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Antisense Approach to Target
MDR Tuberculosis
Diane Meas
Michael Nguyen
Michael DeSalvio
Michael Boateng-Antwi
Agenda
• Introduction & Objectives
• Background & Significance
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Overview of MDR TB
Impact and Importance
• Research Design & Methods
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Previous studies and findings
Mechanism to new approach
Assay Methods
• Conclusion
Introduction
• TB – Overview
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Infectious airborne disease caused by Mycobacterium tuberculosis
2009 incident cases 9.4 million
2009 prevalent cases 14 million
Mortality: - 1.8 million
Funding : $5 billion
Estimated Funding for 2011: $6 billion
(source: WHO Global TB Report, 2010)
TB – Global Distribution
Interventions
• Anti-TB drugs (www.cdc.gov/tb/publications)
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Frontline: rifampicin, isoniazid, pyrizinamide, and ethambutol
Second line: fluoroquinolones, amikacin, kanamycin, or capreomycin
• Drug Resistance: 250,000 reported (WHO-TB,
2010)
• Options for Disease control
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Development of new line of drugs
Reversal of drug resistance
• Antisense Technology
Objectives
• Design an antisense molecule against a gene
in mycobacterium.
• Develop in vitro assay to test the maximum
effect of antisense molecule in
mycobacterium
Background &
Significance
Antibiotics
Mechanism of
Action (Michel J.
Cloutier2, 1995)
• Protein Synthesis
• Folate
Metabolism
• Cell wall
Synthesis
• Cell Membrane
• DNA gyrase
• DNA-directed
RNA-polymerase
Background and Significance
Mechanisms of Antibiotic Resistance (Morris et al,
1995)
• Antibiotic modification by bacterial enzymes
• Preventing the antibiotic from entering the
cell or pumping it out (efflux) faster than it
can flow in.
• Production of an alternative target (usually
an enzyme) that is resistant to inhibition
• Alterations in the primary site of action
Background &
Significance
β-lactam antibiotics
• broad class of drugs
with β-lactam ring as
nucleus of molecular
structure
• Inhibit 4 – 8 enzymes
(PBP) engaged in cell
wall biosynthesis.
β-lactamases cleave βlactam ring in antibiotic to
make drug ineffective
1. Penicillin 2. Cephalosporin
Red Structure - β-lactam core
ring
Antisense Overview
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Made up of RNA
Generally short strands
Complementary to the mRNA strand
Intercept and bind mRNA
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Prevent Translation
No Gene Expression!
http://cdn.venturebeat.com/wp-content/uploads/2007/11/800px-antisense_dna_oligonucleotide.jpg
Antisense Treatments
• Used to treat various treatments
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Cytomegalovirus retinitis
Hemorrhagic fever viruses
Cancer (TGF-beta2)
HIV/AIDS
High cholesterol (mipobersen, 2010 ph-IV)
Proof of Principle
• Harth et.al:
Used phophorothioate-modified
oligodeoxyribonucleotides (PS-ODNs)
o targeted mycolyl transferases to inhibit
essential genes
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Proof of Principle
• Harth et.al:
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Saw a reduction in antigen 85A, 85B and 85C
 (Refered to as 32A, 30 and 32B)
 Reduction in expression also reduced bacterial
growth
 Demonstrated successfully that antisense strategy
is effective
 Successfully inhibited growth in M. tuberculosis
(human)
Proof of Principle
• Dasgupta et al:
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Knocked out Penicillin Binding Proteins (PBPA)
 serine acyl transferases involved in cell wall expansion, cell
shape maintenance, septum formation and cell division
Relied on mutation of PknB precursor proteins responsible for
the phosphorylation of the PBPA
Inactivation of PnkB results in no phosphorylation of PBPA
Cell death
Current Solutions
• Clavulanic Acid
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GlaxoSmithKline
B-lactamase inhibitor
Competitive inhibition
 Binds to active site, causing irreversible covalence
Derived from S. clavuligerus
Concurrent Administration with Amoxicillin
Current Solutions
• Adverse Effects!
