1. Antisense Approach to Target MDR Tuberculosis
Diane Meas
Michael Nguyen
Michael DeSalvio
Michael Boateng-Antwi

2. Agenda Introduction & Objectives Background & Significance
Overview of MDR TB
Impact and Importance
Research Design & Methods
Previous studies and findings
Mechanism to new approach
Assay Methods
Conclusion

3. Introduction TB – Overview
Infectious airborne disease caused by Mycobacterium tuberculosis
2009 incident cases 9.4 million
2009 prevalent cases 14 million
Mortality: million
Funding : $5 billion
Estimated Funding for 2011: $6 billion
(source: WHO Global TB Report, 2010)

4. TB – Global Distribution

5. Interventions Anti-TB drugs (www.cdc.gov/tb/publications)
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
Development of new line of drugs
Reversal of drug resistance
Antisense Technology

6. Objectives
Design an antisense molecule against a gene in mycobacterium.
Develop in vitro assay to test the maximum effect of antisense molecule in mycobacterium

7. 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

8. 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

9. Background & Significance
Penicillin 2. Cephalosporin
Red Structure - β-lactam core ring
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 β-lactam ring in antibiotic to make drug ineffective

10. Antisense Overview Made up of RNA Generally short strands
Complementary to the mRNA strand
Intercept and bind mRNA
Prevent Translation
No Gene Expression!

11. Antisense Treatments Used to treat various treatments
Cytomegalovirus retinitis
Hemorrhagic fever viruses
Cancer (TGF-beta2)
HIV/AIDS
High cholesterol (mipobersen, 2010 ph-IV)

12. Proof of Principle Harth et.al:
Used phophorothioate-modified oligodeoxyribonucleotides (PS-ODNs)
targeted mycolyl transferases to inhibit essential genes

13. Proof of Principle Harth et.al:
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)

14. Proof of Principle Dasgupta et al:
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

15. Current Solutions Clavulanic Acid GlaxoSmithKline
B-lactamase inhibitor
Competitive inhibition
Binds to active site, causing irreversible covalence
Derived from S. clavuligerus
Concurrent Administration with Amoxicillin
streptomyces clavuligerus

16. Current Solutions Adverse Effects! Increased Cholestatic Jaundice
Acute hepatitis
Some microbial resistance
Allergy

17. 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

18. Overview of PknB Proposal
PknB prevents the synthesis of PBPA (penicillin binding protein)
PknB phosphorlyates b-lactamase for insertion into the cell membrane
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

19. Research Design & Methods
Target other essential genes:
Target a Serine/ Threonine protein kinase (STPK)
PknB
Indirectly affects synthesis of B-Lactamases
Effectively causes bacteria to be sensitive to B-Lactam Class antibiotics

20. Gene Identification
PknB = transmembrane serine/threonine-protein kinase B
From M. tuberculosis H37Rv

21. Nucleotide Sequence
ATGACCACCCCTTCCCACCTGTCCGACCGCTACGAACTTGGCGAAATCCTTGGATTTGGGGGCATGTCCGAGGTCCACCTGGCCCGCGACCTCCGGTTGCACCGCGACGTTGCGGTCAAGGTGCTGCGCGCTGATCTAGCCCGCGATCCCAGTTTTTACCTTCGCTTCCGGCGTGAGGCGCAAAACGCCGCGGCATTGAACCACCCTGCAATCGTCGCGGTCTACGACACCGGTGAAGCCGAAACGCCCGCCGGGCCATTGCCCTACATCGTCATGGAATACGTCGACGGCGTTACCCTGCGCGACATTGTCCACACCGAAGGGCCGATGACGCCCAAACGCGCCATCGAGGTCATCGCCGACGCCTGCCAAGCGCTGAACTTCAGTCATCAGAACGGAATCATCCACCGTGACGTCAAGCCGGCGAACATCATGATCAGCGCGACCAATGCAGTAAAGGTGATGGATTTCGGCATCGCCCGCGCCATTGCCGACAGCGGCAACAGCGTGACCCAGACCGCAGCAGTGATCGGCACGGCGCAGTACCTGTCACCCGAACAGGCCCGGGGTGATTCCGTCGACGCCCGATCCGATGTCTATTCCTTGGGCTGTGTTCTTTATGAAGTCCTCACCGGGGAGCCACCTTTCACCGGCGACTCACCCGTCTCGGTTGCCTACCAACATGTGCGCGAAGACCCGATCCCACCTTCGGCGCGGCACGAAGGCCTCTCCGCCGACCTGGACGCCGTCGTTCTCAAGGCGCTGGCCAAAAATCCGGAAAACCGCTATCAGACAGCGGCGGAGATGCGCGCCGACCTGGTCCGCGTGCACAACGGTGAGCCGCCCGAGGCGCCCAAAGTGCTCACCGATGCCGAGCGGACCTCGCTGCTGTCGTCTGCGGCCGGCAACCTTAGCGGTCCGCGCACCGATCCGCTACCACGCCAGGACTTAGACGACACCGACCGTGACCGCAGCATCGGTTCGGTGGGCCGTTGGGTTGCGGTGGTCGCCGTGCTCGCTGTGCTGACCGTCGTGGTAACCATCGCCATCAACACGTTCGGCGGCATCACCCGCGACGTTCAAGTTCCCGACGTTCGGGGTCAATCCTCCGCCGACGCCATCGCCACACTGCAAAACCGGGGCTTCAAAATCCGCACCTTGCAGAAGCCGGACTCGACAATCCCACCGGACCACGTTATCGGCACCGACCCGGCCGCCAACACGTCGGTGAGTGCAGGCGACGAGATCACAGTCAACGTGTCCACCGGACCCGAGCAACGCGAAATACCCGACGTCTCCACGCTGACATACGCCGAAGCGGTCAAGAAACTGACTGCCGCCGGATTCGGCCGCTTCAAGCAAGCGAATTCGCCGTCCACCCCGGAACTGGTGGGCAAGGTCATCGGGACCAACCCGCCAGCCAACCAGACGTCGGCCATCACCAATGTGGTCATCATCATCGTTGGCTCTGGTCCGGCGACCAAAGACATTCCCGATGTCGCGGGCCAGACCGTCGACGTGGCGCAGAAGAACCTCAACGTCTACGGCTTCACCAAATTCAGTCAGGCCTCGGTGGACAGCCCCCGTCCCGCCGGCGAGGTGACCGGCACCAATCCACCCGCAGGCACCACAGTTCCGGTCGATTCAGTCATCGAACTACAGGTGTCCAAGGGCAACCAATTCGTCATGCCCGACCTATCCGGCATGTTCTGGGTCGACGCCGAACCACGATTGCGCGCGCTGGGCTGGACCGGGATGCTCGACAAAGGGGCCGACGTCGACGCCGGTGGCTCCCAACACAACCGGGTCGTCTATCAAAACCCGCCGGCGGGGACCGGCGTCAACCGGGACGGCATCATCACGCTGAGGTTCGGCCAGTAG

