The Biological Activity of a Novel Class of Insecticides, Sterol Carrier Protein Inhibitors SCPIs

1
The Biological Activity of a Novel Class of
Insecticides, Sterol Carrier Protein Inhibitors
(SCPIs)    

• Ryan Larson
• U.S. Navy Entomology HSCP
• University of Wisconsin-Madison
• Dept. of Entomology
• Advisor Que Lan

2
Overview

• Background
• Cholesterol transport and metabolism
• Methods
• High throughput screening
• Biological activity
• Results
• Larval bioassays
• Future Directions

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Insects and Cholesterol

• Unable to synthesize cholesterol
• Must obtain cholesterol from diet
• Cholesterol is hydrophobic
• Protein needed for cholesterol transport
• Lipoproteins do not enter midgut or fat body

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Aedes Sterol Carrier Protein-2

• Aedes Sterol Carrier Protein-2 (AeSCP-2)
  partially responsible for cellular cholesterol
  transfer

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AeSCP2 Cholesterol Shuttle
Insect Fat Body
Lipophorin
Hemolymph
Basal Side
Midgut Cell
AeSCP-2
Lumenal Side
Cholesterol
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Objectives

• Select sterol carrier protein
• Screen for AeSCP-2 inhibitors
• Determine biological activity of inhibitors

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Selecting AeSCP-2
Cholesterol Affinity
AeSCP-2 has the highest affinity for cholesterol
within the SCP-2 family
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Knockdown of SCP-2
Mortality in newly emerged adults Aedes aegypti
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Knockdown of SCP-2
3H cholesterol (DPM) mg total protein
Cholesterol uptake in 4th instars Aedes aegypti
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Uptake of Cholesterol in 4th instars
Midgut
Midgut
Total Cholesterol (ng/animal)
Fat Body
Hours Post Ecdysis
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Chemical Library Screening

• Primary screening 40,000 compounds
• 140 positive hits
• Secondary screening in cell culture
• 70 positive hits
• Biological activity screening in larvae
• 12 positive hits

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High Throughput Screening

• NBD Cholesterol Competitive Binding Assay
• NBD (fluorescent cholesterol analog)
• AeSCP-2 and NBD cholesterol added
• Library compounds compete for binding with NBD
  Cholesterol to AeSCP-2

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Bioassay

• 60 larvae/cup
• 60ml of ddH20
• SCPIs dissolved in DMSO serial diluted
• Mortality recorded after 72h

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Mortality 1st vs. 4th Ae. aegypti Larvae
(µg/ml)ppm
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Mortality 4015 vs. SCPI-1

• Bioassays in 3rd instars
• 4015 is a natural compound
• Less expensive to register

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Future Directions

• Computer modeling
• Semi-field trials
• High throughput cell culture screen

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Summary

• Insects need cholesterol from diet
• SCP-2 partially responsible for intracellular
  cholesterol transport
• SCP-2 inhibitors reduce cholesterol transfer
• Natural 4015 was most effective

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Acknowledgments

• Support
• -DOD (DWFP)
• U.S. Navy
• Dr. Q. Lan
• Dr G. White
• CAPT S. Cope