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Increased Cholestatic Jaundice
Acute hepatitis
Some microbial resistance
Allergy
Midpoint Recap
• Rifampicin resistance in M. tuberculosis
• PS-ODNs and gene knockouts were
shown as effective means of bypassing
drug resistance and restore drug
sensitivity to microorganism
• Current approach can develop serious
side effects
• New Antisense approach will have
reduced side effects
Overview of PknB Proposal
• PknB prevents the synthesis of PBPA (penicillin binding
protein)
• PknB phosphorlyates b-lactamase for insertion into the
cell membrane
o No PknB means no lactamase expression
• Antisense mRNA peptide nucleotides (PNAs) bind to the
active site of PknB and prevent PknB synthesis by steric
hindrance
• Downstream effects would be the loss of B-lactamase
synthesis leading drug sensitivity
• No b-lactamase may also weaken cell wall structure
leading to cell death
Research Design & Methods
• Target other essential genes:
Target a Serine/ Threonine protein kinase
(STPK)
o PknB
o Indirectly affects synthesis of B-Lactamases
o Effectively causes bacteria to be sensitive to
B-Lactam Class antibiotics
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Gene Identification
• PknB = transmembrane serine/threonineprotein kinase B
• From M. tuberculosis H37Rv
Nucleotide Sequence
ATGACCACCCCTTCCCACCTGTCCGACCGCTACGAACTTGGCGAAATCCTTGGATTTGGGGGCATGTCCGAGGT
CCACCTGGCCCGCGACCTCCGGTTGCACCGCGACGTTGCGGTCAAGGTGCTGCGCGCTGATCTAGCCCGCGATC
CCAGTTTTTACCTTCGCTTCCGGCGTGAGGCGCAAAACGCCGCGGCATTGAACCACCCTGCAATCGTCGCGGTC
TACGACACCGGTGAAGCCGAAACGCCCGCCGGGCCATTGCCCTACATCGTCATGGAATACGTCGACGGCGTTAC
CCTGCGCGACATTGTCCACACCGAAGGGCCGATGACGCCCAAACGCGCCATCGAGGTCATCGCCGACGCCTGCC
AAGCGCTGAACTTCAGTCATCAGAACGGAATCATCCACCGTGACGTCAAGCCGGCGAACATCATGATCAGCGCG
ACCAATGCAGTAAAGGTGATGGATTTCGGCATCGCCCGCGCCATTGCCGACAGCGGCAACAGCGTGACCCAGAC
CGCAGCAGTGATCGGCACGGCGCAGTACCTGTCACCCGAACAGGCCCGGGGTGATTCCGTCGACGCCCGATCCG
ATGTCTATTCCTTGGGCTGTGTTCTTTATGAAGTCCTCACCGGGGAGCCACCTTTCACCGGCGACTCACCCGTC
TCGGTTGCCTACCAACATGTGCGCGAAGACCCGATCCCACCTTCGGCGCGGCACGAAGGCCTCTCCGCCGACCT
GGACGCCGTCGTTCTCAAGGCGCTGGCCAAAAATCCGGAAAACCGCTATCAGACAGCGGCGGAGATGCGCGCCG
ACCTGGTCCGCGTGCACAACGGTGAGCCGCCCGAGGCGCCCAAAGTGCTCACCGATGCCGAGCGGACCTCGCTG
CTGTCGTCTGCGGCCGGCAACCTTAGCGGTCCGCGCACCGATCCGCTACCACGCCAGGACTTAGACGACACCGA
CCGTGACCGCAGCATCGGTTCGGTGGGCCGTTGGGTTGCGGTGGTCGCCGTGCTCGCTGTGCTGACCGTCGTGG
TAACCATCGCCATCAACACGTTCGGCGGCATCACCCGCGACGTTCAAGTTCCCGACGTTCGGGGTCAATCCTCC
GCCGACGCCATCGCCACACTGCAAAACCGGGGCTTCAAAATCCGCACCTTGCAGAAGCCGGACTCGACAATCCC
ACCGGACCACGTTATCGGCACCGACCCGGCCGCCAACACGTCGGTGAGTGCAGGCGACGAGATCACAGTCAACG
TGTCCACCGGACCCGAGCAACGCGAAATACCCGACGTCTCCACGCTGACATACGCCGAAGCGGTCAAGAAACTG
ACTGCCGCCGGATTCGGCCGCTTCAAGCAAGCGAATTCGCCGTCCACCCCGGAACTGGTGGGCAAGGTCATCGG
GACCAACCCGCCAGCCAACCAGACGTCGGCCATCACCAATGTGGTCATCATCATCGTTGGCTCTGGTCCGGCGA
CCAAAGACATTCCCGATGTCGCGGGCCAGACCGTCGACGTGGCGCAGAAGAACCTCAACGTCTACGGCTTCACC
AAATTCAGTCAGGCCTCGGTGGACAGCCCCCGTCCCGCCGGCGAGGTGACCGGCACCAATCCACCCGCAGGCAC
CACAGTTCCGGTCGATTCAGTCATCGAACTACAGGTGTCCAAGGGCAACCAATTCGTCATGCCCGACCTATCCG
