1、Probing Nature for Antibiotics,Irosha Nayanthika Nawarathne Michigan State University 04/30/08,,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., Kat
2、sumata, R., 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
3、the antibiotics act against bacteria?,Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 ,Cell Wall Biosynthesis-lactams,Cyclosporins,Glycopeptides,How do the antibiotics act against bacteria?,Walsh, C., Antibiotics Actions origins and Resistance, 2003, ,Protein Biosynthesis Aminoglycos
4、ides,Macrolides, Tetracyclines,Oxazolidinones,How do the antibiotics act against bacteria?,Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 publications.nigms.nih.gov, ,DNA Biosynthesis Quinolones,RNA Biosynthesis Rifampicin,How do the antibiotics act against bacteria?,Walsh, C., Anti
5、biotics Actions origins and Resistance, 2003, ,Metabolic pathwaysFolic Acid Metabolism Trimethoprim, SulfonamidesFatty Acid Biosynthesis Triclosan, Isoniazid, Ethionamide,Why do we need more antibiotics?,- Developing antimicrobial resistanceBacterial species Common types of Antimicrobial Types of In
6、fections Resistance Streptococcus pneumoniae -lactams, cephalosporins, macrolides Otitis media, pneumonia,Tetracyclines sinusitis, meningitis Staphylococcus aureus Community-associated Meticillin, cephalosporins, macrolides Skin, soft tissue, sepsis pneumoniaHealthcare-associated Meticillin, cephalo
7、sporins, quinolones, Endocarditis, pneumonia,aminoglycosides, macrolides sepsisEnterococcus 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.,
8、Nature Biotechnology, 2006, 24, 1497,Where do the antibiotics come from?,NATURE,Where do the antibiotics come from?,NATURE,NP,SS,TS,Where do the antibiotics come from?,NATURE,NP,SS,TS,Helps in designingthe molecules,Natural products as antibiotics,Naturally occurring compounds that are end products
9、of secondary metabolism. Mostly extracted from plants, marine organisms, or microorganisms.,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,
10、marine organisms, or microorganisms. Eg:,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Pal, S., Tetrahedron, 2006, 62, 3171,Erythromycin,Isolation - Streptomyces erythreus in 1952Uses - Respiratory tract diseases, genital infectionsMOA - Inhibition of protein synthesis,Antibio
11、tics which are semi-synthesized,Synthetically modified chemical compounds which are originatedfrom natural products.,Walsh, C., Antibiotics Actions origins and Resistance, 2003, 4,Erythromycin is,Acid unstable,Pal, S., Tetrahedron, 2006, 62, 3171,Antibiotics which are semi-synthesized,Clarithromycin
12、,Azithromycin,HMR3647,TE802,Pal, S., Tetrahedron, 2006, 62, 3171,Antibiotics which are totally from synthesis,Totally synthesized molecules which are potent as antibiotics. Three main types.1. Sulfa drugs 2. Quinolones3. Oxazolidinones,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71,
13、1006,Antibiotics which are totally from synthesis,Sulfa drugs (Sulphonamides),Uses - Urinary tract infections, pneumonia etc.MOA - Inhibition of folate synthesis,Harold, P.L., OGrady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272,Sulfamethoxazole,Singh, S.B., Barrett, J.F., Biochemical Pharmac
14、ology, 2006, 71, 1006,Antibiotics which are totally from synthesis,Sulfa drugs (Sulphonamides) Naturally occurring,Uses - Urinary tract infections, pneumonia etc.MOA - Inhibition of folic acid biosynthesis,Harold, P.L., OGrady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272,Sulfamethoxazole,Sin
15、gh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82,p-aminobenzoic acid,Antibiotics which are totally from synthesis,Quinolones,Ciprofloxacin,Uses - Urinary tract infections, Lower respiratory infections, Gastrointestin
16、alinfectionsMOA - Inhibition of DNA synthesis,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006,Antibiotics which are totally from synthesis,Quinolones Naturally occurring,Ciprofloxacin,Uses - Urinary tract infections, Lower respiratory infections, GastrointestinalinfectionsMOA -
17、Inhibition of DNA synthesis,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Kunze, B., Hofle, G., Reichenbach, H., J. Antibiotics, 1987, 40, 258,Aurachin C,Aurachin D,Antibiotics which are totally from synthesis,Oxazolidinones,Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current
18、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 occurring,Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current Drug Tar
19、gets-Infectious Disorders, 2001, 1,181 Zappia, 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,Sources of antibacterial drugs from 1981 to 200
20、2,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 antibioticsNew architectural scaffolds,Approach A,Conventional way of NP discovery,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71
21、, 1006 www.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 discovery Why isnt it successful?Problems associated with the growth or the availability of the sourceRe
22、plication 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, 1006 Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotec
23、hnol., 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,Di
