SPROUT in the Design of PotentialAnthelmintic Agents

By Michael Briggs

Supervisors Prof. A. P. Johnson* Dr. K. Yeap**

*Ph.D. Supervisor, University of Leeds.**Industrial Supervisor, Pfizer, Sandwich, Kent.

Introduction– Paraherquamide Family of Natural Products– Mode of Action of the Paraherquamides

Results and Discussion– SPROUT Design Work

Summary Acknowledgements

Outline of Presentation

Introduction

Introduction

Only three groups of broad spectrum anthemintic drugs available Resistance has appeared and increased in frequency. The paraherquamides have shown the ability to render immotile worms

present in the intestines of infected animals. Found to have activity against nematodes which are resistant to current

anthelmintic drugs.

Paraherquamide Family

•Paraherquamide A was first isolated in 1981 by Yamazaki et al. from Penicillium paraherquei.•Subsequently isolated from Penicillium charlesii along with six other analogues (Paraherquamides B-G) by a group at Merck in 1990.•Structural differences occur at the C-14 position and in the structure of ring A.

N

X

O

N

N

O

R2

R1

Me O H

MeMe

Me Me

24

25

14

A

Compound/Paraherquamide

analogue

R1 R2 X

A OH CH3 OB H H OC =CH2 - OD -OCH2- - OE H CH3 OF H CH3 -G OH CH3 -

C24-C25Dihydro

OH CH3 O

M. Yamazaki et al, Tetrahedron Lett. 1981, 22, 135.

• Paraherquamide A is the most active of the analogues with the synthetic dihydro analogue being the least active.

• Some sort of alkyl substitution at C-14 position may be required for activity, although the C-14 hydroxy group may not be required for high activity.

Results of biological testing of the paraherquamidesagainst the strain of nematode Caenorhabditis elegans.

Biological Activity of the Paraherquamide Family

N

X

O

N

N

O

R2

R1

Me O H

MeMe

Me Me

14

25

24

Compound/Paraherquamide

analogue

R1 R2 X LD50(g / ml)

A OH CH3 O 2.5B H H O 100C =CH2 - O 40D -OCH2- - O 160E H CH3 O 6F H CH3 - 65G OH CH3 - 20

C24-C25Dihydro

OH CH3 O >200

Mode of Action of the Paraherquamides

•Paraherquamide A binds to the invertebrate nicotinic acetylcholine receptor with Ki’s of 2 pM and 1.7 nM in insect and nematode binding assays.•nACh receptors are ligand-gated ion channel found in the postsynaptic membrane of muscle cells.•A nerve signal causes acetylcholine to be released at the pre-synaptic junction. Activation of the nAChR by ACh causes the ion channel to open.•The flow of Na cations though the open ion channel initiates an electrical signal that causes the muscle to contract.

•N. Unwin et al reported the structure of nACh receptor at 4.6 Å resolution using electron microscopy

Mode of Action of the Paraherquamides

N. Unwin et al, J. Mol. Biol. 1999, 288, 765-786.

•Acetylcholine and nicotine are agonists of the nACh.•Share a common pharmacophore.•Paraherquamide is able to bind to the receptor, but since it is a larger molecule than acetylcholine, it is also capable of binding to other groups outside the acetylcholine binding site.•While paraherquamide is bound, acetylcholine is unable to bind and open the ion channel resulting in paralysis of the nematode.

Mode of Action of the Paraherquamides

Overlay of Paraherquamide A with Acetylcholine Overlay of Paraherquamide A with Nicotine

Chemical Modifications of Paraherquamide A and Marcfortine A

•Four important interaction centres- 14 - CH3

- Basic nitrogen- Oxindole portion of the molecule- Hydrophobic region of the dioxepin ring

O

O

N

N

OH

CH3O

H

CH3

CH3

NO

CH3Me

CH3

14

•Number of reports regarding the structural modifications of paraherquamide A and Marcfortine A.•Testing of the analogues has supplied additional information on the structure activity relationship of the paraherquamides.

Design of Analogues of Paraherquamide A using SPROUT

An X-Ray crystal structure of Paraherquamide A was used to provide the spatial distance between the important interaction centres.

SPROUT is a de novo ligand design program– Generates molecules that fit the steric and chemical constraints of a pharmacophore or a specific protein

receptor. The SPROUT design work has been restricted to a pharmacophore comprising the oxindole ring and the basic

nitrogen.

Design of Analogues of Paraherquamide A using SPROUTCont…

Starting templates were placed at the oxindole ring and the basic nitrogen so as to satisfy the criteria of that site.

Nitrogen was orientated so that its lone pair is in the same position, relative to oxindole, as in paraherquamide A.

Summary

• >15 SPROUT designed analogues have been synthesised

•Screening by industrial partner showed low micro molar activity (< 10) for 3 of the 6 compounds that have so far been tested.

•Testing of further analogues should further probe the SAR

Acknowledgements

Prof. Peter JohnsonDr. Kuen YeapDr. Chris DuttonDr. James Eshelby

EPSRCPfizer

AshDimitrisJamesJennyKeithMarkMattPeterSajSam