Finding the Sweet Spot- Mechanism Guided Design of Glycosidase Inhibitors. Jahnabi Roy CHEM 575 Seminar 11/01/12

Transcription

1 Finding the Sweet Spot- Mechanism Guided Design of Glycosidase Inhibitors Jahnabi Roy CHEM 575 Seminar 11/01/12

2 Glycans and Glycosyl Hydrolases

3 Restricting the Spread of Influenza Virus Moscona A. N. Eng. J. Med. 2005, 353,

4 Current Drugs Acting as Glycosidase Inhibitors- Influenza Zanamivir- Brand Name : Relenza Oseltamivir- Brand Name : Tamiflu Viral Neuraminidase Zanamivir Bound to Neuraminidase

5 Resistance to Oseltamivir Neuraminidase with sialic acid Neuraminidase with oseltamivir Mutated neuraminidase with oseltamivir Collins P. et al. Nature, 2008, 453,

6 Classification of Glycoside Hydrolases Sequence Based Classification: Endo & Exo Acting Hydrolases: Carbohydrate Chemistry & Biochemistry, Michael Sinnott,

7 Classification of Glycoside Hydrolases Mechanism Based Classification: Gebler, J. et al. J. Biol. Chem. 1992, 267, 18,

8 Glycosidases: Mechanism & Inhibition Mechanism Based on Carboxylate Residues Alternate Mechanisms Inverting Retaining Glycosidases Non-covalent Mechanism Based Inhibitors Affinity Based Covalent Mechanism Based

9 Mechanism with Inversion of Configuration β- glycosidases with inverting mechanism: Transition State Acid/base assistance from amino acid side chains, especially aspartic acid and glutamic acid. Oxocarbenium ion transition state with flattened ring structure. McCarter, J. & Withers, S. Curr Opin Struc Biol. 1994, 4,6, ; Davies, G. et al. Structure 2002, 10,

10 Mechanism with Retention of Configuration Classical Koshland Retaining Mechanism: Transition State Glycosyl enzyme intermediate Koshland, D., Biol. Rev. 1953, 28, 416

11 pka of the Carboxylate Groups of a Glycosidase Cycles During Catalysis Nucleophile Xylanase Enzyme from Bacillus Circulans Acid/ Base Catalyst MacIntosh, L. et al. Biochemistry 1996, 35,

12 Evidence Supporting Oxocarbenium Transition State Vocaldlo, D. et al. Nature, 2002, 412,

13 Key Questions Towards Designing Inhibitors Is TS conformation same in all members of a family? Effect of cationic character on TS? Conformation of ring before and during TS? Role of O-H in stabilization? Stereo-electronic requirements at this bond?

14 Approaches Towards Inhibition of Glycoside Hydrolases Mechanism Based on Carboxylate Residues Alternate Mechanisms Inverting Retaining Glycosidases Non-covalent Mechanism Based Inhibitors Affinity Based Covalent Mechanism Based

15 Natural Products Used as Glycosidase Inhibitors Nojirimycin (1966) Antibiotic product of Streptomyces 1-Deoxynojirimycin (DNJ) (1968) Natural product of Streptomyces, Bacillus and Morus mulberry trees 2,5-dideoxy-2,5-imino-D-mannitol (DMDP) (1976) Isolated from the leaves of legume Derris elliptica. N-butyl-1-deoxynojirimycin (1994) Used for Treatment of Gaucher s disease Asano, N. Curr. Top. Med. Chem. 2003, 3,

16 Transition State Conformation Analysis for Inhibitor Design Mimicking the positively charged exocyclic oxygen Inhibitor of mannosidases Mimicking the boat conformation Inhibitor of glucosidases R K i (µm) H 0.41 Me Bn 1.0 R K i (µm) H Me 1.3 Bn 0.5 R Group K i value (µm) IC 50 Value (µm) Skew boat ( 1 S 3, a) and boat ( 1,4 B, b) conformers of a β-dmannopyranoside and isoquinuclidines Bn H Vasella, A. et al. Chem Commun. 2000, , Farrr, R. et al. Tetrahedron Lett. 1990, 31,

