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Probing Nature for Antibiotics. Irosha Nayanthika Nawarathne Michigan State University 04/30/08. health.howstuffworks.com. Struggle for living. dansaper.blogspot.com, www.photos-screensaver-maker.com, tecnocientista.info.com, www.creswell-crags.org.uk.
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Probing Nature for Antibiotics Irosha Nayanthika Nawarathne Michigan State University 04/30/08 health.howstuffworks.com
Struggle for living dansaper.blogspot.com, www.photos-screensaver-maker.com, tecnocientista.info.com, 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., Biosci. Biotechnol. Biochem., 2006,70,1060
Survival against infectious diseases dodd.cmcvellore.ac.in, www.ayurvedicmedicine4u.com, www.rootsweb.com
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 against bacteria? Cell Wall Biosynthesis β-lactams,Cyclosporins,Glycopeptides Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.jacksofscience.com
How do the antibiotics act against bacteria? Protein Biosynthesis Aminoglycosides,Macrolides, Tetracyclines,Oxazolidinones Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.istockphoto.com
How do the antibiotics act against bacteria? DNA Biosynthesis Quinolones RNA Biosynthesis Rifampicin Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 publications.nigms.nih.gov, www.istockphoto.com
How do the antibiotics act against bacteria? Metabolic pathways Folic Acid Metabolism Trimethoprim, Sulfonamides Fatty Acid Biosynthesis Triclosan, Isoniazid, Ethionamide Walsh, C., Antibiotics Actions origins and Resistance, 2003, 19 www.istockphoto.com
Why do we need more antibiotics? - Developing antimicrobial resistance Bacterial speciesCommon types of AntimicrobialTypes ofInfectionsResistance Streptococcus pneumoniaeβ-lactams, cephalosporins, macrolides Otitis media, pneumonia, Tetracyclines sinusitis, meningitis Staphylococcus aureus Community-associated Meticillin, cephalosporins, macrolides Skin, soft tissue, sepsis pneumonia Healthcare-associated Meticillin, cephalosporins, quinolones, Endocarditis, pneumonia, aminoglycosides, macrolides sepsis Enterococcus 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 structures • New modes of action Fernandes, P., Nature Biotechnology, 2006, 24, 1497
NATURE Where do the antibiotics come from?
NATURE Where do the antibiotics come from? TS NP SS
NATURE Where do the antibiotics come from? Helps in designing the molecules TS NP SS
Natural products as antibiotics • Naturally occurring compounds that are end products 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, marine organisms, or microorganisms. • Eg: Isolation- Streptomyces erythreus in 1952 Uses - Respiratory tract diseases, genital infections MOA - Inhibition of protein synthesis Erythromycin Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Pal, S., Tetrahedron, 2006, 62, 3171
Antibiotics which are semi-synthesized • Synthetically modified chemical compounds which are originated from 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 Azithromycin TE802 HMR3647 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. Quinolones 3. Oxazolidinones Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis • Sulfa drugs (Sulphonamides) Sulfamethoxazole Uses - Urinary tract infections, pneumonia etc. MOA - Inhibition of folate synthesis Harold, P.L., O’Grady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272 Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis • Sulfa drugs (Sulphonamides) Naturally occurring Sulfamethoxazole p-aminobenzoic acid Uses - Urinary tract infections, pneumonia etc. MOA - Inhibition of folic acid biosynthesis Harold, P.L., O’Grady, F.W., Antibiotic and Chemotherapy, 1992, 6, 268-272 Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82
Antibiotics which are totally from synthesis • Quinolones Ciprofloxacin Uses - Urinary tract infections, Lower respiratory infections, Gastrointestinal infections MOA - Inhibition of DNA synthesis Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Antibiotics which are totally from synthesis • Quinolones Naturally occurring Aurachin D Ciprofloxacin Aurachin C Uses - Urinary tract infections, Lower respiratory infections, Gastrointestinal infections MOA - 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
Antibiotics which are totally from synthesis • Oxazolidinones Linezolid Uses - Soft tissue infections, skin infections, Tuberculosis etc. MOA - Inhibition of protein synthesis Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current Drug Targets-Infectious Disorders, 2001, 1,181
Antibiotics which are totally from synthesis • Oxazolidinones Naturally occurring (-)-Cytoxazone Linezolid (+)-Sreptazolin Uses - Soft tissue infections, skin infections, Tuberculosis etc. MOA - Inhibition of protein synthesis Ford, C.W., Zurenko, G.E., Barbachyn, M.R., Current Drug Targets-Infectious Disorders, 2001, 1,181 Zappia, G., et al., Mini-Reviews in Medicinal Chemistry, 2007, 7, 389
