探索抗生素的本质（英文PPT）Probing Nature for Antibiotics.ppt

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1、Probing Nature for Antibiotics,Irosha Nayanthika NawarathneMichigan State University04/30/08,Struggle for living,www.photos-screensaver-,www.creswell-crags.org.uk,“History of humankind can be regarded from a medicinal point of view as a struggle against infectious diseases”,Yoneyama,H.,Katsumata,R.,

2、Biosci.Biotechnol.Biochem.,2006,70,1060,Survival against infectious diseases,dodd.cmcvellore.ac.in,What are antibiotics?,Molecules that stop the microbial growth(both bacteria and fungi)or kill them outright,Walsh,C.,Antibiotics Actions origins and Resistance,2003,4,How do the antibiotics act agains

3、t bacteria?,Walsh,C.,Antibiotics Actions origins and Resistance,2003,Cell Wall Biosynthesis-lactams,Cyclosporins,Glycopeptides,How do the antibiotics act against bacteria?,Walsh,C.,Antibiotics Actions origins and Resistance,2003,19,Protein BiosynthesisAminoglycosides,Macrolides,Tetracyclines,Oxazoli

4、dinones,How do the antibiotics act against bacteria?,Walsh,C.,Antibiotics Actions origins and Resistance,2003,19 publications.nigms.nih.gov,DNA BiosynthesisQuinolones,RNA BiosynthesisRifampicin,How do the antibiotics act against bacteria?,Walsh,C.,Antibiotics Actions origins and Resistance,2003,19,M

5、etabolic pathways Folic Acid MetabolismTrimethoprim,SulfonamidesFatty Acid BiosynthesisTriclosan,Isoniazid,Ethionamide,Why do we need more antibiotics?,-Developing antimicrobial resistanceBacterial species Common types of Antimicrobial Types of Infections ResistanceStreptococcus pneumoniae-lactams,c

6、ephalosporins,macrolidesOtitis media,pneumonia,Tetracyclines sinusitis,meningitis Staphylococcus aureusCommunity-associated Meticillin,cephalosporins,macrolides Skin,soft tissue,sepsis pneumoniaHealthcare-associated Meticillin,cephalosporins,quinolones,Endocarditis,pneumonia,aminoglycosides,macrolid

7、essepsisEnterococcus spp.Ampicillin,vancomycin,aminoglycosides Sepsis,urinary tract,Furuya,E.Y.,Lowy,F.D.,Nature,2006,4,36,What should be targeted?,The compounds with,Novel structuresNew modes of action,Fernandes,P.,Nature Biotechnology,2006,24,1497,Where do the antibiotics come from?,NATURE,Where d

8、o the antibiotics come from?,NATURE,NP,SS,TS,Where do the antibiotics come from?,NATURE,NP,SS,TS,Helps in designing the molecules,Natural products as antibiotics,Naturally occurring compounds that are end products of secondary metabolism.Mostly extracted from plants,marine organisms,or microorganism

9、s.,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006,Natural products as antibiotics,Naturally occurring compounds that are end products of secondary metabolism.Mostly extracted from plants,marine organisms,or microorganisms.Eg:,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,10

10、06Pal,S.,Tetrahedron,2006,62,3171,Erythromycin,Isolation-Streptomyces erythreus in 1952Uses-Respiratory tract diseases,genital infectionsMOA-Inhibition of protein synthesis,Antibiotics which are semi-synthesized,Synthetically modified chemical compounds which are originated from natural products.,Wa

11、lsh,C.,Antibiotics Actions origins and Resistance,2003,4,Erythromycin is,Acid unstable,Pal,S.,Tetrahedron,2006,62,3171,Antibiotics which are semi-synthesized,Clarithromycin,Azithromycin,HMR3647,TE802,Pal,S.,Tetrahedron,2006,62,3171,Antibiotics which are totally from synthesis,Totally synthesized mol

12、ecules which are potent as antibiotics.Three main types.1.Sulfa drugs 2.Quinolones 3.Oxazolidinones,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006,Antibiotics which are totally from synthesis,Sulfa drugs(Sulphonamides),Uses-Urinary tract infections,pneumonia etc.MOA-Inhibition of fola

13、te synthesis,Harold,P.L.,OGrady,F.W.,Antibiotic and Chemotherapy,1992,6,268-272,Sulfamethoxazole,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006,Antibiotics which are totally from synthesis,Sulfa drugs(Sulphonamides)Naturally occurring,Uses-Urinary tract infections,pneumonia etc.MOA-In

14、hibition of folic acid biosynthesis,Harold,P.L.,OGrady,F.W.,Antibiotic and Chemotherapy,1992,6,268-272,Sulfamethoxazole,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Walsh,C.,Antibiotics Actions origins and Resistance,2003,80-82,p-aminobenzoic acid,Antibiotics which are totally from s

