A molecular dynamics approach to identify an oxysterol-based hedgehog pathway inhibitor.
Multiple oxysterols (OHCs) have demonstrated the ability to act as agonists or antagonists of the hedgehog (Hh) signaling pathway, a developmental signaling pathway that has been implicated as a potential therapeutic target in a variety of human diseases. These OHCs are known to modulate Hh signaling through direct binding interactions with the N-terminal cysteine rich domain (CRD) of Smoothened, a key regulator of Hh signal transduction.



Homology modeling, molecular dynamics simulations, and MM/GBSA energy calculations were utilized to explore binding interactions between the OHC scaffold and the human Smoothened CRD. Follow-up cellular assays explored the in vitro activity of potential Hh pathway modulators.



Structural features that govern key molecular interactions between the Smoothened CRD and the OHC scaffold were identified. Orientation of the iso-octyl side chain as well as the overall entropy of the OHC-CRD complex are important for determining activity against the Hh pathway. OHC 9, which was previously thought to be inactive because it was not an Hh agonist, was identified as an inhibitor of Hh signal transmission.



Calculated MM/GBSA binding energies for OHCs in complex with the CRD of Smoothened correlate well with in vitro Hh modulatory activity. Compounds with high affinity stabilize Smoothened and are antagonists, whereas compounds with reduced affinity allow a conformational change in Smoothened that results in pathway activation.



Computational modeling and molecular dynamics simulations can be used to predict whether a small molecule that binds the Smoothened CRD will be an agonist or antagonist of the pathway.
Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd., Unit 3092, Storrs, CT, 06269-3092, United States.
Radha Charan
RC
Dash
Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd., Unit 3092, Storrs, CT, 06269-3092, United States.
Chad R
CR
Maschinot
Department of Pharmaceutical Sciences, University of Connecticut, 69 N Eagleville Rd., Unit 3092, Storrs, CT, 06269-3092, United States. Electronic address: kyle.hadden@uconn.edu.
M Kyle
MK
Hadden
D000595:Amino Acid Sequence; D003545:Cysteine / Q000378:metabolism; D053823:Hedgehog Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D056004:Molecular Dynamics Simulation; D000072376:Oxysterols / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D000072417:Protein Domains; D015398:Signal Transduction / Q000187:drug effects* / Q000502:physiology*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D053823:Hedgehog Proteins; D000072376:Oxysterols; D054852:Small Molecule Libraries; D003545:Cysteine
Cysteine-rich domain; Hedgehog signaling; Molecular dynamics; Oxysterols; Smoothened