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An#bio#cs
Lark J. Perez
50S Inhibitors -‐ Chloramphenicol
Protein Synthesis Inhibitors
Nature Rev Microbiol. 2005 3(11):870-‐881
Protein Synthesis Inhibitors
Nature Rev Microbiol. 2005 3(11):870-‐881
212 Cell 139, October 2, 2009 ©2009 Elsevier Inc. DOI 10.1016/j.cell.2009.08.009
SnapShot: Antibiotic Inhibition of Protein
Synthesis II
Daniel Sohmen,1 Joerg M. Harms,2 Frank Schlünzen,3 and Daniel N. Wilson1,4
1
University of Munich, Germany; 2MPSD, University of Hamburg, Germany; 3DESY, Hamburg, Germany; 4CiPS-M, Munich, Germany
See online version for legend and references.
Part I appeared in the September 18 issue.
50S Ribosome PTC Inhibitors
Nature Rev Microbiol. 2005 3(11):870-‐881
Chloramphenicol
Thiamphenicol
Chloramphenicol
Florfenicol
Chloramphenicol – Mechanism of Ac#on
Chloramphenicol – Mechanism of Ac#on
Chloramphenicol
Chloramphenicol – Mechanism of Ac#on
Chloramphenicol
Nature 2001 413, 814-‐821
Chloramphenicol – Mechanism of Ac#on
YouTube: h=ps://www.youtube.com/watch?v=0VINqUF-‐r5I
Chloramphenicol – Acquired Resistance
1) Reduced membrane permeability (wide-‐spread and generally low level resistance)
2) Binding site mutaTon (rare, not clinically significant)
3) Drug inacTvaTon (significant or complete drug resistance mediated by
chloramphenicol acetyltransferase)
Chloramphenicol – Acquired Resistance
1) Reduced membrane permeability (wide-‐spread and generally low level resistance)
2) Binding site mutaTon (rare, not clinically significant)
3) Drug inacTvaTon (significant or complete drug resistance mediated by
chloramphenicol acetyltransferase)
CH 3
OH OH
Cl
HN
O2N
OH O
Chloramphenicol
Acetyltransferase
Cl
Cl
O
O
S
CoA
HS
O
CoA
HN
O2N
Cl
non-enzymatic
O
HO CH 3
O
O
Cl
O
H 3C
O
O
O
O
CH 3
Cl
O2N
HN
Cl
O
H 3C
Chloramphenicol
Acetyltransferase
HS
O2N
S
O
CoA
HN
HN
non-enzymatic
Cl
O
Cl
O
OH
Cl
O
CoA
O2N
An#bio#cs
Lark J. Perez
50S Inhibitors -‐ Macrolides
Macrolides – Introduc#on
Macrolides -‐ Classes
Universally macrocyclic lactones, typically more effecTve against gram posiTve bacteria.
Erythromycin (14-‐member ring)
Azithromycin (15-‐member ring)
Clarithromycin (14-‐member ring)
Tylosin (16-‐member ring)
Macrolides – Mechanism of Ac#on
Macrolides – Mechanism of Ac#on
Macrolides – Mechanism of Ac#on
YouTube: h=ps://www.youtube.com/watch?v=oC21vLFtsjo
Macrolides – Mechanism of Ac#on
Molecular Cell, 2002,10 (1), 117–128.
Macrolides – Mechanism of Ac#on
Macrolides – Mechanism of Ac#on
Proceedings of the Na;onal Academy of Sciences, USA 2014, 111 (27), 9804-‐9809.
Macrolides – Mechanism of Ac#on
Macrolides – Mechanism of Ac#on Details
Cell, 2012, 151, 469-471.
Molecular Cell, 2002,10 (1), 117–128.
Macrolides – Resistance
Gram-‐negaTve inherently have resistance (low drug permeability), clinically significant
resistance largely from altered drug binding site.
Molecular Cell, 2002,10 (1), 117–128.
