Antibiotic discovery - improving the chances.

1. Antibiotic discovery - improving the chances. Dr Jem Stach BIO3030-BIO8041

2. Antibiotics a brief history • Approximately 60 years ago the life expectancy of a child at birth was 45 years. Childhood infection and mortality was very high, and the top 10 causes of death were infection related. • The discovery and implementation of antibiotics in the ‘antibiotic era’ (1940 - 1969) reversed that trend, so that in the the year 2000 the average life expectancy of a child at birth was 75 years. The top 10 causes of death are no longer infections. • Our current situation is described as the post antibiotic era…microbes are increasingly becoming resistant to antibiotics, and there is little industrial incentive to discover new antimicrobials. • Some speculate that the average life expectancy will decrease with the return of drug-resistant microbial infection

3. A very worrying trend....

4. Before we consider why... Need to know how the majority of antimicrobial discovery was done in the “golden age of discovery” The majority of antibiotics on the market are microbial in origin. Process of discovery: isolate bacteria and fungi from soil, grow them, screen them against other microbes. Chemically isolate active fraction, determine structure and commence pre-clinical and clinical screening

5. A brief history Penicillin first described as a potential medicine in Alexander Fleming’s reports 1928. Infant patient cured of gonococcal infection in 1930. Outbreak of World War caused UK and US governments to collaborate on mass-production (effectively forced companies to research mass production). Initial growth in flasks, moved to submerged culture. Initial yield 0.002% - early mass production enough to treat 10 patients! Optimization of strains (UV and X-ray mutation) and fermentation conditions greatly increased yields - by the time of the invasion of Normandy (1944) there was enough to treat 3 million patients. Message: bacteria and fungi generally produce antimicrobials in very small quantities.

6. Why the decline in discovery? Many companies doing the same thing = a high rate of rediscovery. Took months to go from hit to identification, often only to discover that the antimicrobial discovered was already patented. Antibiotics work too well! A short course (1-5 days) clears infection - Pharma companies would prefer drugs that people take every day = bigger profits. Emerging resistance - a new drug may only have a very short clinical window before resistance develops - reduces value of antibiotic. Patent life is ca. 20 years - file at discovery - 15 years (and maybe $12 billion in development) to have exclusivity on the market for 2-4 years before patent expires - certain markets ignoring patents. In short - ECONOMICS: the antibiotic market was viewed not to be an area for significant growth. UNFORTUNATELY: massive underestimation of the ability of microbes to become resistant to antimicrobials.

7. Quick summary 1 Antibiotics have saved millions of lives and continue to do so - their discovery was a health care miracle. Antibiotics come from bacteria and fungi. They are produced during growth in varying quantities (often small). Resistance to antimicrobials threatens to return us to the pre-antibiotic era of high mortality rates. Economics for antimicrobial discovery are not favourable Need low cost ways of discovering new antibiotic compounds.

8. We’ll concentrate on improved detection of antimicrobials i.e. those present in fermentation cultures in very small quantities Hypothesis: the reason that the discovery rate of antimicrobials declined, was because only high-abundance compounds could be detected. Need to think about how the screens actually work...

9. Essential enzyme inhibitor - antibiotic inhibited enzyme Antibiotic assay 10 units of inhibitor Enzyme inhibition kills cell In this example 8 units of inhibitor are required to kill the bacterium

10. Essential enzyme inhibitor - antibiotic inhibited enzyme Antibiotic assay 10 units of inhibitor What if there is more enzyme? Same amount of inhibitor would fail to kill the cell. It would require 12 units of inhibitor to kill the cell

11. Essential enzyme inhibitor - antibiotic inhibited enzyme Antibiotic assay 5 units of inhibitor Antibiotic assay 10 units of inhibitor Bacterial cells are sensitized to the antibiotic - could use half the amount to detect activity What if there is less enzyme? More than the required amount of inhibitor is present. It would require 4 units of enzyme to kill the cell

12. Relationship is an approximation. Not all inhibitor/enzyme relationships will be linear. In most cases 100% inhibition of enzyme is not required for cell death. However, principal holds that sensitivity will be affected by the amount of protein. Generalized relationship Amount of inhibitor required for cell death Amount of protein

13. Hypothesis: If we can control the amount of protein in the cell, we can control its sensitivity to antimicrobial compounds Need to know what the target of the antimicrobial in order to control the amount of the correct target protein. Theoretically we can then improve the sensitivity of the screen, and increase the chances of detecting low abundance compounds

14. So how do we control the abundance of a specific protein in a bacterial cell? Ideas...

15. Antisense in action First discovered in Eukaryotes, different mechanisms involved in Prokaryotic antisense mechanism, but principal is similar: targeted mRNA is enzymatically degraded.

