**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION. The title,
authors, and abstract for this completion report are provided below. For a copy of the full completion report,
please contact the author via e-mail at KuhnL@msu.edu
or via telephone at 517-353-8745. Questions? Contact the GLFC via email at
frp@glfc.org or via telephone at 734-662-3209.**
DEVELOPMENT OF 3KPZS ANTAGONISTS FOR SEA LAMPREY CONTROL
Leslie A. Kuhn1,2,3, Nan Liu1,4, Sebastian Raschka1,
Santosh K. Gunturu1,2,
Weiming Li3, Anne
Scott3, and Mar Huertas3
1Department
of Biochemistry & Molecular Biology Michigan State University
502C Biochemistry Building, 603 Wilson Road East
Lansing, MI 48824
2Department
of Computer Science & Engineering Michigan State University
East Lansing, MI 48824
3Department
of Fisheries & Wildlife Michigan State University
142 Giltner Hall, 293 Farm
Lane East Lansing, MI 48824
4Department
of Chemistry Michigan State University East Lansing, MI 48824
December 2014
ABSTRACT:
In spawning streams, sexually mature males release 3kPZS (7α,
12α, 24-trihydroxy-5α-cholan-3-one- 24-sulfate), the main component
of the mating pheromone that attracts ovulatory females and is indispensable
for sea lamprey reproduction. The Li lab
recently identified a repertoire of 60 olfactory receptor genes from the draft
sea lamprey genome and characterized a receptor (SLOR1) that specifically
detects 3kPZS. We hypothesized that an antagonist that mimics 3kPZS would
tightly interact with the binding pocket of SLOR1, block 3kPZS binding by the
receptor, and interrupt olfactory detection and behavioral responses of female
lampreys to 3kPZS. To identify such
antagonists for use in integrated sea lamprey control, computational screening
of 16 million organic molecules was carried out with ScreenLamp
software developed in the Kuhn lab. ScreenLamp allowed us to prioritize compounds for
experimental testing as 3kPZS antagonists based on their shape and electrostatic
similarity to 3kPZS, matching of key chemical groups known to influence 3kPZS
potency, and goodness of fit following chemical docking into the ligand binding
site of an atomic model of the SLOR1 receptor.
More than 300 compounds were procured and tested for their ability to
block sea lamprey olfactory (electro-olfactogram;
EOG) responses to 3kPZS. Seven compounds were discovered that blocked at least
50% of the olfactory response to 3kPZS, three of which are non-steroidal
compounds. The 3-hydroxyl analog of 3kPZS, petromyzonol
sulfate (PZS), was found to block 92% of the olfactory response to 3kPZS.
Behavioral effects of PZS were tested in two-choice maze trials in flowing
stream water, with 3kPZS alone, 3kPZS + PZS, or PZS alone (each at 10-12M
concentration) applied on one randomly selected side of the maze and the other
side used as a control (applying only the 50% methanol vehicle). The amount of time spent in the treated
versus control channel was monitored for 55 ovulated female sea lampreys. The results indicate that 3kPZS alone is a
significant attractant, PZS alone is a significant repellant, and when mixed
together, there is neither significant attraction nor repulsion of sea lamprey;
that is, PZS apparently neutralizes the attractant effect of 3kPZS. Follow-up two-choice maze experiments with
PZS at 10-10M concentration showed that at this higher concentration, PZS
significantly repelled sea lamprey when mixed with 3kPZS at 10-12M. Tetrasulfonated PZS significantly repelled sea lamprey both
alone and with 3kPZS at 10-12M concentration, whereas a double-bonded analog of
3kPZS significantly attracted sea lamprey, both alone and in combination with
3kPZS at 10-12M. In a further test of
the potential for PZS to control sea lamprey behavior by neutralizing the
attraction to 3kPZS released from nesting sites, stream trials were performed
in 2014 on the upper Ocqueoc River in Michigan. The stream trials were configured similarly
to the maze trials, with treated and control 1m2 nest areas placed 1.5m apart. Ovulated female sea lampreys were released
45m downstream of the nests. When 3kPZS
and PZS were mixed equally at 5 x 10-13M concentration and applied to the
treatment nest, the proportion of females that moved upstream and entered the
treatment nest decreased to 19%, compared to 52% entering the nest when 3kPZS
was applied alone. The females also spent more time in the nest baited with
only 3kPZS compared to that with the 3kPZS and PZS mixture. Together, the results indicate that PZS in
low concentration has the ability to significantly offset female sea lamprey
attraction to 3kPZS in a native setting. These results provide proof of concept
for the application of high-throughput computational screening combined with
EOG assays for discovering antagonists of sea lamprey pheromones that influence
lamprey behavior. Additional compounds
show promise as behavioral antagonists or agonists in maze trials. Screening software and a 3-dimensional
structural database of steroid analogs, G-protein coupled receptor ligands, and
compounds with pheromone-matching functional groups have been prepared and will
also be useful in future research. This
hypothesis-driven strategy has integrated computational and organismal biology
to discover pheromone receptor antagonists, providing a ground-breaking
application of structure-based drug discovery techniques to aquatic invasive
species control.