50S Inhibitors -‐ Recent Innova#ons
Erythromycin red
Nacent pepTde chain blue
PDB 3OFO-‐R
Chloroamphenicol (red, 2 sites)
Erythromycin overlaid in yellow
PDB’s 3OFA-‐D, 1NJI and 3OFO-‐R
Erythromycin-‐Chloramphenicol Conjugate
Inhibitors of Nucleic Acid Processes
and Metabolism:
DNA/RNA Metabolism Background
caputo@rowan.edu
DNA Synthesis Inhibitors
• Prevent the replicaEon of DNA, thereby
blocking cell division/replicaEon
Nucleic Acid Inhibitors
• Prevent the synthesis of DNA nucleoEdes,
prevenEng DNA replicaEon
RNA Synthesis Inhibitors
• Block RNA polymerases (transcripEon of
genes)
• Four classes of RNAPol inhibitors
– rifamycins -‐ blocks RNA extension
– sorangicin -‐ blocks RNA extension
– streptolydigin -‐ blocks RNAPol catalyEc recycling
– myxopyronin. -‐ Blocks RNAPol interacEon with
DNA
Rifamycin
Streptolydigin
myxopyronin
DNA Background
• Source of geneEc informaEon
• “long term” storage of informaEon in genes
• DNA is a biopolymer of nucleoEdes
DNA Synthesis -‐ Background
• Required for duplicaEon of cells
• Need to pass on a copy of DNA to each
“daughter” cell
• MulEple proteins involved in duplicaEon
process
• Bind DNA
• Unwind DNA
• Separate DNA
• Prime DNA
• Synthesize DNA
Fig. 7-10
Semiconservative
replication
Parental strand
New strand
Table 7-2
Table 7-2
Fig. 7-13
RNA primer
Lagging strand
Primase
Single-strand
binding protein
Helicase
Free 3ʹ′-OH
DNA polymerase III
Leading strand
RNA primer
Fig. 7-17
Origin (DnaA
binding site)
Replication
forks
Lagging
Leading
Leading
Lagging
Direction
Direction
Origin
Replication
fork
Table 7-2
Fig. 7-15
DNA polymerase III
3ʹ′-OH
RNA primer
5ʹ′-P
DNA polymerase I
3ʹ′-OH 5ʹ′-P
DNA ligase
Table 7-2
Fig. 7-16
Origin of
replication
Replication
forks
Newly
synthesized
DNA
Theta
structure
Fig. 7-19
Newly synthesized strand
DNA polymerase III
RNA primer
DNA helicase
Leading strand template
DNA gyrase
Tau
Parental DNA
RNA primer
DNA polymerase III
DNA primase
Newly synthesized strand
Lagging
strand
template
Single-strand
DNA-binding
proteins
Table 7-2
Fluoroquinolones
RNA Synthesis
• Varies significantly from eukaryoEc cells
• Consists of two subunits
– RNA Polymerase (RNA extension)
– Sigma factor (promoter binding)
• The complete funcEonal complex consists of 6
proteins: β, β’, αΙ, αΙΙ, ω,σ
• Sigma factors vary based on which promoter
the polymerase is binding to
Fig. 7-21
RNA polymerase
(core enzyme)
Promoter region
Sigma factor
Gene(s) to be transcribed
(light green strand)
Sigma recognizes
promoter and
initiation site
Sigma
RNA
Transcription begins; sigma
released. RNA chain growth
continues to termination site
Termination site
reached; chain
growth stops
Release of
polymerase
and RNA
DNA
Short transcripts
Longer transcripts
Fig. 7-22
RNA polymerase
(core enzyme)
Transcription
Sigma
mRNA start
1.
2.
3.
4.
5.
6.