16. There has been a great deal of emphasis on screening a small number of targets (pathways). Mostly because they prove to be very susceptible to inhibition. However, screening in these proteins may increase the chances of finding a known compound We can use antisense methods to reduce the abundance of a protein...But which targets should we choose?

17. Antisense techniques can also aid in the choice of targets In general what makes a good target for an antibacterial development? Many aspects will contribute to this, but simply put: 1. It should be essential i.e. if inhibited the bacterium should die or stop growing 2. The protein should not be present in humans

18. Essential genes Fairly easy to identify genes that are essential. Transposons (or other techniques) can be used to disrupt or knockout a specific gene. Genes that can’t be deleted are considered to be essential. Databases exist describing the essential genes of many bacterial species.

19. Transposon insertion coupled to massively parallel sequencing allows for high-density mapping of essential genes. Genome wide analysis

20. Protein 1 Protein 3 Protein 2

21. Example of target selection using antisense We took four essential genes: acpP, fabI, ftsZ and murA Antisense strains were constructed for each and their growth profiles assessed as the amount of antisense RNA was increased (N.B. more antisense RNA = less essential protein) Results demonstrated that antisense RNA could affect growth of bacteria, high levels of antisense prevented detectable growth antisense RNA

22. small reduction of acpP mRNA (a thus AcpP protein) results in large decrease in growth rate. Thus AcpP is stringently required by the cell for growth - small reduction in protein or inhibition is highly growth inhibitory. Quantification of target mRNA enables determination of the stringency of requirement acpP fabI ftsZ murA

23. We can plot the relative growth rate against the relative mRNA abundance of the gene target to give an indication of the stringency requirement. Gives a minimum transcript level for a 50% reduction in growth MTL50 Hierarchy of essentiality?

25. Quick summary 2 • The sensitivity of a bacterium to an antibacterial compound will be dictated by a number of factors • The stringency of requirement (i.e. the degree of essentiality) can be used for target selection • A small degree of inhibition of a stringently required protein will theoretically give inhibitors which require small doses to kill the bacterium • Antisense methods can be used to make sensitive strains and to select targets.

26. How does a sensitised strain help in discovering new antibacterials? Ideas...

27. It’s all about quantities Remember that most antimicrobial compounds are produced in small amounts in fermentation. What if a fermentation produces a novel antimicrobial compound in very small quantities? Wild-type strain would not be susceptible - compound inhibits the protein, but there is not sufficient amounts to kill the bacteria. Antisense sensitized strain has less protein and so is sensitive to the small quantity of inhibitor Wild-type strain Fermentation extract Antisense strain

28. Increasing sensitivity to enzyme inhibition Example: strain expressing antisense RNA to fatty acid biosynthesis protein (fabF) Cerulenin is a specific inhibitor of fabF Cells expressing antisense RNA should have increased sensitivity to inhibitors of the cognate protein....

29. Antisense fabF expressing S. aureus was used in an agar diffusion screening method. Mix cells with agar, spot natural product extracts into wells When asfabF is induced inhibition is detected with some compounds Antisense screening for FabF inhibitors

30. We used the anti-FtsZ strain to screen berberine, a compound found in Berberis plants and commonly used in Chinese traditional medicine. Reported to be an FtsZ inhibitor - no genetic evidence to support claim. Screening using antisense strains

31. We have shown that berberine is an inhibitor of FtsZ. Decreasing the abundance of FtsZ increases the susceptibility to berberine. Strain can be used to screen for new inhibitors. Screening using antisense strains

32. Summary • Novel antimicrobial compounds are urgently required • New targets and screening methods are needed to prevent rediscovery of known compounds • Undiscovered compounds may be present in fermentations at very low concentration • Antisense methods can be used to aid in the selection of new targets and to improve the likelihood of detection in screening platforms • Antisense methods can be used on libraries of extracts that have been made previously - not need for big re-investment.