment containing yy¢FG (18033 fo 186292 in Gen Bark BSUBOO21) This work was ample by PCR with B. subi 168 genomic DNA as template, te primer ("-GGG- CTAACAAGATCOQCACA-3°), the primer (5ATTCTTTGCCGGT AGCCTGC-3"), and KOD Dash (Toyobo), then digested with £082 it was ligated into Nor site of pBlueserin- isk PYyc0 {6G was cloned into pQE30 as described intext “This work DYVeGHSEGR, Y5eG H386R, pY¥eG This work PYYEGHSHOA YyeG H386A, p¥veG This work pYveF Ate the ype (15308 to 158321 in GenBank BSUBIOL! was amped by PCR with PBYS3 This work ts template, the primer ('-ATGGGATCCAAGATC CTTGT-3°, the primer (5-ATTCTG: CAGGCCATTAGTCC-1, thea digested with Bam HI and Pet, was gated into the coe responding sites of pQE30. PHO Borii-Hind 11 fragment containing phoQ amplied by PCR was lone into the cor- (2) ponding sites of FOE. (0 mM Tris-HCI pH7.5, 50 mM KCI, and 10 mM MgCl containing 2.5 404 of cold ATP at room temperature. To stop the reaction, 2 sample vereeeewne bute (120 mot Tris-HCI pH6.8, 20% glycerol, 4% mmm] 6 SDS, 10% femercaptocthanol, 0.1% BPB) was ad- 5 ded. After SDS-polyacrylamide gel electrophoresis, the gel was dried and analyzed with BAS 1000 Mac i (Fuji Film Co. Japan). z YyeG showed the maximum activity of autophos- é phorylation 15 min. after addition of [y-"PJATP in SEIS GEESE’ IEEE” ERTET” EEE vitro. (Fig. 1A), whereas YyeGH386R and Timon) YycGH386A did not (Fig. 1B). In addition, YycG @) «© \was able to transfer the phosphate group 10 YycF when combined with YyeF (Fig. 1C). These results indicate that YycG is a histidine kinase with autophosphorylation activity, and that YycG-YycF bP eyo constitutes a two-component system. The results also @ =e show a specificity of the sensor kinase (YyeG) For its we cognate phosphorylation partner (YycF). Next we examined the imidazole"'® and zerum- bone”? derivatives for their inhibitory activities Tats a against the YyoG-YyeF system, and subsequently their antibacterial activities In an earlier paper, Roychoudhury e¢ al. disco- vered a lead compound B (J-benzyl-2-methyl-3- myristylimidazolium bromide) that significantly in- hibited the autophsphorylation activities of Che, NRII, and KinA, but could not effectively block bac- terial growth.'® In this study, we clarified the effect ‘on autophosphorylation of YycG by using such im- idazole derivatives (Fig. 2). We found NHI25, NH126, and NH127 inhibited the incorporation of phosphate from ATP into YycG at 50 ug/ml Fig. 1, Avtophosphorylation of YyeG and Phosphoiransfer to Yyer. “Aatophosphorylation (A. YycG; B lenel, YyeG; C lane YYyeGs B lane2, YyeGH3B6R; B lane’, VyeGH386A; C lane2, ‘YyeF) was done as described in the text. YyeF (10 0 and ATP (10 mn) were added to the autophosphorylated Y¥eG and react- fed for Smin, at room temperature (C lanes). The radioactivity ‘of the bands corresponding to YyeG (A) was measured with 3 liquid scintlation counter (Aloca Co. Japan). (Fig. 3A), with a one-half maximal inhibitory con- centration (IC50) of 6.6-40 yat (Table 2), Further-2308 K. Yawaworo e a (99 tac deraives Crigeenwn~~~~-~> oma Cate ox Na fa ‘NHI28 oman NIII29 NHI2s (®) Zerumbone derivatives and synthetic pathway esse : Xo [. ce Neos NHO898——_NHO89 Fig. 2, Imidazole Derivatives andl Symthetie