Studies of Amino Acid Mutations in Drug Resistance of the SMO Protein Eunice Wintona 11/14/2018

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. 11/14/2018

Hedgehog Signaling Pathway Abbreviations: Hh: Hedgehog Ptch: Patched Smo: Smoothened 11/14/2018

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. 11/14/2018

Extracellular domain (5L7I) Intracellular Domain 7-TM Domain Color code: Orange: 5L7I Cyan: 4QIM Dark red: 4QIN Yellow: 4O9R Coffee: 4N4W Extracellular domain (4N4W) 11/14/2018

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

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 11/14/2018

Vismodegib & Sonidegib The drugs interactions with amino acids 11/14/2018

Importance The importance of this research is that it provides guidance in drugs treatment of the various cancer diseases associated with SMO 11/14/2018

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 11/14/2018

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. 11/14/2018

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) 11/14/2018

Mutational Locations 11/14/2018

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 11/14/2018

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 11/14/2018

Asp473His Asp473Met 11/14/2018

We predict drug resistance in all mutants except mutants D473Pro, Tyr, to which Sonidegib may still be sensitive 11/14/2018

Asp473Tyr Asp473Met 11/14/2018

Mutants at position E518 would be resistant to Vismodegib treatment except for mutant E518Tyr 11/14/2018

Here, we can see that all the values are negative; so, we predict that mutants at position E518 are resistant to Sonidegib 11/14/2018

At position C469, all mutants are resistant to Vismodegib 11/14/2018

At position C469, all mutants are resistant to Sonidegib except for mutant C469Tyr 11/14/2018

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 11/14/2018

Acknowledgements Dr. Andy Zhong Chemistry Department FUSE Grant 11/14/2018

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