–35 sequence
Pribnow box
Consensus
Promoter sequence
Table 7-3
Fig. 9-7
arg Promoter arg Operator
argC
RNA
polymerase
argB
argH
Transcription proceeds
Repressor
arg Promoter arg Operator
RNA
polymerase
argC
argB
argH
Corepressor
Transcription blocked
(arginine)
Repressor
Fig. 9-8
lac Promoter lac Operator
lacZ
RNA
polymerase
lacY
lacA
Transcription proceeds
Repressor
lac Promoter lac Operator
lacZ
RNA
polymerase
lacY
lacA
Transcription proceeds
Repressor
Inducer
Fig. 9-9
Activator
binding site mal Promoter
malE
malF
malG
No transcription
RNA
polymerase
Maltose activator protein
Activator
binding site mal Promoter
malE
RNA
polymerase
Maltose activator protein
Inducer
malF
malG
Transcription proceeds
Fig. 9-10
DNA
Protein
Fig. 9-11
Activator
binding site
Promoter
RNA
polymerase Transcription
proceeds
Activator protein
Promoter
Activator protein
RNA
polymerase Transcription
proceeds
Activator
binding site
Differences
• There are significant differences between
prokaryoEc and eukaryoEc polymerases
• ProkaryoEc transcripts contain mulEple gene
products
• EukaryoEc polymerases are more complex
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
h^p://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of AnEmicrobial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
h^p://www.microbiologybook.org/mayer/anEbiot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7
2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
h^p://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/NucleoEdeBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
Brock – biology of microorganisms
PMID: 19926275
PMID: 11313498
Inhibitors of Nucleic Acid Processes
and Metabolism:
DNA Synthesis Inhibitors
caputo@rowan.edu
DNA Synthesis
• Replica@on of DNA is required for cell division
• Provides necessary gene@c informa@on for all
cellular processes
• Cells cannot survive without in-‐tact DNA
DNA Synthesis Inhibitors
• Prevent the replica@on of DNA, thereby
blocking cell division/replica@on
Table 7-2
Fluoroquinolones
Fluoroquinolones Nalidixic Acid
• Original molecule, nalidixic acid,
isolated as a side product from
the synthesis of chloroquine in
the 1960s
• Nalidixic acid is primarily
bactericidal
• Later deriva@ves include the
fluorinated core known as
fluoroquinolone
DRUG
CAS REGISTRY
NO
MOLECULAR FORMULA STRUCTURE
Ciprofloxacin HCl
86393-‐32-‐0
C17-‐H18-‐F-‐N3-‐O3.Cl-‐
H.H2-‐O
Gemifloxacin
175463-‐14-‐6
C18-‐H20-‐F-‐N5-‐O4
Levofloxacin
100986-‐85-‐4
C18-‐H20-‐F-‐N3-‐O4
Moxifloxacin HCl
186826-‐86-‐8
C21-‐H24-‐F-‐N3-‐O4.Cl-‐H
Norfloxacin
70458-‐96-‐7
C16-‐H18-‐F-‐N3-‐O3
Ofloxacin
82419-‐36-‐1
C18-‐H20-‐F-‐N3-‐O4
Synthesis
Solid-‐Phase Synthesis
Structural Aspects
Mechanism of Ac@on
• Act by disrup@ng DNA synthesis
• Targets are DNA gyrase and Topoisomerases
• Disrupt the progression of the replica@on fork
• Ac@on can be bactericidal or bacteriosta@c,
depending on concentra@on
Resistance
• Two iden@fied mechanisms of resistance
• Muta@ons in parC and gyrA reduce efficacy
• Muta@ons affec@ng accumula@on of
fluoroquinolones have also been iden@fied
(mul@drug efflux pumps)
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
hap://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of An@microbial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
hap://www.microbiologybook.org/mayer/an@biot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7
2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
hap://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/Nucleo@deBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
PMC3250697 -‐ Can J Infect Dis. 1999 May-‐Jun; 10(3): 207–238.
Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S113-‐9.