Pathway of Zarur. ‘bone Derivatives. ‘All imidazole derivatives were synthesized as described previ cusly." Zerumbone was isolated by simple dislation and recrystalizaion from fresh thizomes of Zinger zerumbet Smith." NHOS84, NHOB9I, NHOLS, NHOS98, and NHOS9 were newly synthesized by Kitayama ef a” more, they had antibacterial activity in B. subtilis with MIC 1,56-6.25 ug/ml (Table 2). NHI25 which has an alkyl chain in length of C16 inhibited the au- tophosphorylation of YyeG most strongly (Fig. 3A). The interference of unsaturated fatty acids in au- tophosphorylation has been reported for Kina from B. subtilis." However, the autophosphorylation of YycG was not inhibited by a cis-unsaturated fatty acid (oleic acid) or saturated fatty acids (palmitic acid and stearic acid) (Fig. 3A). Zerumbone (Fig. 2) is a cyclic sesquiterpene which is the main component of the essential oil of a wild ginger, Zingiber zerumbet Smith, which is widespread in Southeast Asia, India, and Okinawa, and is used as a spice and ethnomedicine. Recently Kitayama et al, succeeded in obtaining zerumbone by simple steam distillation and recrystallizatio which enabled us to synthesize a large number of zerumbone derivatives which can be tested as new an- tibacterial agents. Although we analyzed the autophosphorylation of YycG with 100 zerumbone derivatives that had the cyclic structure, we observed no inhibitor. If the a) (Ie 123456789 @ 1234567 ae Fig. 3. Inhibition of the Autophosphorylation of YyeG. (A) YyeG was incubated in the absence lane 1) of the presence of a essunsarurated fatty acid (a oleic acid, saturated Tatty acids (b, palmitic acid, stearic acid), and imidazole com pounds (d, NHI25; e, NHI26; f, NHI27; g, N25; h, NHI29) ft diferent concentrations (ane 2, 0.39yus/ml; lane 3, O.7Bug/al; lane 4, 1-56yg/ml; lane 5, 3.13 us/a; lane 6, 6.25 p/m; lane 7, 12.5 g/ml; lane 8, 25.0ug/a; lane 9, '$0,0,ue/m for Smin., and then autophosphorylated and ans ized as described in Fig. 1A. (B) Autophosphoryiation of ¥yoG was done inthe presence of 500 ug/ml of zerumbone derivatives (lane 1, DMSO; lane 2, zerambone; lane 3, NHOIS; lane 4, INHO894; lane 5, NHO898; lane 6, NHOS91: lane 7, NHOS9), Table 2. IC, and MIC MIC (ue/mb) Dn Hep GN) - Ecol B. subi Imidazoes NHI2s 343 1.56 NHI26 123 635 NHL? ns 156 NHI28 50 nse NHI29 soe Bse Zerumbones Zerumbone woo 1000 NHOIs ico 1000 HOS coo 1000 HORS coo 1000 NHO891 woos 2s NHOS9 1000< 500 Moteur weet of compounds: NEI2S, 27; NHI26 499; NHLZT $71: [NHI2g, 415; N29, 359, zerumion, 218, NHOIS, 234, NHO8S4, 378; [NiO 394; HOSS 295; NHOGS, 31. MIC was measured as dest previously cyclic structure of zerumbone is cleaved, it can be converted into new zerumbone derivatives. To de- velop such new compounds, bromide was added to the C2 conjugated double bond of zerumbone and zerumbone-oxide (NHOIS), giving dibromidesInhibitors of Histidine Protein Kinase YycG 2309 (NHO894 and NHO898), respectively (Fig. 2). After that, they were treated with KOH in the presence of a-cyclodextrin to obtain NH0891 and _NHO89." NHO891((2E, 6E,10(E/Z)]-11-bromo-4,4,7-trimethyl- 2,6,10-dodecatrienoie acid) was found to be an ini- bitor of YycG (Fig. 3B, Table 2). 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