Sources of antibacterial drugsfrom1981 to 2002 Newman, D.J., Cragg, G.M., Snader, K.M., J. Nat. Prod., 2003, 66, 1022
NATURE Approach B Generating the Nature Mimics Approach A By exploring the novel Natural Products Ways of probing nature for antibiotics
NATURE Approach B Generating the Nature Mimics Approach A By exploring the novel Natural Products Ways of probing nature for antibiotics New antibiotics New architectural scaffolds
Approach A Conventional way of NP discovery Extraction to the solvents Natural materials Isolation and Structure Elucidation Bioassay guided fractionation Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006 www.spc.int, www.oceanexplorer.noaa.gov, www.nature.com, www.textbookofbacteriology.net
Approach A Conventional way of NP discovery Why isn’t it successful? • Problems associated with the growth or the availability of the source • Replication of the hits • Do not distinguish novel from old • Mostly miss the novel compounds due to the lack of sensitivity • No hints about MOA • Cannot 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. Biotechnol.,2006, 24, 1541
Approach A What are the new strategies to explore nature for NPs Novel culturing techniques Heterologous expression of biosynthetic genes & Metagenomics Molecular Biology based Techniques 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., 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 NPs Novel culturing techniques Heterologous expression of biosynthetic genes & Metagenomics Molecular Biology based Techniques 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., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol.,2006, 24, 1541 Donadio, S., Chemistry & Biology, 2006, 13, 560
Precursor Directed Biosynthesis & Mutasynthesis Approach A Extraction to the Solvents Producing organisms found in nature Pathogen Wild type Mutant type
Approach A Precursor Directed Biosynthesis & Mutasynthesis Wild type Natural Biosynthetic pathway Kennedy, J., Nat. Prod. Rep.,2008, 25, 25 Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol.,2005, 68, 141
Approach A Precursor Directed Biosynthesis and Mutasynthesis Wild type Precursor-Directed Biosynthesis Kennedy, J., Nat. Prod. Rep.,2008, 25, 25 Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol.,2005, 68, 141
Approach A Mutant Precursor Directed Biosynthesis and Mutasynthesis Mutasynthon Mutasynthesis Kennedy, J., Nat. Prod. Rep.,2008, 25, 25 Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol.,2005, 68, 141
Approach A Mutasynthesis Ring A Ring B Ring C Novobiocin (Albamycin) Ring A Ring B Ring C Clorobiocin Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901 Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol.,2005, 68, 141
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 Eustảquio, A.S., et al, Arch. Microbiol., 2003, 180, 25
Approach A Mutasynthesis Clorobiocin CloQ-mutant Galm, U., et al, Chemistry & Biology, 2004, 11, 173 Pojer, F., Li, S.M., Heide, L., Microbiology, 2002, 148, 3901
Approach A Mutasynthesis Clorobiocin CloQ-mutant 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 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 to explore nature for NPs Novel culturing techniques Heterologous expression of biosynthetic genes & Metagenomics Molecular Biology based Techniques 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., Fischbach, M.A., Walsh, C., Nat. Rev. Biotechnol.,2006, 24, 1541 Donadio, S., Chemistry & Biology, 2006, 13, 560
Approach A Differential sensitivity screening approach Producing organism from nature Pathogen Expression of certain protein/s Wild type Normal Extraction to the solvents Low Disabled type Increased sensitivity Target the pathway Couzin, J., Nature, 2006, 314, 34, Forsyth R.A., Molecular Biology, 2002, 43, 1387 Wang, J., et al, Antimicrob. Agents Chemother., 2006, 50, 519
Approach A Fatty Acid Biosynthesis… A good target Differential sensitivity screening approach FAB Type 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
Biosynthesis of Saturated Fatty Acids Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305
Differential sensitivity screening approach Antisense RNA Sense DNA Antisense DNA 5` ………ATGGCCTGGACTTCA…………3` 3` ………TACCGGACCTGAAGT…………5` Transcription mRNA 5` ………AUGGCCUGGACUUCA…………3` Translation Peptide Met - Ala - Trp - Thr - Ser - Approach A RNA-mediated gene silencing technique 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 A Differential sensitivity screening approach RNA-mediated gene silencing technique In Prokaryotes- ds RNA 5`……… AUGGCCUGGACUUCA………3` 3`……… UACCGGACCTGTTGU ………5` Degradation of fabF mRNA or inhibition of translation Reduced or No FabF expression Higher sensitivity towards FabF inhibitors 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