GCATGTTCTGGGTCGACGCCGAACCACGATTGCGCGCGCTGGGCTGGACCGGGATGCTCGACAAAGGGGCCGAC
GTCGACGCCGGTGGCTCCCAACACAACCGGGTCGTCTATCAAAACCCGCCGGCGGGGACCGGCGTCAACCGGGA
CGGCATCATCACGCTGAGGTTCGGCCAGTAG
Amino Acid Sequence
MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARD
PSFYLRFRREAQNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGV
TLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMIS
ATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDAR
SDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSA
DLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERT
SLLSSAAGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLT
VVVTIAINTFGGITRDVQVPDVRGQSSADAIATLQNRGFKIRTLQKPDS
TIPPDHVIGTDPAANTSVSAGDEITVNVSTGPEQREIPDVSTLTYAEAV
KKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAITNVVIIIVGS
GPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNP
PAGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLD
KGADVDAGGSQHNRVVYQNPPAGTGVNRDGIITLRFGQ
Kinase Domain
RNA Active Site w/ Domains
UACGAACUUGGCGAA
CUCCGGUUGCACCGC
AGUUUUUACCUUCGC
AUCGUCGCGGUCUAC
GUCAUGGAAUACGUC
ACGCCCAAACGCGCC
CAGAACGGAAUCAUC
GCAGUAAAGGUGAUG
ACCCAGACCGCAGCA
GAUUCCGUCGACGCC
ACCGGGGAGCCACCU
GAAGACCCGAUCCCA
GUUCUCAAGGCGCUG
GCCGACCUGGUC
AUCCUUGGAUUUGGG
GACGUUGCGGUCAAG
UUCCGGCGUGAGGCG
GACACCGGUGAAGCC
GACGGCGUUACCCUG
AUCGAGGUCAUCGCC
CACCGUGACGUCAAG
GAUUUCGGCAUCGCC
GUGAUCGGCACGGCG
CGAUCCGAUGUCUAU
UUCACCGGCGACUCA
CCUUCGGCGCGGCAC
GCCAAAAAUCCGGAA
GGCAUGUCCGAGGUC
GUGCUGCGCGCUGAU
CAAAACGCCGCGGCA
GAAACGCCCGCCGGG
CGCGACAUUGUCCAC
GACGCCUGCCAAGCG
CCGGCGAACAUCAUG
CGCGCCAUUGCCGAC
CAGUACCUGUCACCC
UCCUUGGGCUGUGUU
CCCGUCUCGGUUGCC
GAAGGCCUCUCCGCC
AACCGCUAUCAGACA
CACCUGGCCCGCGAC
CUAGCCCGCGAUCCC
UUGAACCACCCUGCA
CCAUUGCCCUACAUC
ACCGAAGGGCCGAUG
CUGAACUUCAGUCAU
AUCAGCGCGACCAAU
AGCGGCAACAGCGUG
GAACAGGCCCGGGGU
CUUUAUGAAGUCCUC
UACCAACAUGUGCGC
GACCUGGACGCCGUC
GCGGCGGAGAUGCGC
Efficiency of PNA
• PNA stands for peptide nucleic acids
• Antisense PNAs are larger than most drugs
PNA size/length is an important parameter for
efficiency
o PNAs targeted to the start codon region of the
chromosomal β-galactosidase gene (lacZ) were
synthesized over 7- to 15-mer size range
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• E. coli outer cell wall is a major barrier to
PNAs, so need to find a more efficient
technique
Concentrations of PNA
(100nM – 500nM)
Concentrations of PNA
(1mM – 5mM)
Efficiency of the KFFKFFKFFK
cap
• Also expressed as (KFF)3K
• This is a synthetic peptide and it is a cell
wall-permeating peptide
• When this cap is conjugated to PNA
oligomers, it could enhance the uptake
and efficiency of antisense PNAs
Efficacy of Cap Peptide
Peptide Nucleic Acids
• Outperforms Oligonucleotides
• 7-15 mer lengths
• Capped with KFFKFFKFFK – synthetic
molecule shown to increase PNA uptake
into cell
• PNA immune to exonuclease activity
Comparison of Nucleotides
mRNA and its Antisense PNA
5’-GACGUUGCGUCAAGGUGUCUGCGCGCUGAU-3’
3’-CUGCAACGCAGUUCGACAGACGCGCGACUA-CAP-5’
5’-CACCGUGACGUCAAGCCGGCGAACAUCAUG-3’
3’-GUGGCACUGCAGUUCGGCCGCUUGUAGUAC-CAP-5’
