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