15、ynthesis,Quinolones,Ciprofloxacin,Uses-Urinary tract infections,Lower respiratory infections,Gastrointestinal infectionsMOA-Inhibition of DNA synthesis,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006,Antibiotics which are totally from synthesis,Quinolones Naturally occurring,Ciprofloxa

16、cin,Uses-Urinary tract infections,Lower respiratory infections,Gastrointestinal infectionsMOA-Inhibition of DNA synthesis,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Kunze,B.,Hofle,G.,Reichenbach,H.,J.Antibiotics,1987,40,258,Aurachin C,Aurachin D,Antibiotics which are totally from s

17、ynthesis,Oxazolidinones,Ford,C.W.,Zurenko,G.E.,Barbachyn,M.R.,Current Drug Targets-Infectious Disorders,2001,1,181,Linezolid,Uses-Soft tissue infections,skin infections,Tuberculosis etc.MOA-Inhibition of protein synthesis,Antibiotics which are totally from synthesis,Oxazolidinones Naturally occurrin

18、g,Ford,C.W.,Zurenko,G.E.,Barbachyn,M.R.,Current Drug Targets-Infectious Disorders,2001,1,181Zappia,G.,et al.,Mini-Reviews in Medicinal Chemistry,2007,7,389,Linezolid,Uses-Soft tissue infections,skin infections,Tuberculosis etc.MOA-Inhibition of protein synthesis,(-)-Cytoxazone,(+)-Sreptazolin,Source

19、s of antibacterial drugs from 1981 to 2002,Newman,D.J.,Cragg,G.M.,Snader,K.M.,J.Nat.Prod.,2003,66,1022,Ways of probing nature for antibiotics,Ways of probing nature for antibiotics,New antibiotics New architectural scaffolds,Approach A,Conventional way of NP discovery,Singh,S.B.,Barrett,J.F.,Biochem

20、ical Pharmacology,2006,71,1006www.spc.int,www.oceanexplorer.noaa.gov,Extraction to the solvents,Isolation and Structure Elucidation,Natural materials,Bioassay guided fractionation,Approach A,Conventional way of NP discoveryWhy isnt it successful?Problems associated with the growth or the availabilit

21、y of the sourceReplication of the hitsDo not distinguish novel from oldMostly miss the novel compounds due to the lack of sensitivityNo hints about MOACannot reveal potency at screening stage,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Clardy,J.,Fischbach,M.A.,Walsh,C.,Nat.Rev.Biote

22、chnol.,2006,24,1541,Approach A,What are the new strategies to explore nature for NPs,Molecular Biology based Techniques,Novel culturing techniques,Heterologous expression of biosynthetic genes&Metagenomics,Genomics and Combinatorial biosynthesis,Precursor directed biosynthesis&Mutasynthesis,Differen

23、tial sensitivity screening approach,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Clardy,J.,Fischbach,M.A.,Walsh,C.,Nat.Rev.Biotechnol.,2006,24,1541Donadio,S.,Chemistry&Biology,2006,13,560,Approach A,What are the new strategies to explore nature for NPs,Molecular Biology based Techniq

24、ues,Novel culturing techniques,Heterologous expression of biosynthetic genes&Metagenomics,Genomics and Combinatorial biosynthesis,Precursor directed biosynthesis&Mutasynthesis,Differential sensitivity screening approach,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Clardy,J.,Fischbach

25、,M.A.,Walsh,C.,Nat.Rev.Biotechnol.,2006,24,1541Donadio,S.,Chemistry&Biology,2006,13,560,Approach A,Extraction to the Solvents,Producing organismsfound in nature,Pathogen,Precursor Directed Biosynthesis&Mutasynthesis,Wild type,Mutant type,Approach A,Precursor Directed Biosynthesis&Mutasynthesis,Natur

26、al Biosynthetic pathway,Kennedy,J.,Nat.Prod.Rep.,2008,25,25Weist,S.,Sssmuth,R.D.,Appl.Microbiol.Biotechnol.,2005,68,141,Wild type,Approach A,Precursor Directed Biosynthesis and Mutasynthesis,Precursor-Directed Biosynthesis,Wild type,Kennedy,J.,Nat.Prod.Rep.,2008,25,25Weist,S.,Sssmuth,R.D.,Appl.Micro

27、biol.Biotechnol.,2005,68,141,Approach A,Precursor Directed Biosynthesis and Mutasynthesis,Mutasynthesis,Mutant,Mutasynthon,Kennedy,J.,Nat.Prod.Rep.,2008,25,25Weist,S.,Sssmuth,R.D.,Appl.Microbiol.Biotechnol.,2005,68,141,Approach A,Mutasynthesis,Pojer,F.,Li,S.M.,Heide,L.,Microbiology,2002,148,3901Galm