J. An#microb. Chemother. (2000) 46 (suppl 3): 17-‐24.
www.intechopen.com/download/pdf/38653
doi:10.1016/S0960-‐894X(99)00326-‐1
Inhibitors of Nucleic Acid Processes
and Metabolism:
RNA Synthesis Inhibitors
caputo@rowan.edu
RNA Synthesis
RNA Synthesis Inhibitors
• Block RNA polymerases (transcripBon of
genes)
• Four classes of RNAPol inhibitors
– rifamycins -‐ blocks RNA extension
– sorangicin -‐ blocks RNA extension
– streptolydigin -‐ blocks RNAPol catalyBc recycling
– myxopyronin. -‐ Blocks RNAPol interacBon with
DNA
Rifamycin
Streptolydigin
myxopyronin
Rifamycin
• Originally isolated from the bacterium
Amycolatopsis rifamycinica
BiosyntheBc Pathway
• MulBple genes involved in synthesis
• 10 step synthesis to create AHBA, a rifampin
precursor
Mechanism of AcBon
• Rif binds to the RNAPol β subunit
• Binds near the DNA/RNA interface
• Does NOT directly bind to the RNAPol acBve
site
Complex of RNAPol and Rif
Three-‐dimensional structure of Taq
core RNAP in complex with Rif,
generated using GRASP (Nicholls et al.,
1991). The backbone of the RNAP
structure is shown as tubes, along with
the color coded transparent molecular
surface (β, cyan; β’, pink; ω, white; the
α subunits are behind the RNAP and
are not visible). The Mg2+ ion chelated
at the acBve site is shown as a
magenta sphere. The Rif is shown as
CPK atoms (carbon, orange; oxygen,
red; nitrogen, blue).
Cell. 2001 Mar 23;104(6):901-‐12
Binding Site InteracBons
• SchemaBc drawing of RNAP β
subunit interacBons with Rif,
modified from LIGPLOT (Wallace et
al., 1995).
• Residues forming van der Waals
interacBons are indicated, those
parBcipaBng in hydrogen bonds
are shown in a ball-‐and-‐sBck
representaBon, with hydrogen
bonds depicted as dashed lines.
• Carbon atoms of the protein are
black, while carbon atoms of Rif
are orange. Oxygen atoms are red
and nitrogen atoms are blue
Cell. 2001 Mar 23;104(6):901-‐12
Mechanism of InhibiBon
• Rif does not affect nt +1
(green) or -‐1
• Phosphates of -‐2 clashes with
Rif
• Nt -‐3 -‐ -‐5 have mulBple steric
clashes with Rif
• “Rif sterically blocks the path
of the elongaBng RNA
transcript at the 5ʹ′ end, and
indicates that the blockage is
a direct consequence of Rif
binding in its site”
Cell. 2001 Mar 23;104(6):901-‐12
Resistance
• High rate of resistance development
• As a result, rif is ofen used in combinatorial
treatments and only against certain types of
infecBons
• Most resistance occurs in rpoB, β-‐subunit of
RNA polymerase
Streptolydigin
• Streptolydigin: polykeBde-‐derived tetramic-‐
acid
• Originally isolated from Streptomyces
griseoflavus
Biosynthesis
Chemical Synthesis
Mechanism of AcBon
• Streptolydigin inhibits nucleoBde addiBon in transcripBon iniBaBon, nucleoBde addiBon in
transcripBon elongaBon, and pyrophosphorolysis
• Tetramic-‐acid contacts the bridge helix and trigger loop; the streptolol moiety contacts the
bridge helix
• Interferes with structural cycling
Myxopyronin
• PolykeBde-‐derived α-‐pyrone
• Isolated from Myxobacterium Myxococcus
fulvus
Biosynthesis
• Less well understood
• Linked to synthesis of corallopyronin A
Mechanism of AcBon
• Myx interacts with the RNAP “switch region” :
the hinge that mediates opening and closing
of the RNAP “clamp“
• This inhibits opening and closing of the acBve
site and unwinding of DNA for progression
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
hkp://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of AnBmicrobial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
hkp://www.microbiologybook.org/mayer/anBbiot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 AnFmicrob. Agents Chemother. July 2004 vol. 48 no. 7 2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
hkp://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/NucleoBdeBiochemistry.html
AnFmicrob. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
PMC3250697 -‐ Can J Infect Dis. 1999 May-‐Jun; 10(3): 207–238.
Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S113-‐9.
J. AnFmicrob. Chemother. (2000) 46 (suppl 3): 17-‐24.
www.intechopen.com/download/pdf/38653
doi:10.1016/S0960-‐894X(99)00326-‐1
hRp://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/central_dogma.html
Chem. Rev., 2005, 105 (2), pp 621–632 DOI: 10.1021/cr030112j
Reviews of InfecFous Diseases
Vol. 5, Supplement 3. The Use of Rifampin in the Treatment of Nontuberculous InfecBons (Jul. -‐ Aug., 1983), pp. S402-‐S406
Cell. 2001 Mar 23;104(6):901-‐12.
doi:10.1016/j.chembiol.2009.09.015
J. Am. Chem. Soc., 2010, 132 (41), pp 14394–14396 DOI: 10.1021/ja107190w
Curr Opin Struct Biol. 2009 Dec; 19(6): 715–723.