17 Modifications at C-2 OH for Non-Covalent Inhibition Vasella, A. et al. Helv Chim Acta. 2000, 83, deoxynojirimycin

18 Approaches Towards Inhibition of Glycoside Hydrolases Mechanism Based on Carboxylate Residues Alternate Mechanisms Inverting Retaining Glycosidases Non-covalent Mechanism Based Inhibitors Affinity Based Covalent Mechanism Based

19 Mechanism Based Covalent Inhibitors- Reactive Aglycons Inhibitor of yeast α-glucosidase Mechanism of Activation: Inhibitor of bacterial phospotriesterase Halazy S. et al. J. Am. Chem. Soc. 1989, 111, ; Lo. L, et al. Bioorg Med Chem Lett 1996,

20 Mechanism Based Covalent Inhibitors- Labelling of Enzymatic Nucleophiles Epoxides & Aziridines a b d e Aziridine based inactivators c Epoxide based inactivators f Covalent attachment of epoxide inhibitor to active site:

21 Covalent Inhibitors in Deducing Mechanism 1. CBE CBE used to confirm that mutation at active site causes inactivation, proving Asp is the catalytic residue. 2. β-glucosidase CBE Cyclophellitol 1,6- epicyclophellitol Inhibits both α & β glucosidases Inhibits β glucosidases Inhibits α glucosidases Tai, V. et al. Biochem Biophys Res Commun, 1995, 213,

22 Mechanism Based Covalent Inhibitors- Labelling of Enzymatic Nucleophiles Activated Fluorinated Glycoside Inhibitors Mechanism Of Inactivation:

23 Alternative Mechanisms of Hydrolysis Neighbouring Group Participation Enzymes Not Relying on Carboxylate Residues for Hydrolysis Alternate Nucleophiles NAD Dependent Hydrolysis

24 Neighboring Group Participation Transition State Terwisscha van Scheltinga AC, et al. Biochemistry. 1995, 34,48,

25 Inhibitors for Enzymes Undergoing NGP Assisted Hydrolysis NAG- thiazoline Withers S, et al. 1996, 118, ; Brameld, K. et al. J. Mol. Biol. 1998, 280, Allosamidine

26 Enzymes Exhibiting NAD- Dependent Hydrolysis Transition State Withers, S. et al.j. Am. Chem. Soc. 2004, 126,

27 Alternative Nucleophiles To Affect Hydrolysis Transition State Withers, S. et al. J. Am. Chem. Soc. 2003, 125,

28 Current Progress in Inhibitor Development for Neuraminidases Peramivir Laninamivir- Currently in Phase III trials Laninamivir Octanoate Yamashita, M. et al Antimicrob. Agents Chemother. 2009, 53, ; A. Watanabe et al. Clin. Inf. Dis. 2010, 51, 1167

29 Summary Glycosyl hydrolases are catabolic enzymes that participate in key life processes. They can be targeted towards therapeutic applications and is currently oseltamivir and zanamivir are being used for treatment of influenza. Low oral-availability, rapid excretion and resistance need better understanding of mechanism for better drugs. Their mechanisms can be classified mainly into retaining and inverting. Aside from a few families, most utilize aspartate and glutamate residues for hydrolysis. Transition state mimics have been designed to identify better inhibitors. Better analogs of influenza drugs like laninamivir have developed as a result of better understanding of active sites of enzymes.

30 Challenges Inhibitors currently developed need to be modified to accommodate mutational changes. Transition state mimics of glycosidases with alternate mechanisms like NAD dependence still unexplored. K i values for TS analogs are 10-9 or M but theoretically can be upto M.

31 Future Directions Strong Inhibitors of Family 33 sialidases Replace with silyl group More affinity and covalent binding to phenol R= hydrophobic group like alkyl chain or aromatic ring

32 Acknowledgements Prof. Doug Mitchell CHEM 575 class Prof. Marty Burke Burke Group Prof. Hergenrother Prof. van der Donk