5’-GCAGUAAAGGUGAUGGAUUUCGGCAUCGCC-3’
3’-CGUCAUUUCCACUACCUAAAGCCGUAGCGG-CAP-5’
5’-AGCGGCAACAGCGUGACCCAGACCGCAGCA-3'
3’-UCGCCGUUGUCGCUCUGGGUCUGGCGUCGU-CAP-5’
5’-AGAUAGCGCAAUGACCACCCCUUCCCACCU-3’
3’-UCUAUCGCGUUACUGGUGGGGUUGGGUGGA-CAP-5’
Whole Cell Assay
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BioSafety Level 1
Mycobacteria smegmatis
Middlebrook 7H9 Broth Media
Middlebrook 7H10 Agar Media
β-Lactam Antibiotic Library
Assay Method
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Grow Mycobacteria for 7 days @ 35oC in 7H9
Take OD reading (A600)
Transfer culture to 96-well plates
Screen against various PNAs (going across)
Vary concentrations of PNAs (doing down)
Screen multiple B-lactam class antibiotics
HTS Method
2-Day OD readings (up to 8 weeks)
o Can change depending on growth rate
Assay Plate
A
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Buffer
PNA1
10 nM
PNA2
PNA3
PNA4
PNA5
PNA6
PNA7
PNA8
PNA9
PNA10
Clavulanic
Acid
B
1:2
C
1:4
D
1:8
E
1:16
F
1:32
G
1:64
H
0 nM
- One B-lactam antibiotic treated across entire plate
- Every well contains M. smegmatis
Antibiotics: Beta-Lactams
• Glycopeptides
– Vancomycin, Teicoplanin
• Penicillins
– Amoxicillin, Ampicillin, Azlocillin, Mecillinam
– Benzylpenicillin, Clometocillin
– *Cloxacillin, *Oxacillin, *Nafcillin (*B-lactamase resistant)
• Cephalosporins
– Cefazolin, Cefapirin, Ceftezole
– Cefamandole, Cefprozil, Cefminox
– Cefixime, Ceftrixone, Cefpimizole
– Ceftiofur
• Monobactams
– Aztreonam, Tigemonam
Expected Results
• No effect on Proliferation in Buffer wells
• Reduction in Mycobacteria growth over time
on PNA wells
• Higher concentration PNA results in lower
OD
• Clavulanic Acid shows greatest change in
growth
Future Studies
• Select Promising PNAs for additional
screening
• Screen Against other microorganisms
• Design PNAs for other essential genes or
pathways
Contingency Assays
In the event Mycobacteria does not grow in 96-well plate or
detection is poor:
Large Scale Assay
• Assay repeated using tubes of 7H9 Media (1mL)
• Smaller β-Lactam library
• Measure OD via spectroscopy
Zone of Inhibition Assay
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Use of 7H10 Agar media
Impregnate with β-Lactam
Spots of various concentration PNAs
Measure inhibition zones
Some Issues with Assay
• PNAs not very well studied
– Mode of transport and toxicity still unclear
• Not much information with in vivo assays
• Assumes Mycobacteria can be sensitized to BLactam
• Assumes β-Lactam will remain active
– Not cleaved or lysed by Lactamases
Summary
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Tuberculosis is a worldwide epidemic
Wide proliferation have created Multi-Drug Resistant Strains
First Line defense, Rifampicin, Ineffective
New Approach: Return sensitivity to B-Lactam
Inhibit Expression of PknB at mRNA level
– Prevents Phosphorylation of Penicllin Binding Proteins
– Prevents expression of PBP on Cell surface (B-Lactamases)
• Synthesize Peptide Nucleic Acids (PNAs) for specificity
• HTS Assays
– Against B-Lactam Library
Questions?