Chembiochem. 2013 Sep 2;14(13):1581-‐9. doi: 10.1002/cbic.201300289. Epub 2013 Aug 26.
Inhibitors of Nucleic Acid
Processes and Metabolism
caputo@rowan.edu
DNA Synthesis Inhibitors
• Prevent the replica?on of DNA, thereby
blocking cell division/replica?on
Nucleic Acid Inhibitors
• Prevent the synthesis of DNA nucleo?des,
preven?ng DNA replica?on
RNA Synthesis Inhibitors
• Block RNA polymerases (transcrip?on of
genes)
• Four classes of RNAPol inhibitors
– rifamycins -‐ blocks RNA extension
– sorangicin -‐ blocks RNA extension
– streptolydigin -‐ blocks RNAPol cataly?c recycling
– myxopyronin. -‐ Blocks RNAPol interac?on with
DNA
Rifamycin
Streptolydigin
myxopyronin
DNA Background
• Source of gene?c informa?on
• “long term” storage of informa?on in genes
• DNA is a biopolymer of nucleo?des
DNA Synthesis -‐ Background
• Required for duplica?on of cells
• Need to pass on a copy of DNA to each
“daughter” cell
• Mul?ple proteins involved in duplica?on
process
Fig. 7-10
Semiconservative
replication
Parental strand
New strand
Table 7-2
Table 7-2
Fig. 7-13
RNA primer
Lagging strand
Primase
Single-strand
binding protein
Helicase
Free 3ʹ′-OH
DNA polymerase III
Leading strand
RNA primer
Fig. 7-17
Origin (DnaA
binding site)
Replication
forks
Lagging
Leading
Leading
Lagging
Direction
Direction
Origin
Replication
fork
Table 7-2
Fig. 7-15
DNA polymerase III
3ʹ′-OH
RNA primer
5ʹ′-P
DNA polymerase I
3ʹ′-OH 5ʹ′-P
DNA ligase
Table 7-2
Fig. 7-16
Origin of
replication
Replication
forks
Newly
synthesized
DNA
Theta
structure
Fig. 7-19
Newly synthesized strand
DNA polymerase III
RNA primer
DNA helicase
Leading strand template
DNA gyrase
Tau
Parental DNA
RNA primer
DNA polymerase III
DNA primase
Newly synthesized strand
Lagging
strand
template
Single-strand
DNA-binding
proteins
Table 7-2
Fluoroquinolones
RNA Synthesis
• Varies significantly from eukaryo?c cells
• Consists of two subunits
– RNA Polymerase (RNA extension)
– Sigma factor (promoter binding)
• The complete func?onal complex consists of 6
proteins: β, β’, αΙ, αΙΙ, ω,σ
• Sigma factors vary based on which promoter
the polymerase is binding to
Fig. 7-21
RNA polymerase
(core enzyme)
Promoter region
Sigma factor
Gene(s) to be transcribed
(light green strand)
Sigma recognizes
promoter and
initiation site
Sigma
RNA
Transcription begins; sigma
released. RNA chain growth
continues to termination site
Termination site
reached; chain
growth stops
Release of
polymerase
and RNA
DNA
Short transcripts
Longer transcripts
Fig. 7-22
RNA polymerase
(core enzyme)
Transcription
Sigma
mRNA start
1.
2.
3.
4.
5.
6.
