Superbug challenger from nature

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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 species          Common types of Antimicrobial                   Types of Infections
                                  Resistance
Streptococcus pneumoniae   β-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
Where do the antibiotics come from?

               NATURE
Where do the antibiotics come from?

               NATURE




          NP     SS      TS
Where do the antibiotics come from?

               NATURE


                              Helps in designing
                                the molecules
          NP     SS      TS
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 drugs
from 1981 to 2002

                                  1%            10%
         21%
                                                                               NP

                                                                               SS

                                                                               TS

                                                                               NM

                                                   68%




      Newman, D.J., Cragg, G.M., Snader, K.M., J. Nat. Prod., 2003, 66, 1022
Ways of probing nature for antibiotics

                         NATURE


          Approach A                  Approach B
     By exploring the novel           Generating
       Natural Products           the Nature Mimics
Ways of probing nature for antibiotics

                         NATURE


          Approach A                               Approach B
     By exploring the novel                        Generating
       Natural Products                        the Nature Mimics



                             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
                                      Genomics and Combinatorial biosynthesis
based Techniques

                                      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
                                      Genomics and Combinatorial biosynthesis
based Techniques

                                      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
 Precursor Directed Biosynthesis & Mutasynthesis



                         Extraction
Producing organisms        to the          Pathogen
  found in nature         Solvents



           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
Precursor Directed Biosynthesis and Mutasynthesis
      Mutant




                                                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                        Ring A      Ring B          Ring C


Novobiocin (Albamycin)                                           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
                                      Genomics and Combinatorial biosynthesis
based Techniques

                                      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

     Extraction                      Wild type                               Normal
       to the
      solvents                                                                Low
                                  Disabled type

                                     Increased                          Target the
                                     sensitivity                         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
Differential sensitivity screening approach
Fatty Acid Biosynthesis… A good target




     FAB Type I                                        FAB Type II
     - In mammals                                      - 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
Approach A
Differential sensitivity screening approach
RNA-mediated gene silencing technique

5` ………ATGGCCTGGACTTCA…………3` Sense DNA                                         Antisense RNA
3` ………TACCGGACCTGAAGT…………5` Antisense DNA


                    Transcription

5` ………AUGGCCUGGACUUCA…………3`                         mRNA

                    Translation


  Met - Ala - Trp - Thr - Ser -                    Peptide


                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-   5`……… AUGGCCUGGACUUCA………3`                               ds RNA
                  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
Approach A
Differential sensitivity screening approach
Results - RNA-mediated gene silencing technique



       Wild type

    fabF Anti-sense


                                    Inhibitor (μg)




                   Wang, J., et al, Nature, 2006, 441, 358
Approach A
Differential sensitivity screening approach
Results - RNA-mediated gene silencing technique

       Wild type

    fabF Anti-sense

                                                                      200 times more potent
                                                                          than Cerulenin
      Wild type

   fabF Anti-sense

                                       Inhibitor (μg)


                         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
Approach A
Differential sensitivity screening approach
Discovery of Platensimycin


                                     OH
                                       O                O
               HO
                                     N
                                     H
                     O       OH
                                             O


                         Platensimycin
         from a strain of Streptomyces platensis



          Singh, S.B., et al, J. Am. Chem. Soc., 2006, 128, 11916
Approach A
Differential sensitivity screening approach
Potency of Platensimycin
 Organism and genotype                            Platensimycin            Linezolid

 Antibacterial activity                                         (MIC, µg/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
       Toxicity                                                     (µg/ml)

   HeLa MTT (IC50)                                       >1,000               >100
    MIC – Concentration of inhibitor used to result no visible growth of the pathogens
    IC50 – Concentration of the inhibitor used to kill 50% population of the living cells

                          Wang, J., et al, Nature, 2006, 441, 358
    Approach A
    Differential sensitivity screening approach
    High FabF selectivity
                                                         O   O                             O    O                     O
                                                                            FabD
                                                    HO           SCoA                HO             S   ACP               CoA