24、fferential sensitivity screening approach,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541 Donadio, S., Chemistry & Biology, 2006, 13, 560,Approach A,What are the new strategies to explore nature for NP
25、s,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., Biochemical Pha
26、rmacology, 2006, 71, 1006 Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541 Donadio, S., Chemistry & Biology, 2006, 13, 560,Approach A,Extraction to the Solvents,Producing organisms found in nature,Pathogen,Precursor Directed Biosynthesis & Mutasynthesis,Wild type,Mutant
27、type,Approach A,Precursor Directed Biosynthesis & Mutasynthesis,Natural Biosynthetic pathway,Kennedy, J., Nat. Prod. Rep., 2008, 25, 25 Weist, S., Sssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141,Wild type,Approach A,Precursor Directed Biosynthesis and Mutasynthesis,Precursor-Directed Bio
28、synthesis,Wild type,Kennedy, J., Nat. Prod. Rep., 2008, 25, 25 Weist, S., Sssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141,Approach A,Precursor Directed Biosynthesis and Mutasynthesis,Mutasynthesis,Mutant,Mutasynthon,Kennedy, J., Nat. Prod. Rep., 2008, 25, 25 Weist, S., Sssmuth, R. D., Ap
29、pl. Microbiol. Biotechnol., 2005, 68, 141,Approach A,Mutasynthesis,Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901 Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Weist, S., Sssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141,Ring A Ring B Ring CNovobiocin (Albamycin),Ring
30、A Ring B Ring CClorobiocin,Approach A,Mutasynthesis,CloQ- mutants,Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901 Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Eustquio, A.S., et al, Arch. Microbiol., 2003, 180, 25,Approach A,Mutasynthesis,Galm, U., et al, Chemistry & Biology, 20
31、04, 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, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901,Approach A,Mutasynthesis,CloQ-mutant,An
32、alogs 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 to explore nature for NPs,Molecular Biology based Techniques,No
33、vel 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, 1006 Clardy, J., F
34、ischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541 Donadio, S., Chemistry & Biology, 2006, 13, 560,Approach A,Differential sensitivity screening approach,Extraction to the solvents,Couzin, J., Nature, 2006, 314, 34, Forsyth R.A., Molecular Biology, 2002, 43, 1387 Wang, J., et al, Antim
35、icrob. Agents Chemother., 2006, 50, 519,Producing organism from nature,Disabled type,Wild type,Increased sensitivity,Low,Normal,Pathogen,Expression of certain protein/s,Target thepathway,Approach A,Differential sensitivity screening approach,Fatty Acid Biosynthesis A good target,FAB Type I - In mamm
36、als,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.
37、, 2001, 55, 305,Biosynthesis 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, 11916 Forsyth, R.A., Molecular Biology, 2002, 43, 1387 Wang, J., et al, Antimicrob. Agents Chemother., 2006, 50, 519,Approach
38、A,RNA-mediated gene silencing technique,Differential sensitivity screening approach,Reduced or No FabF expression,5 AUGGCCUGGACUUCA3 3 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.
39、Chem. Soc., 2006, 128, 11916 Forsyth, R.A., Molecular Biology, 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 - RN
40、A-mediated gene silencing technique,Wild typefabF Anti-sense,Wang, J., et al, 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 potentthan Cerule
41、nin,Wang, J., et al, Nature, 2006, 441, 358 Price, A.C., et al, The Journal of Biological Chemistry, 2001, 276, 6551 Heath, R.J., White, S.W., Rock, C.O., Progress in Lipid Research, 2001, 40, 467,Inhibitor (g),Approach A,Differential sensitivity screening approach,Platensimycin from a strain of Str
42、eptomyces platensis,Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916,Discovery of Platensimycin,Approach A,Differential sensitivity screening approach,Potency of Platensimycin,Organism and genotype Platensimycin LinezolidAntibacterial activity (MIC, g/ml)S. aureus (MSSA) 0.5 4S. aureus (MRSA)
43、 0.5 2S. aureus (MRSA, macrolideR) 0.5 2S. aureus (MRSA, linezolidR) 1 32Enterococcus faecium (VRE) 0.1 2,MIC Concentration of inhibitor 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 inhibi
44、tor 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,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 probin
45、g nature for antibiotics,New antibioticsNew architectural scaffolds,Approach B,Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Clardy, J., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol., 2006, 24, 1541,Generating Nature Mimics,Biosynthetic pathway,Designing theoretical chemic
46、al 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,Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458 Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Revi
47、ews in Medicinal Chemistry, 2003, 3,115,Essential for the bacterial growthDoes not exist in mammals,Generating Nature Mimics,Biosynthesis of lysine A good target,isoleucine,threonine,methionine,Hutton, C.A., Perugini, M.A., Gerrard, J.A., Mol. Biosyst., 2007, 3, 458 Hutton, 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, 458 Hutton, C.A., Southwood, T.J., Turner, J.J., Mini-Reviews in Medicinal Chemistry, 2003, 3,115,