–35 sequence
Pribnow box
Consensus
Promoter sequence
Table 7-3
Fig. 9-7
arg Promoter arg Operator
argC
RNA
polymerase
argB
argH
Transcription proceeds
Repressor
arg Promoter arg Operator
RNA
polymerase
argC
argB
argH
Corepressor
Transcription blocked
(arginine)
Repressor
Fig. 9-8
lac Promoter lac Operator
lacZ
RNA
polymerase
lacY
lacA
Transcription proceeds
Repressor
lac Promoter lac Operator
lacZ
RNA
polymerase
lacY
lacA
Transcription proceeds
Repressor
Inducer
Fig. 9-9
Activator
binding site mal Promoter
malE
malF
malG
No transcription
RNA
polymerase
Maltose activator protein
Activator
binding site mal Promoter
malE
RNA
polymerase
Maltose activator protein
Inducer
malF
malG
Transcription proceeds
Fig. 9-10
DNA
Protein
Fig. 9-11
Activator
binding site
Promoter
RNA
polymerase Transcription
proceeds
Activator protein
Promoter
Activator protein
RNA
polymerase Transcription
proceeds
Activator
binding site
Differences
• There are significant differences between
prokaryo?c and eukaryo?c polymerases
• Prokaryo?c transcripts contain mul?ple gene
products
• Eukaryo?c polymerases are more complex
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
h\p://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of An?microbial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
h\p://www.microbiologybook.org/mayer/an?biot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7
2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
h\p://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/Nucleo?deBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
Brock – biology of microorganisms
PMID: 19926275
PMID: 11313498
Inhibitors of Nucleic Acid Processes
and Metabolism:
DNA Synthesis Inhibitors
caputo@rowan.edu
DNA Synthesis
• Replica?on of DNA is required for cell division
• Provides necessary gene?c informa?on for all
cellular processes
• Cells cannot survive without in-‐tact DNA
DNA Synthesis Inhibitors
• Prevent the replica?on of DNA, thereby
blocking cell division/replica?on
Table 7-2
Fluoroquinolones
Fluoroquinolones Nalidixic Acid
• Original molecule, nalidixic acid,
isolated as a side product from
the synthesis of chloroquine in
the 1960s
• Nalidixic acid is primarily
bactericidal
• Later deriva?ves include the
fluorinated core known as
fluoroquinolone
DRUG
CAS REGISTRY
NO
MOLECULAR FORMULA STRUCTURE
Ciprofloxacin HCl
86393-‐32-‐0
C17-‐H18-‐F-‐N3-‐O3.Cl-‐
H.H2-‐O
Gemifloxacin
175463-‐14-‐6
C18-‐H20-‐F-‐N5-‐O4
Levofloxacin
100986-‐85-‐4
C18-‐H20-‐F-‐N3-‐O4
Moxifloxacin HCl
186826-‐86-‐8
C21-‐H24-‐F-‐N3-‐O4.Cl-‐H
Norfloxacin
70458-‐96-‐7
C16-‐H18-‐F-‐N3-‐O3
Ofloxacin
82419-‐36-‐1
C18-‐H20-‐F-‐N3-‐O4
Synthesis
Solid-‐Phase Synthesis
Structural Aspects
Mechanism of Ac?on
• Act by disrup?ng DNA synthesis
• Targets are DNA gyrase and Topoisomerases
• Disrupt the progression of the replica?on fork
• Ac?on can be bactericidal or bacteriosta?c,
depending on concentra?on
Resistance
• Two iden?fied mechanisms of resistance
• Muta?ons in parC and gyrA reduce efficacy
• Muta?ons affec?ng accumula?on of
fluoroquinolones have also been iden?fied
(mul?drug efflux pumps)
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
h\p://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of An?microbial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
h\p://www.microbiologybook.org/mayer/an?biot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7
2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
h\p://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/Nucleo?deBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
PMC3250697 -‐ Can J Infect Dis. 1999 May-‐Jun; 10(3): 207–238.
Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S113-‐9.
