Antifungal drug discovery.

1. Antifungal drug discovery. • Jem Stach

2. Fungal Infections • Fungi have emerged as the fourth most common hospital bloodstream infections. • Nearly 40% of which prove fatal - particularly significant in patients that are immunocompromised. • Resistance to existing classes of drugs is on the increase • Only one new antifungal agents with novel mechanism of action has been discovered in the past decade

3. New infections • Until the 1970’s fungal infections were treated as largely treatable and the need for novel drugs was very small • The antifungal standard, amphotericin B, was developed in the 1950s • Environmental isolates (Fusarium, Aspergillus) are becoming serious medical threats - due to rise in immunocompromised patients. • Such strains are often highly resistant to antifungal compounds

4. Unlike bacteria, fungi belong to the Eucarya. Because of their phylogenetic similarity, fungi and humans have homologous metabolic pathways for energy production, protein synthesis and cell division. Consequently, there is a greater difficulty in developing selective antifungal agents than selective antibacterials why is it so difficult to identify antifungal compounds?

5. Candida albicans an emerging threat • Candida albicans is the principal fungus causing human infection. • Genome sequence available • Difficult to use parallel gene disruption techniques to identify essential genes • C. albicans is not normally capable of mating (genetic crosses are made by mating) • Random mutagenesis techniques can not be used as there is a paucity of functional transposable elements • C. albicans (unlike S. cerevisiae [Baker's Yeast]) doesn't have a haploid form

6. If constructing gene knockouts is difficult, how else can we identify new targets for drug development?In other words how do we identify novel essential genes? • Think about previous lectures...

7. The diploid nature of Candida albicans doesn’t prevent antisense methods from working. It is still possible to assay gene essentiality: lower the amount of the protein in the cell and assay growth Antisense method

8. The gene of interest is placed in front of a promoter in the opposite orientation (when transcribed an antisense RNA is produced. The construct can integrate into the host genome at two locations: at the gene of interest or the promoter Integration at the promoter results in antisense production Integration at the gene of interest also produces antisense RNA, but also “promoter collision” The theory...different from bacterial antisense previously covered

9. Monitor growth when cells are producing antisense RNA against non-antisense producing condition Identify growth affect transformants - gene affected is essential (in theory) Compare induced and non-induced C. albicans transformants

11. Authors knock out one copy of the gene of interest resulting in a heterozygous strain (i.e. has only one copy of the gene in the genome).In theory, this strain will have half the amount of the specific protein. Half the amount of protein should render the strain more sensitive to inhibition of that protein - could of also used the antisense expressing strain... • How can you use these strains to identify novel antifungal compounds?

12. Authors identify compounds that inhibit the growth of the “crippled strains” modification of those compounds can be used to improve efficacy - novel inhibitors - novel target

13. Part 1 Summary • Candida albicans is an emerging threat • Difficulty to do classical yeast genetics to identify essential genes • Antisense method shows promise in diploid organism • Novel essential genes can be identified and used as targets from drug development.

14. We have concentrated on identifying new drugs, what about improving known ones? • When single proteins are targeted (as is the case with current antifungals) high doses can be required can cause side effects and select for resistance - targeting multiple pathways with codrugs may reduce the need for high dosages and decrease resistance

15. B I E A 2 E C D

16. Pump 2 B I E E A Hypothetical co-drug mechanism

17. Combination therapy • Recent studies on antifungal combination therapy have demonstrated efficacy • e.g. combination of fungistatic and analgesic agents were shown to generate fungicidal activity against drug-resistant Candida albicans but did not significantly affect human cells • Very limited study with known antifungals - potential numbers of combinations is effectively limitless • Can take a currently active antifungal compound and screen natural product or combi-chem libraries for potential codrugs:

18. Authors took ketoconazole (KTC) as their test antifungal KTC inhibits lanosterol 14-demethylase. Primarily used to treat Candida infections. At clinical doses it can cause toxic side effects, including hepatitis Lower the effective concentration of this drug was very desirable Search for codrugs

