Glycosyltransferases and more, important tools for drug design A. Bechthold, Universität Freiburg. - презентация

1 Glycosyltransferases and more, important tools for drug design A. Bechthold, Universität Freiburg

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4 Compounds with anticancer activity

5 Compounds with antimicrobial activity

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7 9 Sugars 6 GTs 6 Sugars 4 GTs 17 Sugars 10 GTs

8 UrdGT2 UrdGT1a UrdGT1c UrdGT1b LanGT2 LanGT1 LanGT4 LanGT3 LanGT1 LanGT4 SaqGT5 SaqGT2 SaqGT1 SaqGT6 SaqGT3 SaqGT4 (?) SaqGT3 SaqGT4 (?)

9 Can we use glycosyltransferases as tools to generate novel antibiotics?

10 LanGT2 LanGT1 LanGT3 LanGT4

11 LanGT1 LanGT3 LanGT4 UrdGT2

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13 More than 50 new compounds by combining landomycin- and urdamycin biosynthetic genes ! Landomycin A Urdamycin A

14 How do glycosyltransferases look like?

15 UrdGT2 MurG OleD OleI GtfA GtfB GtfD

16 UrdGT2

17 Can we combine N- and C- terminal parts of different glycosyltransferases?

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19 +

20 LanGT1/LndGT1: 390 aa (25% non - identical aa)

21 LanGT1-like activity: Landomycin with 5 and 6 sugars LndGT1-like activity: Landomycin with 2, 3 and 4 sugars no activity: Landomycin with 1 sugar

22 LanGT1 like activity % LndGT 1 like activity % No activity % < < N-term.C-term.

23 Which amino acids are responsible for substrate specificity?

24 Mutations affecting the acceptor substrate specificity of a glycosyltransferase Hoffmeister et al., 2001, ChemBiol Hoffmeister et al., 2002, ChemBiol Hancock et al., 2006, Curr Opin Chem Biol Williams et al., 2007, Nat Chem Biol Williams et al., 2008, ChemBiol

25 ß1 α1α1 ß2 α2α2 ß3 α3α3 H4 ß4 α5α5 ß5 α6a α6b ß6 ß7 α7α7 α8α8 ß8 α9α9 ß9 α10 ß10 H11 ß11 ß12 α12 αH13 ß13 α14 α15 α16 N C α4α4 v1a: β1-α3 v1b: region between α3 and α4 v2a: α4 v2b: β4-β5 v3: α6-α7

26 N-term.C-term. v1a v1b v2a v2b v3 v1a (64 aa): β1-α3 v1b (19 aa): region between α3 and α4 v2a (23 aa): α4 v2b (35 aa): β4-β5 v3 (68 aa): α6-α7

27 LanGT1 like activity % LndGT 1 like activity % No activity % < C

28 ß1 α1α1 ß2 α2α2 ß3 α3α3 H4 ß4 α5α5 ß5 α6a α6b ß6 ß7 α7α7 α8α8 ß8 α9α9 ß9 α10 ß10 H11 ß11 ß12 α12 αH13 ß13 α14 α15 α16 N C α4α4 v1a: β1- α3 v1b: region between α3 and α4 v2a: α4 v2b: β4- β5 v3: α6-α7

29 LanGT1 like activity % LndGT 1 like activity % No activity % 3466< <

30 ß1 α1α1 ß2 α2α2 ß3 α3α3 H4 ß4 α5α5 ß5 α6a α6b ß6 ß7 α7α7 α8α8 ß8 α9α9 ß9 α10 ß10 H11 ß11 ß12 α12 αH13 ß13 α14 α15 α16 N C α4α4 v1a: β1-α3 v1b: region between α3 and α4 v2a: α4 v2b: β4- β5 v3: α6-α7

31 LanGT1 like activity % LndGT 1 like activity % No activity % < <0.1 I 64 V 67 A 68 V 64 L 67 A 68 A 73 E 74 A 75 L 73 Q 74 S 75 D 82 E 82 v1b

32 LanGT1 like activity % LndGT 1 like activity % No activity % G 107 R 110 R 107 G 110 L 119 Y 121 V 122 F 124 I 119 F 121 I 122 L 124 T 129 D 129 v2b

33 LanGT1 like activity % LndGT 1 like activity % No activity % G 107 R 110 R 107 G 110 L 119 Y 121 V 122 F 124 I 119 F 121 I 122 L 124 T 129 D 129 v2b

34 LanGT1 like activity % LndGT 1 like activity % No activity % < < T 89 S 89 R 93 P 94 D 97 P 93 Q 94 E 97 E 101 A 101 A 104 D 104 v2a

35 LanGT1 like activity % LndGT 1 like activity % No activity % < I 64 V 67 A 68 R 93 P 94 D 97 L 119 Y 121 V 122 F 124 V 64 L 67 G 68 P 93 Q 94 E 97 I 119 F 121 I 122 L 124

36 ß1 α1α1 ß2 α2α2 ß3 α3α3 H4 ß4 α5α5 ß5 α6a α6b ß6 ß7 α7α7 α8α8 ß8 α9α9 ß9 α10 ß10 H11 ß11 ß12 α12 αH13 ß13 α14 α15 α16 N C α4α4

37 α3α3 α4α4 β5β5 β4β4 ERecognition Positioning

38 Glycosyltransferases and more, important tools for drug design A. Bechthold, Universität Freiburg

39 Weitnauer et al., ChemBiol,2001 Weitnauer et al., Microbiol, 2002 Mosbacher et al., J Mol Biol 2003 Treede et al., Mol. Microbiol 2003 Weitnauer et al., ChemBiol 2004 Mosbacher et al., J Mol Biol, 2005 Treede et al., Ap Env Mic, 2005 Hofmann et al., ChemBiol, 2005 Boll et al., JBC, 2006

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41 Avilamycin AGavibamycin L1 Activity against MRSA (MIC) 42 logP (pH: 7.4) Protein binding (%) Survival rate of mice(1.25 mg/kg) 33%67% Successful co- crystallization with rRNA NoYes

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44 AviE2 AviQ1

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48 Gavibamycin L1 Gavibamycin N1 Activity against MRSA (MIC) 216 Activity against Streptococcus pyrogenes (MIC) 28 Activity against Enterococcus faecalis (MIC) 18

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51 AviX12

52 AviG6

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55 Search for compounds acting like avilamycins Development of a two-component assay using the FACS technology (in cooperation with N.Q, Le, Dr. I. Mecklenbräuker, Prof. Dr. D. Bumann)

56 no avilamycin 6µg/ml Detection of strains producing compounds acting like avilamycin A

57 Glycosyltrasferase s OxygenasesMembrane proteins (White biotechnology) Recombineering A.ChargeM. DaumU. HardterS. Hermann A.GüntherA.LinnenbrinkK. ProbstK. Petzke J. HärleI.PeintnerS. EbelingE. Welle C. KrauthH. J. SchnellM. FedoryshynT. Strobel M. FedoryshynA. Schandelmeier C. Jilg H. WeißM. Weber S. Gaiss A. Luzhetskyy, G. Weitnauer, H. Yu Thank you very much!!

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