1. Studies of Amino Acid Mutations in Drug Resistance of the SMO Protein
Eunice Wintona
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2. BACKGROUNDS
Hedgehog (Hh) signaling pathway regulates cell growth by transmitting required information to embryonic cells
Hh is initiated when a family of Hh ligand (Desert Dhh, Indian Ihh or Sonic Shh) interacts with a cell surface transmembrane receptor Patched (PTCH)
Aberrant activation of Hh pathway drives human tumors
The first association of Hh pathway in cancer was observed in leukemia, breast, gastric, pancreatic, prostate, and lung cancers.
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3. Hedgehog Signaling Pathway
Abbreviations:
Hh: Hedgehog
Ptch: Patched
Smo: Smoothened
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4. BACKGROUNDS (cont.)
Smoothened protein (SMO) is a main component of the hedgehog signaling pathway.
Hh pathway antagonists target a seven-transmembrane (7-TM) domain of SMO protein.
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5. Extracellular domain (5L7I) Intracellular Domain
7-TM Domain
Color code:
Orange: 5L7I
Cyan: 4QIM
Dark red: 4QIN
Yellow: 4O9R
Coffee: 4N4W
Extracellular domain (4N4W)
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6. Close-up 7-TM Domain
First superposition of SMO receptors to identify binding pockets.
We find out most ligands including GDC-0449 bind at the top blue box while the ligand in 4N4W binds at lower portion (orange box, i.e., deeper)
Thus the docking of GDC-0449 and its analogues were docked to the pocket whose centroid was defined by ligand in 5L7I (blue box).
Color code for Ligand:
Pink: 5L7I (Vis, GDC-0449)
Pale: 4QIM (A8T)
Green: 4QIN (SG8)
Blue: 4O9R (CY8)
Orange: 4N4W

7. Backgrounds (cont.)
GDC-0449 is the first FDA-approved Hh pathway inhibitor for the treatment of metastatic or locally advanced BCC (Basal Cell Carcinoma)
Soon after approval of GDC-0449 (Vismodegib), Sonidegib was approved by the FDA as the second clinically prescribed SMO antagonist
The clinical use of GDC-0449 and Sonidegib is severely restricted due to numerous side effects including constipation, diarrhea, decreased appetite, hair loss, muscle spasms, and tiredness
Development of resistance to these drugs has become a major hurdle for their continued advancement
Preliminary researches show that SMO mutants D473H and W281C significantly rendered resistance to GDC-0449 by reducing its binding to the protein
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8. Vismodegib & Sonidegib
The drugs interactions with amino acids
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9. Importance
The importance of this research is that it provides guidance in drugs treatment of the various cancer diseases associated with SMO
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10. Objective Systemically mutate residues in the binding pocket of SMO
Run docking scores and validate the method by comparing docking scores of mutants
Predict the sensitivity, binding of drugs to new mutants
Analyzing residues that are responsible for drug binding
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11. SMO/Ligand interactions for MDB5 MDB2 GDC_0449
Binding pocket of SMO receptors as defined by superposition of five human SMO receptors
SMO/Ligand interactions for MDB5
MDB2
GDC_0449
The H-bond interactions are depicted as orange dotted lines.
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12. Methods
First, I downloaded the SMO protein, superpose the different proteins using MOE and visually inspect the structural change
Helped me identify mutation effects on SMO structures in the drug binding regions
Minimized proteins with backbone fixed
With assumption the mutations would not cause drastic change to the whole structures of the protein
To maintain the bound conformation of protein, only side chains and ligand were minimized due the consideration that without water support a full minimization of protein may cause some
Prepared protein mutants by mutating amino acid residues to others, one at a time using Maestro
Mutant proteins were built using Wild-Type protein with PDB of 4QIM at positions 321, 518, 469, and 473 (at each position the original amino acid will be mutant to all other 19 mutants)
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13. Mutational Locations
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14. Methods (cont.)
Docking of drugs molecules to binding pocket then followed
Grid files were generated using Glide dock protein, and then all ligands were docked to the grid files, which represent the binding pocket.
Then, ligands were evaluated based on their docking scores or the change of binding affinity, which are calculated by
Docking WT – Docking Mutant
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15. Vismodegib is ineffective to SMO mutants D473H (exp);
We predict drug resistance in all mutants except mutants D473Tyr, Pro, Phe, Cys, to which Vismodegib may still be sensitive
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16. Asp473His
Asp473Met
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17. We predict drug resistance in all mutants except mutants D473Pro, Tyr, to which Sonidegib may still be sensitive
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18. Asp473Tyr
Asp473Met
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19. Mutants at position E518 would be resistant to Vismodegib treatment except for mutant E518Tyr
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20. Here, we can see that all the values are negative; so, we predict that mutants at position E518 are resistant to Sonidegib
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21. At position C469, all mutants are resistant to Vismodegib
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22. At position C469, all mutants are resistant to Sonidegib except for mutant C469Tyr
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23. Conclusions
For GDC_0449, all mutants except mutants D473Tyr, Pro, Phe, and D473Cys may enhance ligand binding and thus may still be sensitive to GDC_0449 treatment
Vismodegib only binds with mutant E518Tyr at position E518
No mutants is sensitive to Vismodegib at position C469
Mutant Tyr is susceptible to treatment with Sonidegib at all three positions (D473, E518, and C469)
Mutant Tyr is important
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24. Acknowledgements Dr. Andy Zhong Chemistry Department FUSE Grant
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25. References
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Hoch, Lucile et al. “MRT-92 Inhibits Hedgehog Signaling by Blocking Overlapping Binding Sites in the Transmembrane Domain of the Smoothened Receptor.” The FASEB Journal 29.5 (2015): 1817–1829. PMC. Web. 1 Mar
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