19. Screening principal resazurin (blue) resorufin (red) • Authors used AlamarBlue as a viability indicator • Dispense Candida strain in microtitre plates and grow in the presence of clinically relevant concentration of KCT (1 ug ml-1) = All wells blue (no growth) • Repeat using 100 X less KCT (0.01 ug ml -1) = All wells red (cells, survive and respire) • 0.01 ug ml -1 is approximately 5 times less effective than 1 ug ml -1 • Repeat, but use natural product extracts in combination with the low KCT dose. metabolic reduction

20. Success! KTC 0.01 ug ml-1 + F101604 KTC 0.01 ug ml-1 KTC 1 ug ml-1 F101604 • Authors screened 20,000 natural product extracts and identified 12 natural product extracts that improved the antifungal efficacy of the KTC low dose to beyond that of the the clinically relevant dose. • Extract alone has no antifungal activity - acting as a codrug • FURTHERMORE: If cells from the 1.0 ug ml-1 KTC wells are transfered to fresh media without the drug, they recover (wells red). However, if the same is done with cells from the combined treatment wells, no recovery is seen (wells blue) • KTC alone is fungistatic, KTC+F101604 is fungicidal -combined therapy changes the mode of action!

21. Synergistic compounds - reduced toxicity

22. All very well in test tubes... • Ultimately, novel treatments, targets, or potential drugs that are discovered in the laboratory need to be validated clinically. • Mouse models are often used to gain data on efficacy in a mammalian background. • In this study the authors developed a mouse model for immunocompromised patients (Candida is usually an opportunistic pathogen in AIDS patients) for testing their codrug treatment - termed an in vivo model

23. Candida infected mice were treated with BEA (F101604) and a low dose (0.5 mg kg-1) of KTC. Survival was significantly prolonged, Candida was reduced in internal organs Better than even a high dose (50 mg kg-1) of KTC alone! It works in vivo!

24. Summary part 2 • Antifungal drugs can have severe side affects due to high dosage needed. • Reducing the required dose of an antifungal can reduce toxicity and the incidence of resistance • Screening for codrugs (compounds that are not antifungal in isolation, but are synergistic with known antifungals) can improve the drug profile of antifungals already in the market • Combination therapy is undergoing a renaissance - used to be common practice.

25. Fungal cell wall • Basically consists of β-1,3-glucan, β-1,6-glucan, chitin and mannoproteins, which are interconnected. • basically consists of β-1,3-glucan, β-1,6-glucan, chitin and mannoproteins, which are interconnected • Mannoprotein glycosylation and processing occur stepwise throughout the secretory pathway, which is highly conserved from yeast to human

26. Fungal cell wall • β-1,3-glucan and chitin are synthesized by the fungal specific polymerases a number of β-1,3-glucan synthesis inhibitors such as echinocandins, papulacandins and enfumafungin, and chitin synthase inhibitors such as Nikkomycin have been discovered. • On the contrary, only a little is known about the synthesis of β-1,6-glucan. • The β-1,6-glucan component is a highly branched polymer comprising of about 10% of the total cell wall.

28. Fungal cell wall • > 10 genes identified in the synthesis of β-1,6-glucan. • Most have homologues in humans. • GPI anchor proteins in Candida sp. have specific signals that target them to the membrane or cell wall.

29. Reporter proteins such as GFP can be targeted to either the cell wall or membrane depending on the targeting signal of the GI-anchor protein Screen for inhibitors that release the reporter into the medium. Screening for inhibitors

30. Inhibitors of cell wall synthesis discovered

31. Efficacy of D21-6076 (A) or C. glabrata ATCC 48435 (2.4 x 108 cells) (B). D21-6076 was administered orally 1, 4, and 7 h after infection.

32. Summary part 3 • Knowledge of cell wall biosynthesis can be exploited to develop screens. • Proteins unique to fungi are used as the molecular targets • Screen is based on disruption of the cell wall biosynthesis. • Specific inhibitors discovered. • Compounds likely work by preventing the invasion process.