J. An#microb. Chemother. (2000) 46 (suppl 3): 17-‐24.
www.intechopen.com/download/pdf/38653
doi:10.1016/S0960-‐894X(99)00326-‐1
Inhibitors of Nucleic Acid Processes
and Metabolism:
RNA Synthesis Inhibitors
caputo@rowan.edu
RNA Synthesis
RNA Synthesis Inhibitors
• Block RNA polymerases (transcrip?on of
genes)
• Four classes of RNAPol inhibitors
– rifamycins -‐ blocks RNA extension
– sorangicin -‐ blocks RNA extension
– streptolydigin -‐ blocks RNAPol cataly?c recycling
– myxopyronin. -‐ Blocks RNAPol interac?on with
DNA
Rifamycin
Streptolydigin
myxopyronin
Rifamycin
• Originally isolated from the bacterium
Amycolatopsis rifamycinica
Biosynthe?c Pathway
• Mul?ple genes involved in synthesis
• 10 step synthesis to create AHBA, a rifampin
precursor
Mechanism of Ac?on
• Rif binds to the RNAPol β subunit
• Binds near the DNA/RNA interface
• Does NOT directly bind to the RNAPol ac?ve
site
Complex of RNAPol and Rif
Three-‐dimensional structure of Taq
core RNAP in complex with Rif,
generated using GRASP (Nicholls et al.,
1991). The backbone of the RNAP
structure is shown as tubes, along with
the color coded transparent molecular
surface (β, cyan; β’, pink; ω, white; the
α subunits are behind the RNAP and
are not visible). The Mg2+ ion chelated
at the ac?ve site is shown as a
magenta sphere. The Rif is shown as
CPK atoms (carbon, orange; oxygen,
red; nitrogen, blue).
Binding Site Interac?ons
• Schema?c drawing of RNAP β
subunit interac?ons with Rif,
modified from LIGPLOT (Wallace et
al., 1995).
• Residues forming van der Waals
interac?ons are indicated, those
par?cipa?ng in hydrogen bonds
are shown in a ball-‐and-‐s?ck
representa?on, with hydrogen
bonds depicted as dashed lines.
• Carbon atoms of the protein are
black, while carbon atoms of Rif
are orange. Oxygen atoms are red
and nitrogen atoms are blue
Mechanism of Inhibi?on
• Rif does not affect nt +1
(green) or -‐1
• Phosphates of -‐2 clashes with
Rif
• Nt -‐3 -‐ -‐5 have mul?ple steric
clashes with Rif
• “Rif sterically blocks the path
of the elonga?ng RNA
transcript at the 5ʹ′ end, and
indicates that the blockage is
a direct consequence of Rif
binding in its site”
Resistance
• High rate of resistance development
• As a result, rif is oxen used in combinatorial
treatments and only against certain types of
infec?ons
• Most resistance occurs in rpoB, β-‐subunit of
RNA polymerase
Streptolydigin
• Streptolydigin: polyke?de-‐derived tetramic-‐
acid a
• Originally isolated from Streptomyces
griseoflavus
Biosynthesis
Chemical Synthesis
Mechanism of Ac?on
• Streptolydigin inhibits nucleo?de addi?on in transcrip?on ini?a?on, nucleo?de addi?on in
transcrip?on elonga?on, and pyrophosphorolysis
• Tetramic-‐acid contacts the bridge helix and trigger loop; the streptolol moiety contacts the
bridge helix
• Interferes with structural cycling
Myxopyronin
• polyke?de-‐derived α-‐pyrone
• Isolated from Myxobacterium Myxococcus
fulvus
Biosynthesis
• Less well understood
• Linked to synthesis of corallopyronin A
Mechanism of Ac?on
• Myx interacts with the RNAP “switch region” :
the hinge that mediates opening and closing
of the RNAP “clamp“
• This inhibits opening and closing of the ac?ve
site and unwinding of DNA
References
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•
•
•
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•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
h\p://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of An?microbial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
h\p://www.microbiologybook.org/mayer/an?biot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7 2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
h\p://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/Nucleo?deBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
PMC3250697 -‐ Can J Infect Dis. 1999 May-‐Jun; 10(3): 207–238.
Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S113-‐9.
J. An#microb. Chemother. (2000) 46 (suppl 3): 17-‐24.
www.intechopen.com/download/pdf/38653
doi:10.1016/S0960-‐894X(99)00326-‐1
hTp://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/central_dogma.html
Chem. Rev., 2005, 105 (2), pp 621–632 DOI: 10.1021/cr030112j
Reviews of Infec#ous Diseases
Vol. 5, Supplement 3. The Use of Rifampin in the Treatment of Nontuberculous Infec?ons (Jul. -‐ Aug., 1983), pp. S402-‐S406
Cell. 2001 Mar 23;104(6):901-‐12.
doi:10.1016/j.chembiol.2009.09.015
J. Am. Chem. Soc., 2010, 132 (41), pp 14394–14396 DOI: 10.1021/ja107190w
Curr Opin Struct Biol. 2009 Dec; 19(6): 715–723.