       Cell - free gel - elongation assay                           ACPSH         CoASH
                                                                                                              FabH
                                                                                     C02 + CoASH


                                                                                                        O     O

 Malonyl-ACP                                                                                                      S    ACP

C4:1(Δ2)-ACP                                                                                   NADPH
                                                                                                              FabG

    C4:0-ACP                                        CO 2 + ACPSH
                                                                                               NADP

                                                                                                        OH    O

                                                                                                                  S    ACP

                                                                           FabF                                FabA
                                                                                                               FabZ
   >6C-ACP                                                                                      H 2O
                                                                                                              O

                                                                                                                   S      ACP

                                                                                                NADPH
                                                         O   O                                                    FabI
                                                                                                                  FabK
                                                    HO           S   ACP                                          FabL
                                                                                                NADP
                                                                                                              O

                                                                                                                   S      ACP


                         Wang, J., et al, Nature, 2006, 441, 358
                        Heath, R.J., Nat.Prod.Rep., 2002, 19, 581
Ways of probing nature for antibiotics

                         NATURE


          Approach A                               Approach B
     By exploring the novel                        Generating
       Natural Products                        the Nature Mimics



                             New antibiotics
                         New architectural scaffolds
Approach B
Generating Nature Mimics

                             Biosynthetic pathway


                           Enzyme purification &
                         3D structural determination


    Designing theoretical chemical space that fits the active site or
                       docking the database structures



                 Translate to a real structure by synthesis



        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 B
Generating Nature Mimics
Biosynthesis of lysine… A good target

 Essential for the bacterial growth


 Does not exist in mammals




            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




methionine

threonine



 isoleucine




                   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




methionine

threonine



isoleucine




                 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
Approach B
Generating Nature Mimics
Proposed mechanism




            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
Approach B
Generating Nature Mimics
Supportive data




                                                                   Acyl-enzyme intermediate
                                                                   (Streptococcus pneumoniae)

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




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




        Cox, R.J., Gibson, J.S., Martín, M.B.M., Chem. Commun., 2001, 1710
        Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255
        Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
Approach B
Generating Nature Mimics
In vitro assays



                               Reverse Biosynthesis




      Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
      Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255
Approach B
Generating Nature Mimics
Competitive assays




       Direct assay                KI (ASA)        KI (Phosphate)


                                           -                -


                                        750 μM          2130μM


                                        214 μM           92μM

       Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
          Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
              Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Approach B
Generating Nature Mimics
Competitive assays



      Direct assay




        Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
           Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
               Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Approach B
Generating Nature Mimics
Competitive assays



      Direct assay




        Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
           Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
               Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Approach B
Generating Nature Mimics
Competitive assays



      Direct assay
                              KI (ASA)         KI (Phosphate)         2nd pKa


                                     -                  -             4.2-5.0


                                  750 μM            2130μM               6.1


                                  214 μM             92μM             6.2-6.4

        Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
           Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
               Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Approach B
Generating Nature Mimics
Time-dependent inhibition assays



  Pre-incubation assay
                                               KI (ASA)


                                                95μM


                                                   -


                                                   -

         Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
            Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
                Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Approach B
Generating Nature Mimics
Time-dependent inhibition assays



  Pre-incubation assay




         Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
            Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
                Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Ways of probing nature for antibiotics

                         NATURE


          Approach A                               Approach B
     By exploring the novel                        Generating
       Natural Products                        the Nature Mimics



                             New antibiotics
                         New architectural scaffolds
Please, Don’t flush!


 Average american receives more than 11 prescriptions a year.

 About 3.3 billion a total.

 Nonprescription drugs !




