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

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

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Antisense Approach to Target MDR Tuberculosis Diane Meas Michael Nguyen Michael DeSalvio Michael Boateng-Antwi Slide 2 Agenda Introduction & Objectives Background & Significance o Overview of MDR TB o Impact and Importance Research Design & Methods o Previous studies and findings o Mechanism to new approach o Assay Methods Conclusion Slide 3 Introduction TB Overview o Infectious airborne disease caused by Mycobacterium tuberculosis o 2009 incident cases 9.4 million o 2009 prevalent cases 14 million o Mortality: - 1.8 million o Funding : $5 billion o Estimated Funding for 2011: $6 billion (source: WHO Global TB Report, 2010) Slide 4 TB Global Distribution Slide 5 Interventions Anti-TB drugs ( www.cdc.gov/tb/publications ) o Frontline: rifampicin, isoniazid, pyrizinamide, and ethambutol o Second line: fluoroquinolones, amikacin, kanamycin, or capreomycin Drug Resistance: 250,000 reported (WHO-TB, 2010) Options for Disease control o Development of new line of drugs o Reversal of drug resistance Antisense Technology Slide 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 Slide 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 Slide 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 Slide 9 Background & Significance 1.Penicillin 2. Cephalosporin Red Structure - -lactam core ring -lactam antibiotics broad class of drugs with -lactam ring as nucleus of molecular structure Inhibit 4 8 enzymes (PBP) engaged in cell wall biosynthesis. -lactamases cleave - lactam ring in antibiotic to make drug ineffective Slide 10 Antisense Overview Made up of RNA Generally short strands Complementary to the mRNA strand Intercept and bind mRNA o Prevent Translation o No Gene Expression! http://cdn.venturebeat.com/wp-content/uploads/2007/11/800px-antisense_dna_oligonucleotide.jpg Slide 11 Antisense Treatments Used to treat various treatments o Cytomegalovirus retinitis o Hemorrhagic fever viruses o Cancer (TGF-beta2) o HIV/AIDS o High cholesterol (mipobersen, 2010 ph-IV) Slide 12 Proof of Principle Harth et.al: o Used phophorothioate-modified oligodeoxyribonucleotides (PS-ODNs) o targeted mycolyl transferases to inhibit essential genes Slide 13 Proof of Principle Harth et.al: o 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) Slide 14 Proof of Principle Dasgupta et al: o Knocked out Penicillin Binding Proteins (PBPA) serine acyl transferases involved in cell wall expansion, cell shape maintenance, septum formation and cell division o Relied on mutation of PknB precursor proteins responsible for the phosphorylation of the PBPA o Inactivation of PnkB results in no phosphorylation of PBPA Cell death Slide 15 Current Solutions Clavulanic Acid o GlaxoSmithKline o B-lactamase inhibitor o Competitive inhibition Binds to active site, causing irreversible covalence o Derived from S. clavuligerus o Concurrent Administration with Amoxicillin Slide 16 Current Solutions Adverse Effects! o Increased Cholestatic Jaundice o Acute hepatitis o Some microbial resistance o Allergy Slide 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 Slide 18 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 Slide 19 Research Design & Methods Target other essential genes: o 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 Slide 20 Gene Identification PknB = transmembrane serine/threonine- protein kinase B From M. tuberculosis H37Rv Slide 21 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 Slide 22 Slide 23 Amino Acid Sequence MTTPSHLSDRYELGEILGFGGMSEVHLARDLRLHRDVAVKVLRADLARD PSFYLRFRREAQNAAALNHPAIVAVYDTGEAETPAGPLPYIVMEYVDGV TLRDIVHTEGPMTPKRAIEVIADACQALNFSHQNGIIHRDVKPANIMIS ATNAVKVMDFGIARAIADSGNSVTQTAAVIGTAQYLSPEQARGDSVDAR SDVYSLGCVLYEVLTGEPPFTGDSPVSVAYQHVREDPIPPSARHEGLSA DLDAVVLKALAKNPENRYQTAAEMRADLVRVHNGEPPEAPKVLTDAERT SLLSSAAGNLSGPRTDPLPRQDLDDTDRDRSIGSVGRWVAVVAVLAVLT VVVTIAINTFGGITRDVQVPDVRGQSSADAIATLQNRGFKIRTLQKPDS TIPPDHVIGTDPAANTSVSAGDEITVNVSTGPEQREIPDVSTLTYAEAV KKLTAAGFGRFKQANSPSTPELVGKVIGTNPPANQTSAITNVVIIIVGS GPATKDIPDVAGQTVDVAQKNLNVYGFTKFSQASVDSPRPAGEVTGTNP PAGTTVPVDSVIELQVSKGNQFVMPDLSGMFWVDAEPRLRALGWTGMLD KGADVDAGGSQHNRVVYQNPPAGTGVNRDGIITLRFGQ Slide 24 Slide 25 Kinase Domain Slide 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 Slide 27 Efficiency of PNA PNA stands for peptide nucleic acids Antisense PNAs are larger than most drugs o PNA size/length is an important parameter for efficiency o PNAs targeted to the start codon region of the chromosomal -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 Slide 28 Concentrations of PNA (100nM 500nM) Slide 29 Concentrations of PNA (1mM 5mM) Slide 30 Efficiency of the KFFKFFKFFK cap Also expressed as (KFF) 3 K 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 Slide 31 Efficacy of Cap Peptide Slide 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 Slide 33 Comparison of Nucleotides Slide 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 Slide 35 Whole Cell Assay BioSafety Level 1 Mycobacteria smegmatis Middlebrook 7H9 Broth Media Middlebrook 7H10 Agar Media -Lactam Antibiotic Library Slide 36 Assay Method Grow Mycobacteria for 7 days @ 35 o C in 7H9 Take OD reading (A 600 ) 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 Slide 37 Assay Plate 123456789101112 A BufferPNA1 10 nM PNA2PNA3PNA4PNA5PNA6PNA7PNA8PNA9PNA10Clavulanic Acid B1:2 C1:4 D1:8 E1:16 F1:32 G1:64 H0 nM -One B-lactam antibiotic treated across entire plate -Every well contains M. smegmatis Slide 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 Slide 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 Slide 40 Future Studies Select Promising PNAs for additional screening Screen Against other microorganisms Design PNAs for other essential genes or pathways Slide 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 -Lactam library Measure OD via spectroscopy Zone of Inhibition Assay Use of 7H10 Agar media Impregnate with -Lactam Spots of various concentration PNAs Measure inhibition zones Slide 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 -Lactam will remain active Not cleaved or lysed by Lactamases Slide 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 Slide 44 Questions?