23. Amino Acid Sequence
MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSAAGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDVQVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNVSTGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAITNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPPAGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQHNRVVYQNPPAGTGVNRDGIITLRFGQ

24. Active site identification with special domains marked
Active site identification with special domains marked. Mer 40 is the ATP binding domain.

25. Kinase Domain

26. RNA Active Site w/ Domains
UACGAACUUGGCGAA AUCCUUGGAUUUGGG GGCAUGUCCGAGGUC CACCUGGCCCGCGAC CUCCGGUUGCACCGC GACGUUGCGGUCAAG GUGCUGCGCGCUGAU CUAGCCCGCGAUCCC AGUUUUUACCUUCGC UUCCGGCGUGAGGCG CAAAACGCCGCGGCA UUGAACCACCCUGCA AUCGUCGCGGUCUAC GACACCGGUGAAGCC GAAACGCCCGCCGGG CCAUUGCCCUACAUC GUCAUGGAAUACGUC GACGGCGUUACCCUG CGCGACAUUGUCCAC ACCGAAGGGCCGAUG ACGCCCAAACGCGCC AUCGAGGUCAUCGCC GACGCCUGCCAAGCG CUGAACUUCAGUCAU CAGAACGGAAUCAUC CACCGUGACGUCAAG CCGGCGAACAUCAUG AUCAGCGCGACCAAU GCAGUAAAGGUGAUG GAUUUCGGCAUCGCC CGCGCCAUUGCCGAC AGCGGCAACAGCGUG ACCCAGACCGCAGCA GUGAUCGGCACGGCG CAGUACCUGUCACCC GAACAGGCCCGGGGU GAUUCCGUCGACGCC CGAUCCGAUGUCUAU UCCUUGGGCUGUGUU CUUUAUGAAGUCCUC ACCGGGGAGCCACCU UUCACCGGCGACUCA CCCGUCUCGGUUGCC UACCAACAUGUGCGC GAAGACCCGAUCCCA CCUUCGGCGCGGCAC GAAGGCCUCUCCGCC GACCUGGACGCCGUC GUUCUCAAGGCGCUG GCCAAAAAUCCGGAA AACCGCUAUCAGACA GCGGCGGAGAUGCGC GCCGACCUGGUC

27. 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
PNAs targeted to the start codon region of the chromosomal β-galactosidase gene (lacZ) were synthesized over 7- to 15-mer size range
E. coli outer cell wall is a major barrier to PNAs, so need to find a more efficient technique

28. Concentrations of PNA (100nM – 500nM)

29. Concentrations of PNA (1mM – 5mM)

30. 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

31. Efficacy of Cap Peptide

32. 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

33. Comparison of Nucleotides

34. 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’

35. Whole Cell Assay BioSafety Level 1 Mycobacteria smegmatis
Middlebrook 7H9 Broth Media
Middlebrook 7H10 Agar Media
β-Lactam Antibiotic Library

36. Assay Method 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)
Can change depending on growth rate
Mycobacteria grown for 7 days. OD is taken then that culture is divided onto 96well plate.  Plate is treated with the PNAs and an antibiotic. Plates then allowed to incubate 35C. Readings are taken every 7 days.
Does the antibiotic need to be refereshed? Possibly at 3 days or something. Might need more research, but out of time. I shall put it in one of the slides of this issue.

37. Assay Plate One B-lactam antibiotic treated across entire plate1 2 3 4 5 6 7 8 9 10 11 12 A
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

38. 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

39. 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
Why not against Essential genes now? As demonstrated by MDR TB, if the essential gene mutates, there may be a specificity issue with the PNA and that gene. We can bypass this by attacking a non essential mechanism that is highly conserved. Crippling this mechanism will disrupt everything downstream, including any essential genes

40. Future Studies Select Promising PNAs for additional screening
Screen Against other microorganisms
Design PNAs for other essential genes or pathways

41. Contingency Assays Large Scale Assay Zone of Inhibition Assay
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
Use of 7H10 Agar media
Impregnate with β-Lactam
Spots of various concentration PNAs
Measure inhibition zones

42. 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 B-Lactam
Assumes β-Lactam will remain active
Not cleaved or lysed by Lactamases

43. Summary 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
   

44. Questions?