Finding the Sweet Spot- Mechanism Guided Design of Glycosidase ...
Transcript of Finding the Sweet Spot- Mechanism Guided Design of Glycosidase ...
Finding the Sweet Spot- Mechanism Guided Design of
Glycosidase Inhibitors
Jahnabi Roy CHEM 575 Seminar
11/01/12
Glycans and Glycosyl Hydrolases
http://cellbiology.med.unsw.edu.au/units/science/lecture0803.htm
Restricting the Spread of Influenza Virus
Moscona A. N. Eng. J. Med. 2005, 353, 1363-1373
Current Drugs Acting as Glycosidase Inhibitors- Influenza
Oseltamivir- Brand Name : Tamiflu®
Zanamivir- Brand Name : Relenza®
Viral Neuraminidase Zanamivir Bound to Neuraminidase
Resistance to Oseltamivir
Neuraminidase with sialic acid Neuraminidase with oseltamivir Mutated neuraminidase with oseltamivir
Collins P. et al. Nature, 2008, 453, 1258-1262
Classification of Glycoside Hydrolases
Endo & Exo Acting Hydrolases:
Carbohydrate Chemistry & Biochemistry, Michael Sinnott,
Sequence Based Classification:
Classification of Glycoside Hydrolases
Mechanism Based Classification:
Gebler, J. et al. J. Biol. Chem. 1992, 267, 18, 12559-12561
Glycosidases
Mechanism
Based on Carboxylate
Residues
Inverting
Retaining
Alternate Mechanisms
Inhibitors
Non-covalent Mechanism
Based
Covalent
Affinity Based
Mechanism Based
Glycosidases: Mechanism & Inhibition
Mechanism with Inversion of Configuration
β- glycosidases with inverting mechanism:
McCarter, J. & Withers, S. Curr Opin Struc Biol. 1994, 4,6, 885-892; Davies, G. et al. Structure 2002, 10, 547-556
• Acid/base assistance from amino acid side chains, especially aspartic acid and glutamic acid.
• Oxocarbenium ion transition state with flattened ring structure.
Transition State
Mechanism with Retention of Configuration
Classical Koshland Retaining Mechanism:
Transition State
Glycosyl enzyme intermediate
Koshland, D., Biol. Rev. 1953, 28, 416
pKa of the Carboxylate Groups of a Glycosidase Cycles During Catalysis
MacIntosh, L. et al. Biochemistry 1996, 35, 9958-9966
Nucleophile Acid/ Base Catalyst Xylanase Enzyme from Bacillus Circulans
Evidence Supporting Oxocarbenium Transition State
Vocaldlo, D. et al. Nature, 2002, 412, 835-838
Role of O-H in stabilization?
Effect of cationic character on TS?
Stereo-electronic requirements at this bond?
Key Questions Towards Designing Inhibitors
Conformation of ring before and during
TS?
Is TS conformation same in all members of a family?
Approaches Towards Inhibition of Glycoside Hydrolases
Glycosidases
Mechanism
Based on Carboxylate
Residues
Inverting
Retaining
Alternate Mechanisms
Inhibitors
Non-covalent Mechanism
Based
Covalent
Affinity Based
Mechanism Based
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, 471-484
Transition State Conformation Analysis for Inhibitor Design
Skew boat (1S3, a) and boat (1,4B, b) conformers of a β-D-mannopyranoside and isoquinuclidines
R Group
Ki
value (µM)
IC50
Value (µM)
Bn 0.17 0.69
H 20 29.4
Vasella, A. et al. Chem Commun. 2000, 1829-1830, Farrr, R. et al. Tetrahedron Lett. 1990, 31, 7109-7112
Mimicking the positively charged exocyclic oxygen
Inhibitor of mannosidases
Inhibitor of glucosidases
R’ Ki (µM)
H 0.41
Me 0.062
Bn 1.0
Mimicking the boat conformation
R’ Ki (µM)
H 0.074
Me 1.3
Bn 0.5
Modifications at C-2 OH for Non-Covalent Inhibition
Vasella, A. et al. Helv Chim Acta. 2000, 83, 513-534
1- deoxynojirimycin
Approaches Towards Inhibition of Glycoside Hydrolases
Glycosidases
Mechanism
Based on Carboxylate
Residues
Inverting
Retaining
Alternate Mechanisms
Inhibitors
Non-covalent Mechanism
Based
Covalent
Affinity Based
Mechanism Based
Mechanism Based Covalent Inhibitors- Reactive Aglycons
Mechanism of Activation:
Halazy S. et al. J. Am. Chem. Soc. 1989, 111, 3484-3485; Lo. L, et al. Bioorg Med Chem Lett 1996, 2117-2120
Inhibitor of yeast α-glucosidase Inhibitor of bacterial phospo-
triesterase
Mechanism Based Covalent Inhibitors- Labelling of Enzymatic Nucleophiles
Epoxide based inactivators Aziridine based inactivators
a b
c
d e
f
Covalent attachment of epoxide inhibitor to active site:
Epoxides & Aziridines
Covalent Inhibitors in Deducing Mechanism
CBE Cyclophellitol 1,6- epicyclophellitol
Tai, V. et al. Biochem Biophys Res Commun, 1995, 213, 175-180
2.
1.
Inhibits both α & β glucosidases
Inhibits β glucosidases
Inhibits α glucosidases
CBE
β-glucosidase
CBE used to confirm that mutation at active site causes inactivation, proving Asp is the catalytic residue.
Mechanism Based Covalent Inhibitors- Labelling of Enzymatic Nucleophiles
Activated Fluorinated Glycoside Inhibitors
Mechanism Of Inactivation:
Alternative Mechanisms of Hydrolysis
Enzymes Not Relying on Carboxylate
Residues for Hydrolysis
Neighbouring Group Participation
Alternate Nucleophiles
NAD Dependent Hydrolysis
Neighboring Group Participation
Terwisscha van Scheltinga AC, et al. Biochemistry. 1995, 34 ,48, 15619-23
Transition State
Inhibitors for Enzymes Undergoing NGP Assisted Hydrolysis
Withers S, et al. 1996, 118, 6804- 6805; Brameld, K. et al. J. Mol. Biol. 1998, 280, 913-923
NAG- thiazoline Allosamidine
Enzymes Exhibiting NAD- Dependent Hydrolysis
Withers, S. et al.J. Am. Chem. Soc. 2004, 126, 8354-8355
Transition State
Alternative Nucleophiles To Affect Hydrolysis
Withers, S. et al. J. Am. Chem. Soc. 2003, 125, 7532-7533
Transition State
Current Progress in Inhibitor Development for Neuraminidases
Laninamivir- Currently in Phase III trials
Peramivir
Yamashita, M. et al Antimicrob. Agents Chemother. 2009, 53, 186-192; A. Watanabe et al. Clin. Inf. Dis. 2010, 51, 1167
Laninamivir Octanoate
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.
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.
• Ki values for TS analogs are 10-9 or 10-10 M but theoretically can be upto 10-22 M.
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
Acknowledgements
Prof. Doug Mitchell CHEM 575 class
Prof. Hergenrother Prof. van der Donk
Prof. Marty Burke Burke Group