Chembiochem. 2013 Sep 2;14(13):1581-‐9. doi: 10.1002/cbic.201300289. Epub 2013 Aug 26.
dx.doi.org/10.1021/ja405949a | J. Am. Chem. Soc. 2013, 135, 10638−10641
Inhibitors of Nucleic Acid
Processes and Metabolism
caputo@rowan.edu
Nucleic Acid Inhibitors
• Prevent the synthesis of DNA nucleo?des,
preven?ng DNA replica?on
Trimethoprim/sulfamethoxazole
• Inhibitors of folate biosynthesis
• Each individual component is
bacteriosta?c, in combina?on act
as a bactericide.
• Sulfamethoxazole acts as a
compe?tor in the synthesis of
folate/folic acid precursors.
• Trimethoprim inhibits DHFR, which
converts folic acid into
tetrahydrofolate
Sulfamethoxazole
• Class of “sulfonamide” drugs
• Originally isolated from coal tar dyes.
• Originally referred to as “sulfa” or “sulpha”
drugs
• Had great success in early/mid 1900’s
Sulfonamides -‐ Synthesis
Sulfonamides -‐ Synthesis
Sulfamethoxazole inhibits
dihydropteroate synthetase
Sulfamethoxazole inhibits
dihydropteroate synthetase
Trimethoprim inhibits
Dihydrofolate Reductase
• DHFR reduces dihydrofolic acid into
tetrahydrofolic acid
Synthesis of Trimethoprin
Trimethoprim inhibits
Dihydrofolate Reductase
Trimethoprim inhibits
Dihydrofolate Reductase
References
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
PMID: 15718136
h\p://www.ncbi.nlm.nih.gov/books/NBK21261/
Journal of An?microbial Chemotherapy (2005) 55, 518–522 doi:10.1093/jac/dki030
DOI: 10.1039/B418460B (Paper) Analyst, 2005, 130, 1032-‐1037
h\p://www.microbiologybook.org/mayer/an?biot.htm
doi: 10.1128/AAC.48.7.2610-‐2616.2004 An#microb. Agents Chemother. July 2004 vol. 48 no. 7 2610-‐2616
Rcsb pdbid 3TZF
DOI:10.2210/pdb2w9h/pdb
Rcsb pdbid 2w9h
h\p://www.d.umn.edu/~jfitzake/Lectures/DMED/FolateB12/Nucleo?deBiochemistry.html
An#microb. Agents Chemother. March 2004 vol. 48 no. 3 799-‐803
Appl. Environ. Microbiol. September 2013 vol. 79 no. 18 5550-‐5558
PMC3250697 -‐ Can J Infect Dis. 1999 May-‐Jun; 10(3): 207–238.
Clin Infect Dis. 2005 Jul 15;41 Suppl 2:S113-‐9.
J. An#microb. Chemother. (2000) 46 (suppl 3): 17-‐24.
www.intechopen.com/download/pdf/38653
doi:10.1016/S0960-‐894X(99)00326-‐1
hTp://www.ncbi.nlm.nih.gov/Class/MLACourse/Modules/MolBioReview/central_dogma.html
Chem. Rev., 2005, 105 (2), pp 621–632 DOI: 10.1021/cr030112j
Reviews of Infec#ous Diseases
Vol. 5, Supplement 3. The Use of Rifampin in the Treatment of Nontuberculous Infec?ons (Jul. -‐ Aug., 1983), pp. S402-‐S406
Cell. 2001 Mar 23;104(6):901-‐12.
doi:10.1016/j.chembiol.2009.09.015
J. Am. Chem. Soc., 2010, 132 (41), pp 14394–14396 DOI: 10.1021/ja107190w
Curr Opin Struct Biol. 2009 Dec; 19(6): 715–723.
Chembiochem. 2013 Sep 2;14(13):1581-‐9. doi: 10.1002/cbic.201300289. Epub 2013 Aug 26.
dx.doi.org/10.1021/ja405949a | J. Am. Chem. Soc. 2013, 135, 10638−10641
