Antisense Approach to Target MDR Tuberculosis

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

2. 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 Agenda

3. TB – Overview • 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) Introduction

4. TB – Global Distribution

5. 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 Interventions

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

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. 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 and Significance

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

10. Made up of RNA • Generally short strands • Complementary to the mRNA strand • Intercept and bind mRNA • Prevent Translation • No Gene Expression! Antisense Overview http://cdn.venturebeat.com/wp-content/uploads/2007/11/800px-antisense_dna_oligonucleotide.jpg

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

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

13. 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) Proof of Principle

14. 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 Proof of Principle

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

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

17. 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 Midpoint Recap

18. 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 Overview of PknB Proposal

19. 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 Research Design & Methods

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

21. ATGACCACCCCTTCCCACCTGTCCGACCGCTACGAACTTGGCGAAATCCTTGGATTTGGGGGCATGTCCGAGGTCCACCTGGCCCGCGACCTCCGGTTGCACCGCGACGTTGCGGTCAAGGTGCTGCGCGCTGATCTAGCCCGCGATCCCAGTTTTTACCTTCGCTTCCGGCGTGAGGCGCAAAACGCCGCGGCATTGAACCACCCTGCAATCGTCGCGGTCTACGACACCGGTGAAGCCGAAACGCCCGCCGGGCCATTGCCCTACATCGTCATGGAATACGTCGACGGCGTTACCCTGCGCGACATTGTCCACACCGAAGGGCCGATGACGCCCAAACGCGCCATCGAGGTCATCGCCGACGCCTGCCAAGCGCTGAACTTCAGTCATCAGAACGGAATCATCCACCGTGACGTCAAGCCGGCGAACATCATGATCAGCGCGACCAATGCAGTAAAGGTGATGGATTTCGGCATCGCCCGCGCCATTGCCGACAGCGGCAACAGCGTGACCCAGACCGCAGCAGTGATCGGCACGGCGCAGTACCTGTCACCCGAACAGGCCCGGGGTGATTCCGTCGACGCCCGATCCGATGTCTATTCCTTGGGCTGTGTTCTTTATGAAGTCCTCACCGGGGAGCCACCTTTCACCGGCGACTCACCCGTCTCGGTTGCCTACCAACATGTGCGCGAAGACCCGATCCCACCTTCGGCGCGGCACGAAGGCCTCTCCGCCGACCTGGACGCCGTCGTTCTCAAGGCGCTGGCCAAAAATCCGGAAAACCGCTATCAGACAGCGGCGGAGATGCGCGCCGACCTGGTCCGCGTGCACAACGGTGAGCCGCCCGAGGCGCCCAAAGTGCTCACCGATGCCGAGCGGACCTCGCTGCTGTCGTCTGCGGCCGGCAACCTTAGCGGTCCGCGCACCGATCCGCTACCACGCCAGGACTTAGACGACACCGACCGTGACCGCAGCATCGGTTCGGTGGGCCGTTGGGTTGCGGTGGTCGCCGTGCTCGCTGTGCTGACCGTCGTGGTAACCATCGCCATCAACACGTTCGGCGGCATCACCCGCGACGTTCAAGTTCCCGACGTTCGGGGTCAATCCTCCGCCGACGCCATCGCCACACTGCAAAACCGGGGCTTCAAAATCCGCACCTTGCAGAAGCCGGACTCGACAATCCCACCGGACCACGTTATCGGCACCGACCCGGCCGCCAACACGTCGGTGAGTGCAGGCGACGAGATCACAGTCAACGTGTCCACCGGACCCGAGCAACGCGAAATACCCGACGTCTCCACGCTGACATACGCCGAAGCGGTCAAGAAACTGACTGCCGCCGGATTCGGCCGCTTCAAGCAAGCGAATTCGCCGTCCACCCCGGAACTGGTGGGCAAGGTCATCGGGACCAACCCGCCAGCCAACCAGACGTCGGCCATCACCAATGTGGTCATCATCATCGTTGGCTCTGGTCCGGCGACCAAAGACATTCCCGATGTCGCGGGCCAGACCGTCGACGTGGCGCAGAAGAACCTCAACGTCTACGGCTTCACCAAATTCAGTCAGGCCTCGGTGGACAGCCCCCGTCCCGCCGGCGAGGTGACCGGCACCAATCCACCCGCAGGCACCACAGTTCCGGTCGATTCAGTCATCGAACTACAGGTGTCCAAGGGCAACCAATTCGTCATGCCCGACCTATCCGGCATGTTCTGGGTCGACGCCGAACCACGATTGCGCGCGCTGGGCTGGACCGGGATGCTCGACAAAGGGGCCGACGTCGACGCCGGTGGCTCCCAACACAACCGGGTCGTCTATCAAAACCCGCCGGCGGGGACCGGCGTCAACCGGGACGGCATCATCACGCTGAGGTTCGGCCAGTAGATGACCACCCCTTCCCACCTGTCCGACCGCTACGAACTTGGCGAAATCCTTGGATTTGGGGGCATGTCCGAGGTCCACCTGGCCCGCGACCTCCGGTTGCACCGCGACGTTGCGGTCAAGGTGCTGCGCGCTGATCTAGCCCGCGATCCCAGTTTTTACCTTCGCTTCCGGCGTGAGGCGCAAAACGCCGCGGCATTGAACCACCCTGCAATCGTCGCGGTCTACGACACCGGTGAAGCCGAAACGCCCGCCGGGCCATTGCCCTACATCGTCATGGAATACGTCGACGGCGTTACCCTGCGCGACATTGTCCACACCGAAGGGCCGATGACGCCCAAACGCGCCATCGAGGTCATCGCCGACGCCTGCCAAGCGCTGAACTTCAGTCATCAGAACGGAATCATCCACCGTGACGTCAAGCCGGCGAACATCATGATCAGCGCGACCAATGCAGTAAAGGTGATGGATTTCGGCATCGCCCGCGCCATTGCCGACAGCGGCAACAGCGTGACCCAGACCGCAGCAGTGATCGGCACGGCGCAGTACCTGTCACCCGAACAGGCCCGGGGTGATTCCGTCGACGCCCGATCCGATGTCTATTCCTTGGGCTGTGTTCTTTATGAAGTCCTCACCGGGGAGCCACCTTTCACCGGCGACTCACCCGTCTCGGTTGCCTACCAACATGTGCGCGAAGACCCGATCCCACCTTCGGCGCGGCACGAAGGCCTCTCCGCCGACCTGGACGCCGTCGTTCTCAAGGCGCTGGCCAAAAATCCGGAAAACCGCTATCAGACAGCGGCGGAGATGCGCGCCGACCTGGTCCGCGTGCACAACGGTGAGCCGCCCGAGGCGCCCAAAGTGCTCACCGATGCCGAGCGGACCTCGCTGCTGTCGTCTGCGGCCGGCAACCTTAGCGGTCCGCGCACCGATCCGCTACCACGCCAGGACTTAGACGACACCGACCGTGACCGCAGCATCGGTTCGGTGGGCCGTTGGGTTGCGGTGGTCGCCGTGCTCGCTGTGCTGACCGTCGTGGTAACCATCGCCATCAACACGTTCGGCGGCATCACCCGCGACGTTCAAGTTCCCGACGTTCGGGGTCAATCCTCCGCCGACGCCATCGCCACACTGCAAAACCGGGGCTTCAAAATCCGCACCTTGCAGAAGCCGGACTCGACAATCCCACCGGACCACGTTATCGGCACCGACCCGGCCGCCAACACGTCGGTGAGTGCAGGCGACGAGATCACAGTCAACGTGTCCACCGGACCCGAGCAACGCGAAATACCCGACGTCTCCACGCTGACATACGCCGAAGCGGTCAAGAAACTGACTGCCGCCGGATTCGGCCGCTTCAAGCAAGCGAATTCGCCGTCCACCCCGGAACTGGTGGGCAAGGTCATCGGGACCAACCCGCCAGCCAACCAGACGTCGGCCATCACCAATGTGGTCATCATCATCGTTGGCTCTGGTCCGGCGACCAAAGACATTCCCGATGTCGCGGGCCAGACCGTCGACGTGGCGCAGAAGAACCTCAACGTCTACGGCTTCACCAAATTCAGTCAGGCCTCGGTGGACAGCCCCCGTCCCGCCGGCGAGGTGACCGGCACCAATCCACCCGCAGGCACCACAGTTCCGGTCGATTCAGTCATCGAACTACAGGTGTCCAAGGGCAACCAATTCGTCATGCCCGACCTATCCGGCATGTTCTGGGTCGACGCCGAACCACGATTGCGCGCGCTGGGCTGGACCGGGATGCTCGACAAAGGGGCCGACGTCGACGCCGGTGGCTCCCAACACAACCGGGTCGTCTATCAAAACCCGCCGGCGGGGACCGGCGTCAACCGGGACGGCATCATCACGCTGAGGTTCGGCCAGTAG Nucleotide Sequence

23. MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSAAGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDVQVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNVSTGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAITNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPPAGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQHNRVVYQNPPAGTGVNRDGIITLRFGQMTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARDPSFYLRFRREAQNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGVTLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMISATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDARSDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSADLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERTSLLSSAAGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLTVVVTIAINTFGGITRDVQVPDVRGQSSADAIATLQNRGFKIRTLQKPDSTIPPDHVIGTDPAANTSVSAGDEITVNVSTGPEQREIPDVSTLTYAEAVKKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAITNVVIIIVGSGPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNPPAGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLDKGADVDAGGSQHNRVVYQNPPAGTGVNRDGIITLRFGQ Amino Acid Sequence

25. Kinase Domain

26. 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 RNA Active Site w/ Domains

27. 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 Efficiency of PNA

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

29. Concentrations of PNA(1mM – 5mM)

30. 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 Efficiency of the KFFKFFKFFK cap

31. Efficacy of Cap Peptide

32. Outperforms Oligonucleotides • 7-15 mer lengths • Capped with KFFKFFKFFK – synthetic molecule shown to increase PNA uptake into cell • PNA immune to exonuclease activity Peptide Nucleic Acids

33. Comparison of Nucleotides

34. 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’ mRNA and its Antisense PNA

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

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

37. Assay Plate • 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

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

41. 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 • 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?