Antifungal Drug Design (HC38) PDF

1 Design of novel antifungal drugs (5052MBP12Y HC38) Dr. Frans Hochstenbach Department of Medical Biochemistry Amsterdam UMC, location AMC University of Amsterdam [email protected] 2 Transition m...

1 Design of novel antifungal drugs (5052MBP12Y HC38) Dr. Frans Hochstenbach Department of Medical Biochemistry Amsterdam UMC, location AMC University of Amsterdam [email protected] 2 Transition metals transition metals 3 Coordinate bonds Insulin hexamer contains two zinc ions, each coordinated by three His residues 4 Coordinate bonds In manganese superoxide dismutase the cofactor manganese is bound by three of its histidine side chains and one aspartate side chain. In addition, a water molecule and the inhibitor azide (N3, –N=N+=N–) can be coordinated to manganese azide 5 Targets of antifungal drug design Mycoses Opportunistic yeasts and fungi Current antifungals Strategy for the development of new antifungals 6 Current antifungals Polyenes – nystatin (1949) – amphotericin B (1958) – candicidin, SPK-843 (Phase III) amphotericin B Azoles – miconazole – itraconazole – fluconazole – voriconazole (2000) voriconazole – posaconazole (2005) – ravuconazole (2007) fluconazole – isavuconazole – albaconazole 7 Amphotericin B Therapy for systemic fungal infections hydroxyl groups (hydrophilic side) seven di-een groups (hydrophobic side) 8 Chemical structure of ergosterol Ergosterol HO Cholesterol HO 9 Amphotericin B interacts with ergosterol amphotericin B ergosterol phospholipid pore -OH HO- -OH HO- -OH HO- Molecular orientation of a pore of ampothericin B and ergosterol 10 Amphotericin B forms a conducting pore A pore is formed in the plasma membrane by two rings of eight amphotericin B molecules linked hydrophobically to ergosterol. In this pore, the hydroxyl residues face inward, creating an aqueous pore through which ions such as K+ and Mg2+ can leak. This altered permeability of the plasma membrane results in death of the fungal cell. Consequently, amphotericin B is fungicidal. 11 New opportunities with Candicidin Candidate drug candicidin is more soluble and in clinical trial phase III testing Candicidin, SPK-843 hydroxyl groups (hydrophilic side) Amphotericin B di-een groups (hydrophobic side) 12 Targets of antifungal drug design Mycoses Opportunistic yeasts and fungi Current antifungals Polyenes Azoles 13 Chemical structure of azoles Azoles are heterocycles of carbon and nitrogen atoms 4 3 4 3 N N 5 2 5 N2 1N 1N H H 1,3-diazole 1,2,4-triazole (imidazole) 14 Chemical structure of diazoles Azoles are heterocycles of carbon and nitrogen atoms 4 3 N N 5 2 N N Cl 1 H 1,3-diazole Cl (imidazole) O Cl Cl miconazole 15 Structures of miconazole and fluconazole Miconazole is a diazole, fluconazole is a triazole N N N N Cl N F N Cl N N O Cl OH F Cl miconazole fluconazole 16 Fluconazole and itraconazole Fluconazole Pfizer, Inc. Drug development: $ 600.000.000 Profit per year: $ 1.000.000.000 Patent ran out in 2002 Water-soluble azole Fungistatic No activity against molds Itraconazole Janssen Pharmaceutica 1978: Drug development 1992: FDA registration 1997: Registration of an oral formulation Fungistatic Active against both yeasts and molds 17 Human cytochrome P450 enzymes The 57 different human CYP-enzymes are divided over 18 families Family Main functions Enzym activity Name of individual CYP-enzymes Xenobiotica metabolism CYP1 hydroxylase CYP1A1, CYP1A2, CYP1B1 Eicosanoid (EET) biosynthesis Xenobiotica metabolism CYP2A6, CYP2A7, CYP2A13, CYP2B6, CYP2C8, CYP2C9, Alcohol metabolism (MEOS) hydroxylase CYP2 CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, Vitamine D3 biosynthesis 25-hydroxylase CYP2J2, CYP2R1, CYP2S1, CYP2U1, CYP2W1 Eicosanoid (EET, HETE) biosynthesis Xenobiotica metabolism CYP3 hydroxylase CYP3A4, CYP3A5, CYP3A7, CYP3A43 Eicosanoid (EET, HETE) biosynthesis Xenobiotica metabolism CYP4A11, CYP4A22, CYP4B1, CYP4F2, CYP4F3, CYP4 Vetzuur metabolism ω-hydroxylase CYP4F8, CYP4F11, CYP4F12, CYP4F22, CYP4V2, Eicosanoid (EET, HETE) biosynthesis CYP4X1, CYP4Z1 CYP5 Thromboxaan synthesis hydroxylase CYP5A1 CYP7 Bile acid biosynthesis 7-hydroxylase CYP7A1, CYP7B1 CYP8 Bile acid biosynthesis hydroxylase CYP8A1, CYP8B1 CYP11 Steroid biosynthesis 11-hydroxylase CYP11A1, CYP11B1, CYP11B2 CYP17 Steroid biosynthesis 17-hydroxylase CYP17A1 CYP19 Steroid biosynthesis