28、,U.,et al,Chemistry&Biology,2004,11,173Weist,S.,Sssmuth,R.D.,Appl.Microbiol.Biotechnol.,2005,68,141,Ring A Ring B Ring CNovobiocin(Albamycin),Ring A Ring B Ring CClorobiocin,Approach A,Mutasynthesis,CloQ-mutants,Pojer,F.,Li,S.M.,Heide,L.,Microbiology,2002,148,3901Galm,U.,et al,Chemistry&Biology,2004

29、,11,173Eustquio,A.S.,et al,Arch.Microbiol.,2003,180,25,Approach A,Mutasynthesis,Galm,U.,et al,Chemistry&Biology,2004,11,173 Pojer,F.,Li,S.M.,Heide,L.,Microbiology,2002,148,3901,CloQ-mutant,Clorobiocin,Approach A,Mutasynthesis,CloQ-mutant,Clorobiocin,Galm,U.,et al,Chemistry&Biology,2004,11,173 Pojer,

30、F.,Li,S.M.,Heide,L.,Microbiology,2002,148,3901,Approach A,Mutasynthesis,CloQ-mutant,Analogs of Clorobiocin,Galm,U.,et al,Chemistry&Biology,2004,11,173 Galm,U.,et al,Antimicrob.Agents Chemother.,2004,48,1307 Pojer,F.,Li,S.M.,Heide,L.,Microbiology,2002,148,3901,Approach A,What are the new strategies t

31、o explore nature for NPs,Molecular Biology based Techniques,Novel culturing techniques,Heterologous expression of biosynthetic genes&Metagenomics,Genomics and Combinatorial biosynthesis,Precursor directed biosynthesis&Mutasynthesis,Differential sensitivity screening approach,Singh,S.B.,Barrett,J.F.,

32、Biochemical Pharmacology,2006,71,1006Clardy,J.,Fischbach,M.A.,Walsh,C.,Nat.Rev.Biotechnol.,2006,24,1541Donadio,S.,Chemistry&Biology,2006,13,560,Approach A,Differential sensitivity screening approach,Extraction to the solvents,Couzin,J.,Nature,2006,314,34,ForsythR.A.,Molecular Biology,2002,43,1387 Wa

33、ng,J.,et al,Antimicrob.Agents Chemother.,2006,50,519,Producingorganism from nature,Disabled type,Wild type,Increased sensitivity,Low,Normal,Pathogen,Expression of certain protein/s,Target the pathway,Approach A,Differential sensitivity screening approach,Fatty Acid Biosynthesis A good target,FAB Typ

34、e I-In mammals,FAB Type II-In bacteria,Campbell,J.W.,Cronan,J.E.Jr.,Annu.Rev.Microbiol.,2001,55,305,Biosynthesis of Saturated Fatty Acids,Campbell,J.W.,Cronan,J.E.Jr.,Annu.Rev.Microbiol.,2001,55,305,Biosynthesis of Saturated Fatty Acids,Campbell,J.W.,Cronan,J.E.Jr.,Annu.Rev.Microbiol.,2001,55,305,Bi

35、osynthesis of Saturated Fatty Acids,Campbell,J.W.,Cronan,J.E.Jr.,Annu.Rev.Microbiol.,2001,55,305,Singh,S.B.,et al,J.Am.Chem.Soc.,2006,128,11916Forsyth,R.A.,Molecular Biology,2002,43,1387 Wang,J.,et al,Antimicrob.Agents Chemother.,2006,50,519,Approach A,RNA-mediated gene silencing technique,Different

36、ial sensitivity screening approach,Reduced or No FabF expression,5 AUGGCCUGGACUUCA33 UACCGGACCTGTTGU 5,ds RNA,Degradation of fabF mRNA or inhibition of translation,In Prokaryotes-,Higher sensitivity towards FabF inhibitors,Singh,S.B.,et al,J.Am.Chem.Soc.,2006,128,11916Forsyth,R.A.,Molecular Biology,

37、2002,43,1387 Wang,J.,et al,Antimicrob.Agents Chemother.,2006,50,519,RNA-mediated gene silencing technique,Differential sensitivity screening approach,Approach A,Approach A,Differential sensitivity screening approach,Results-RNA-mediated gene silencing technique,Wild typefabF Anti-sense,Wang,J.,et al

38、,Nature,2006,441,358,Inhibitor(g),Approach A,Differential sensitivity screening approach,Results-RNA-mediated gene silencing technique,Wild typefabF Anti-sense,Wild typefabF Anti-sense,200 times more potent than Cerulenin,Wang,J.,et al,Nature,2006,441,358Price,A.C.,et al,The Journal of Biological Ch

