**ABSTRACT NOT FOR CITATION WITHOUT AUTHOR PERMISSION.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.**




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




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.