                    Halford, B., C & EN News, 2008, 86, 13
                    Halford, B., C & EN News, 2008, 86, 16
Acknowledgement

Dr. Walker
Dr. Hausinger
Dr. Arnosti
Dr. Stoltzfus
Dr. Stephen Soisson, Dr. Jun Wang (Merck)

Labmates - Behnaz, Danielle, Joshua, Mark, Washington, Yemane

Friends - Samantha, Sue, Tharanga, Xiaofei
Thank you all !
Back-up slides
Approach A
Differential sensitivity screening approach
In vivo studies of Platensimycin

In a mouse model of disseminated S. aureus infection




                   Wang, J., et al, Nature, 2006, 441, 358
Timeline of discovery of novel classes of
antibiotics and introduction in clinic




      Singh, S.B., Barrett, J.F., Biochemical Pharmacology, 2006, 71, 1006
Approach B
Generating the Nature Mimics




 Faehnle, C.R., Coq, J.L., Liu, X., Viola, R.E., Journal of Biological Chemistry, 2006, 281, 31031
             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
Antibiotics which are totally from synthesis

   Sulfa drugs (Sulphonamides)                           Naturally occurring




        Sulfamethoxazole
                                                                          p-aminobenzoic acid


 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
            Walsh, C., Antibiotics Actions origins and Resistance, 2003, 80-82
Approach A
Precursor Directed Biosynthesis




       Penicillium                                            6-APA
      Chrysogenum
    (Penicillium notatum)




                Nayer, J.H.C., Trends. Biochem. Sci., 1991, 16, 195
                Nayer, J.H.C., Trends. Biochem.Sci., 1991, 16, 234
                    Kennedy, J., Nat. Prod. Rep., 2008, 25, 25
Approach A
Precursor Directed Biosynthesis




                                                                   Penicillin G


     Penicillium
    Chrysogenum
  (Penicillium notatum)


                                                                   Penicillin V
                 Nayer, J.H.C., Trends. Biochem. Sci., 1991, 16, 195
                 Nayer, J.H.C., Trends. Biochem.Sci., 1991, 16, 234
                     Kennedy, J., Nat. Prod. Rep., 2008, 25, 25
Approach A
Mutasynthesis




               Nov L                                                     Clo L




  Ring A   Ring B      Ring C                         Ring A       Ring B    Ring C


Novobiocin (Albamycin)                                        Clorobiocin

             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
Where do the antibiotics come from?

                                  Kekule stucture of benzene
        NATURE




   NP     SS          TS


               www.boomeria.org
Approach A
Precursor Directed Biosynthesis
Drawbacks

   Involves complex purification procedures

   Require high concentrations of synthetic precursor

   Only few intermediates will incorporate into the product




                        Kennedy, J., Nat. Prod. Rep., 2008, 25, 25
          Weist, S., Sϋssmuth, R. D., Appl. Microbiol. Biotechnol., 2005, 68, 141
Inhibitors of Fatty Acids Biosynthesis



                                                                                 Triclosan,
                                                                                 Isoniazid,
                                                                                 Ethionamide
                            Continues...




                         Cerulenin,
                       Thiolactomycin
          Campbell, J.W., Cronan, J.E.Jr., Annu.Rev.Microbiol., 2001, 55, 305
         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
Where do the antibiotics come from?




           NATURE
Approach B
Generating the Nature Mimics




      Cox, R.J., Gibson, J.S., Martín, M.B.M., Chem. Commun., 2001, 1710
      Cox, R.J., Gibson, J.S., Hadfield, A.T., ChemBioChem, 2005, 6, 2255
      Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
Approach B
Generating the Nature Mimics




     Direct assay




       Cox, R.J., Gibson, J.S., Martín, M.B.M., ChemBioChem, 2002, 3, 874
          Blanco, J., Moore, R.A., Viola, R.E., PNAS, 2003, 100, 12613
              Han, S., Moore, R.A., Viola, R.E., Synlett, 2003, 6, 845
Biosynthesis of Saturated Fatty Acids




                             ACP                                                 FabI / K / L




  FabD

                                                                                 FabZ




                            FabH                   FabG

         Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305
Biosynthesis of Saturated Fatty Acids




                                                                                 FabI / K / L

                            Continues...

  FabD



                                                                                 FabZ




                            FabF                   FabG

         Campbell, J.W., Cronan, J.E.Jr., Annu. Rev. Microbiol., 2001, 55, 305

				
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