39、emistry,2001,276,6551Heath,R.J.,White,S.W.,Rock,C.O.,Progress in Lipid Research,2001,40,467,Inhibitor(g),Approach A,Differential sensitivity screening approach,Platensimycinfrom a strain of Streptomyces platensis,Singh,S.B.,et al,J.Am.Chem.Soc.,2006,128,11916,Discovery of Platensimycin,Approach A,Di

40、fferential sensitivity screening approach,Potency of Platensimycin,Organism and genotype Platensimycin LinezolidAntibacterial activity(MIC,g/ml)S.aureus(MSSA)0.5 4 S.aureus(MRSA)0.5 2 S.aureus(MRSA,macrolideR)0.5 2 S.aureus(MRSA,linezolidR)1 32 Enterococcus faecium(VRE)0.1 2,MIC Concentration of inh

41、ibitor used to result no visible growth of the pathogens,Wang,J.,et al,Nature,2006,441,358,Toxicity(g/ml)HeLa MTT(IC50)1,000 100,IC50 Concentration of the inhibitor used to kill 50%population of the living cells,Cell-free gel-elongation assay,Wang,J.,et al,Nature,2006,441,358,Malonyl-ACP,C4:1(2)-ACP

42、,C4:0-ACP,6C-ACP,Approach A,Differential sensitivity screening approach,High FabF selectivity,Heath,R.J.,Nat.Prod.Rep.,2002,19,581,Ways of probing nature for antibiotics,New antibiotics New architectural scaffolds,Approach B,Singh,S.B.,Barrett,J.F.,Biochemical Pharmacology,2006,71,1006Clardy,J.,Fisc

43、hbach,M.A.,Walsh,C.,Nat.Rev.Biotechnol.,2006,24,1541,Generating Nature Mimics,Biosynthetic pathway,Designing theoretical chemical space that fits the active site or docking the database structures,Translate to a real structure by synthesis,Enzyme purification&3D structural determination,Approach B,H

44、utton,C.A.,Perugini,M.A.,Gerrard,J.A.,Mol.Biosyst.,2007,3,458Hutton,C.A.,Southwood,T.J.,Turner,J.J.,Mini-Reviews in Medicinal Chemistry,2003,3,115,Essential for the bacterial growth Does not exist in mammals,Generating Nature Mimics,Biosynthesis of lysine A good target,isoleucine,threonine,methionin

45、e,Hutton,C.A.,Perugini,M.A.,Gerrard,J.A.,Mol.Biosyst.,2007,3,458Hutton,C.A.,Southwood,T.J.,Turner,J.J.,Mini-Reviews in Medicinal Chemistry,2003,3,115,Biosynthesis of lysine,Hutton,C.A.,Perugini,M.A.,Gerrard,J.A.,Mol.Biosyst.,2007,3,458Hutton,C.A.,Southwood,T.J.,Turner,J.J.,Mini-Reviews in Medicinal

46、Chemistry,2003,3,115,Biosynthesis of lysine,Approach B,Generating Nature Mimics,Hutton,C.A.,Perugini,M.A.,Gerrard,J.A.,Mol.Biosyst.,2007,3,458Hutton,C.A.,Southwood,T.J.,Turner,J.J.,Mini-Reviews in Medicinal Chemistry,2003,3,115,Proposed mechanism,Approach B,Generating Nature Mimics,Supportive data,F

47、aehnle,C.R.,Coq,J.L.,Liu,X.,Viola,R.E.,Journal of Biological Chemistry,2006,281,31031 Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,ChemBioChem,2002,3,874Hutton,C.A.,Southwood,T.J.,Turner,J.J.,Mini-Reviews in Medicinal Chemistry,2003,3,115,Approach B,Generating Nature Mimics,Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,Ch

48、em.Commun.,2001,1710Cox,R.J.,Gibson,J.S.,Hadfield,A.T.,ChemBioChem,2005,6,2255Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,ChemBioChem,2002,3,874,Inhibitors of lysine biosynthesis,Approach B,Generating Nature Mimics,Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,Chem.Commun.,2001,1710Cox,R.J.,Gibson,J.S.,Hadfield,A.T.,Chem

49、BioChem,2005,6,2255Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,ChemBioChem,2002,3,874,Inhibitors of lysine biosynthesis,Approach B,Generating Nature Mimics,Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,ChemBioChem,2002,3,874Cox,R.J.,Gibson,J.S.,Hadfield,A.T.,ChemBioChem,2005,6,2255,Reverse Biosynthesis,In vitro assays,Ap

50、proach B,Cox,R.J.,Gibson,J.S.,Martn,M.B.M.,ChemBioChem,2002,3,874Blanco,J.,Moore,R.A.,Viola,R.E.,PNAS,2003,100,12613Han,S.,Moore,R.A.,Viola,R.E.,Synlett,2003,6,845,Generating Nature Mimics,KI(ASA)KI(Phosphate),Direct assay,Competitive assays,Approach B,Generating Nature Mimics,Direct assay,Cox,R.J.,