Identification of Compounds that Induce the Human Cathelicidin Gene Through the Vitamin D and/or Farnesoid X Receptors Brenda Niu Dr. Adrian F. Gombart Dept. of Biochemistry and Biophysics Linus Pauling Institute Oregon State University HHMI 2010

VITAMIN D Obtained through UVB radiation or food Obtained through UVB radiation or food Beneficial for bones Beneficial for bones Recently linked to innate immune system Recently linked to innate immune system Regulates cathelicidin Regulates cathelicidin antimicrobial peptide (CAMP) gene

THE CAMP GENE Regulated by vitamin D only in humans and primates Regulated by vitamin D only in humans and primates Promoter contains vitamin D response element (VDRE) Promoter contains vitamin D response element (VDRE) Produces protein (cathelicidin)  interferes with cell membrane of bacteria  pathogen death Produces protein (cathelicidin)  interferes with cell membrane of bacteria  pathogen death

FXR AND VDR Farnesoid X Receptor (FXR) Ligand: primary bile acids or xenobiotics Vitamin D Receptor (VDR) Ligand: 1,25(OH) 2 D 3 or secondary bile acids (Gombart, 2009)

ACTIVATION OF CAMP VDRE or FXR Activation of CAMP 1,25-Dihydroxy- vitamin D 3 1. VDR or FXR binds to ligand 2. Forms heterodimers by binding retinoid X receptors (RXRs) 3. Binds to VDRE 4. Activates CAMP gene

HYPOTHESIS Since the VDR and FXR can bind to other ligands, compounds that resemble either vitamin D or bind to FXR will activate the VDR or FXR, respectively, and lead to induction of the CAMP gene. Since the VDR and FXR can bind to other ligands, compounds that resemble either vitamin D or bind to FXR will activate the VDR or FXR, respectively, and lead to induction of the CAMP gene.

METHODS High throughput screening High throughput screening Test library of drugs used in NIH clinical trials for CAMP activation Test library of drugs used in NIH clinical trials for CAMP activation Transfect cells through electroporation Transfect cells through electroporation TSTA-1, RL, and GFP plasmids TSTA-1, RL, and GFP plasmids Dual-glo luciferase assay to test for activation of the CAMP gene Dual-glo luciferase assay to test for activation of the CAMP gene

TSTA-1 CONSTRUCT Two Step Transcriptional Amplification Two Step Transcriptional Amplification hCAMP Promoter GAL4-VP16 Fusion Protein 5X GAL4 Binding Sites Firefly Luciferase Minimal Promoter

DUAL-GLO LUCIFERASE ASSAY Quantifies gene expression by measuring luminescence from reactions catalyzed by firefly and Renilla luciferase Luciferase reagent  Stop & glo reagent  Controls: DMSO (-), EtOH (-), 1,25 D 3 (+) Renilla luciferaseFirefly luciferase

HTS PROGRESS Duplicate screenings of library Triplicates if > 2 fold change TSTA- empty

RESULTS OF HTS Compound Triplicate 1 Fold Change Triplicate 2 Fold Change Triplicate 3 Fold Change Average Fold Change Cytarabine ± 0.25 Disulfarim ± 0.41 Calcipotriol ± 0.03 Linezolid ± 0.82 Nitazoxanide ± 0.88 Pterostilbene ± 0.03 Resveratrol ± 0.15

QRT-PCR

DISULFARIM & RESVERATROL

RESVERATROL

2’ only, Resv 10-5M Control Resveratrol 10-5M hCAP18 LEVELS

SYNERGY/SUPPRESSION 1,25 D 3

RESVERATROL + 1,25 D3

CONCLUSIONS 20 compounds in the NIH library may suppress or synergistically activate the CAMP gene with vitamin D Resveratrol and pterostilbene activate the endogenous CAMP gene as well as hCAP18 protein, while also acting synergistically with Vit. D

FUTURE WORK Test resulting 20 compounds with QRT-PCR to confirm synergy with vitamin D or suppression Determine the mechanism(s) underlying activation of the CAMP gene by resveratrol and pterostilbene

ACKNOWLEDGEMENTS Dr. Adrian Gombart The Gombart Lab A special thank you to Brian Sinnott and Dr. Malcolm Lowry Dr. Kevin Ahern OSU Biochemistry and Biophysics Department Linus Pauling Institute National Institute of Health Howard Hughes Medical Institute