hydroxylase CYP19A1 CYP20 Functie onbekend CYP20A1 CYP21 Steroid biosynthesis 21-hydroxylase CYP21A2 CYP24 Vitamin D metabolism 24-hydroxylase CYP24A1 CYP26 Vitamin A metabolism hydroxylase CYP26A1, CYP26B1, CYP26C1 Bile acid biosynthes 27-hydroxylase CYP27 CYP27A1, CYP27B1, CYP27C1 Vitamin D3 biosynthesis 1-hydroxylase CYP39 Bile acid biosynthesis 7-hydroxylase CYP39A1 CYP46 Steroid biosynthesis 24-hydroxylase CYP46A1 CYP51 Cholesterol biosynthesis 14a-hydroxylase CYP51A1 Nelson DR. The Cytochrome P450 Home page. Human Genomics 2009; 4: 59−65. http://drnelson.uthsc.edu/CytochromeP450.html 18 Lanosterol 14a-methyl demethylation Reaction catalyzed by CYP51 H3C CH3 H3C CH3 CH3 CH3 CH3 CH3 CH3 14 CYP51 CH3 14 CH3 HO HO H3C CH3 H3C CH3 lanosterol 19 Azoles are inhibitors of fungal CYP51 Voriconazole Posaconazole 20 The biosynthetic pathway for ergosterol acetyl-CoA The ergosterol + 3 O2 biosynthetic pathway Squalene azoles serves as a target for Lanosterol two distinct classes of + O2 terbinafine Ergosterol antifungal drugs: Terbinafine Squalene-2,3- (Lamisil, Novartis) epoxide + 3 O2 + 3 O2 Lanosterol Azoles + O2 + O2 21 Current antimycotic drugs target ergosterol Ergosterol acts as a bioregulator of plasma membrane fluidity and asymmetry, and consequently of membrane integrity Polyenes (amphotericin B) Bind ergosterol in the plasma membrane, forming pores Cause leakage of K+ and Mg2+ and consequently cell lysis Are fungicidal Azoles (itraconazole, fluconazole) Inhibit ergosterol biosynthesis Alter structure and function of the plasma membrane Are generally fungistatic 22 Fluconazole resistance in an AIDS patient A 30-year-old man with AIDS Effective daily dose of fluconazole Effective Daily Dose of Fluconazole (CD4 cell count of 9/mm3 Fluconazole resistance of during relapse 5 and remained low). clinical isolate Recurring episodes of oropharyngeal candidosis were followed during a 2-year period. (mg/day) Each infection was treated with oral MIC (μg/ml) fluconazole for 14 days at the indicated dose. Episode 13 lasted for 5 months. The infection failed to respond to 400 mg/day of fluconazole or 200–400 mg/day of itraconazole. Episode 13 and 14 were treated successfully with 800 mg/day of Sequential relapses of oropharyngeal candidosis fluconazole. Subsequently, the patient failed to respond to fluconazole and required amphotericin B (30 mg/day). Redding S et al. Clinical Infectious Diaeases 1994; 18: 240 White TC. Antimicrobial Agents and Chemotherapy 1997; 41: 1482 23 Drawbacks of current antifungals Only a limited number of antifungal drugs are available Amphotericin B is (reversibly) nephrotoxic Resistance against azoles is increasing Conclusion There is a growing need for novel and safe antifungals 24 2015: Fungal meningitis outbreak in the US http://www.cdc.gov/fungal/outbreaks.html 25 Cause of infection: Exserohilum rostratum US Centers for Disease Control (CDC): 749 cases, 76 deaths http://www.cdc.gov/hai/outbreaks/clinicians/index.html Patients received spinal injections of steroids as pain relieve. The steroids were contaminated with fungi. Standard treatment: Voriconazole, 6 mg/kg every 12 hours (initially intravenously) In serious cases: Voriconazole and amphotericin B, 7.5 mg/kg daily (intravenously) Exserohilum rostratum New England Compounding Center co-founder Barry Cadden was sentenced to 9 years in prison for his role in the deadly meningitis outbreak. http://www.cdc.gov/hai/outbreaks/meningitis-facilities-map.html 26 Targets of antifungal drug design Mycoses Opportunistic yeasts and fungi Current antifungals Strategy for the development of new antifungals 27 Different stage in drug discovery and development Target Identification Target Validation in vitro-Assay new therapeutic drug Research institutes High-Throughput EMA,FDA Assay Registration Clinical trials Pharmaceutical companies Phase III Lead Compound Phase II Phase I Combinatorial Chemistry Candidate Drug 28 Why is the cell wall a good target for the development of novel antifungal drugs? 29 An ideal antifungal target An enzyme that synthesizes a reaction product essential for viability of the fungal cell. Inhibition of the enzyme by an antifungal drug leads to lysis of the fungal cell. essential reaction product essential antifungal enzyme drug substrate An enzyme that is specific for fungal cells and absent from human cells. This property decreases chances of side effects of the antifungal drug in patients. 30 Therapeutic margins of antifungals Humans Fungi nucleic acid synthesis nucleic acid synthesis protein synthesis protein synthesis cholesterol ergosterol extracellular matrix cell wall a-glucan synthesis b-glucan synthesis chitin synthesis 31 Cell morphology of model yeasts Saccharomyces cerevisiae Schizosaccharomyces pombe 32 Ultrastructure of the S. pombe cell wall cell wall plasma membrane cytoplasm 33 Model of the S. pombe Cell Wall cell wall proteins b-glucan a-glucan ex plasma membrane in 34 Structural polysaccharides of the fungal cell wall The fungal cell wall contains three major structural polysaccharides, namely a-glucan, b-glucan, and chitin OH OH OH OH OH OH ~ O O O O O O HO HO HO HO HO HO HO OH OH O OH O OH O OH O OH O OH (1,3)-a-glucan OH OH OH OH OH OH O O O O ~ HO HO HO O O HO O HO HO HO O O O OH OH OH OH O OH OH OH (1,3)-b-glucan OH OH O OH HO O OH OH HO O O NH HO O O ~ NH HO O O C O NH HO O C O NH HO OH CH3 C O NH CH3 C O CH3 C O CH3 CH3 chitin 35 Synthases for cell wall polysaccharides The cell wall of yeasts and fungi contains three major structural polysaccharides, namely a-glucan, b-glucan, and chitin. Each polysaccharide is synthesized by a specialized synthase. Thus, most fungi bear a-glucan synthases, b-glucan synthases, and chitin synthases. Identification of the genes for cell-wall polysaccharide synthases: chitin synthase by Phillips Robbins at MIT in 1986, b-glucan synthase by Merck Research Laboratories in 1994, and a-glucan synthase by Frans Hochstenbach at NIH in 1998. 36 Topology of chitin synthases and b-glucan synthases The putative catalytic domains are located intracellularly chitin synthase b-glucan synthase ex N in C C N UDP-N-acetylglucosamine UDP UDP-glucose UDP Energetically enriched substrates Chitin synthase: UDP-N-acetylglucosamine b-Glucan synthase: UDP-glucose 37 Nikkomycin is a competitive inhibitor of chitin synthase Nikkomycin is a peptidyl nucleoside produced by the bacterium Streptomyces tendae. Nikkomycin acts as a Nikkomycin Z competitive inhibitor of chitin synthases by mimicking UDP-GlcNAc. Nikkomycin has potent antimycotic activity. UDP-GlcNAc uridine 38 Model for b-glucan synthase caspofungin ex in UDP-glucose UDP 39 Echinocandins Echinocandins are non-competitive inhibitors of b-glucan synthase. Glarea lozoyensis, the fungus that produced the progenitor echinocandin, was a new species isolated from a water sample from the Lozoya River, close to Madrid, Spain in 1985. It took Merck 15 years from the discovery of the progenitor compound to FDA approval of Caspofungin for clinical use in 2002. Two other echinocandins are currently in clinical use, namely Anidulafungin (2004; Pfizer) and Micafungin (2006; Astellas Pharma), and a third is in Clinical Trial Phase I (Aminocandin, Indevus Pharmaceuticals) 40 Echinocandins are non-competitive inhibitors of b-glucan synthases Echinocandins are lipopeptides. Echinocandin B 41 Cancidas (caspofungin) van Merck Cancidas, to treat invasive candidiasis 42 Morphology of a temperature-sensitive a-glucan synthase mutant 19ºC 34ºC 37ºC 43 Topology of a-glucan synthase domain for linking of a-glucan ex pore for transport of a-glucan in across the membrane domain for synthesis of a-glucan 44 Topology of polysaccharide synthases The putative catalytic domains are located intracellularly N ex a-glucan synthase in C ex b-glucan synthase in C N ex chitin synthase in N C 45 Binding of capsule requires cell-wall a-glucan Electron micrographs of a wild-type cell (A) and an AGSi cell (B) Wild type Decreased Ags1 expression (AGSi) Reese AJ, Doering TL. Scale bar, 0.5 μm Mol Microbiol. 2003; 50: 1401−1409. 46 Conclusion on cell-wall a-glucan Synthases for cell-wall a-glucan are conserved in evolution Chemical structures of cell-wall a-glucan are conserved in evolution Function of cell-wall a-glucan: in S. pombe: required for maintaining structural integrity in Cryptococcus neoformans: required for normal growth and capsule binding Proposal a-Glucan synthase is a target for antifungal drug design 47 The cell wall as a target for new antifungals Designing novel antifungal drugs is engaging in medical biochemistry Mycoses Opportunistic yeasts and fungi Current antifungals Strategy for the development of new antifungals