PRACTICAL EVOLUTION: HARNESSING VARIATION AND SELECTION IN RATIONAL - PowerPoint PPT Presentation

PRACTICAL EVOLUTION: HARNESSING VARIATION AND SELECTION IN RATIONAL DRUG DISCOVERY Robin Pals Rylaarsdam Benedictine University, Lisle, IL

“Evolution” in hearts and minds

Useful principles of evolution  Protein homologies  Conservation of biochemical pathways  Variation and selection

McCune-Albright Syndrome  Mosaicism for constitutively active G α s  Precocious puberty,  Café-au-lait hyperpigmentation,  Polyostotic fibrous dysplasia of bone  Other hyperendocrinopathies  3 possible mutations in Gs protein: R201H, R201C, R201S

Normal Gs Signaling Hormone Adenyly cyclase GPCR α α βγ βγ GTP MAPK ATP cAMP GDP cascade RESPONSE RESPONSE

Cells carrying the MAS mutation have constitutively active signaling Hormone Adenyly cyclase GPCR α α βγ βγ GTP MAPK ATP cAMP GDP cascade RESPONSE RESPONSE

Useful principles of evolution  Setting up a model system  Conservation of biochemical pathways  Protein homologies  Finding drug targets  Variation and selection

Homologous biochemical pathways – proteins are conserved between yeast and humans

Homologous biochemical pathways – proteins are conserved between yeast and humans Pheromone Normal yeast signaling α α βγ βγ GTP MAPK GDP cascade Temporary cell-cycle arrest G α -GDP inactivates βγ to restore cell division

Protein similarities  Blue = different  Green & warmer = similar to identical. Yeast 3 human G-protein genes Nematode Fruit fly

Common sequence, common structure

Homologous mutation in yeast G α causes same biochemical defect as in human protein Yeast with overactive G α α * βγ MAPK GTP cascade Permanent cell-cycle arrest No G α -GDP inactivation of βγ without a suppressor mutation

The MAS mutation activates yeast G α like the human gene FOA Transfected plasmid:

Useful principles of evolution  Setting up a model system  Protein homologies  Conservation of biochemical pathways  Finding drug targets  Variation and selection

Homologous mutation in yeast G α causes same biochemical defect as in human protein Yeast with overactive G α α * α ** βγ α ** MAPK GTP GTP βγ cascade GXP Permanent cell-cycle arrest No G α -GDP inactivation of βγ without a suppressor mutation Inactivation of βγ Restoration of cell cycle progression Colony formation

Variation

Randomly mutated library screened for intragenic suppressor mutations of an “MAS allele” Library: 7800 unique mutants in Gpa1 R297H 55,000 clones screened 39 Clones remained FOA R 424 colonies grew on FOA , plasmids were sequenced

14/39 suppressor mutation sites are conserved between Gs and the yeast GPA1

Do the mutations that suppress the yeast G- protein also suppress the human G-protein? Hormone Adenyly cyclase GPCR α α βγ βγ GTP MAPK ATP cAMP GDP cascade RESPONSE RESPONSE

Homology is not identity, but it works sometimes! 100 80 (% forskolin response) Basal cAMP 60 * * * 40 * ** 20 ** 0 T H S T S R C A I G R T T 6 2 6 3 W 2 3 1 0 1 2 9 0 5 8 6 8 0 4 1 2 3 4 0 2 5 1 1 2 2 3 2 2 2 2 2 3 V I L I R F Q H R T K E R201H +

Examining the variations at each site that can suppress the MAS mutation L266 100 cAMP (% forskolin response) 75 50 * 25 F142 * * F146 * 0 WT R201H R231C R231S R231A R231E R231I R231K F140 R201H + E268 R231

McCune-Albright Syndrome  Mosaicism for constitutively active G α s  Precocious puberty,  Café-au-lait hyperpigmentation,  Polyostotic fibrous dysplasia of bone  Other hyperendocrinopathies  3 possible mutations in Gs protein: R201H, R201C, R201S

Other MAS alleles are suppressed by changes at these three sites 100 (% forskolin response) 80 Basal cAMP 60 40 * * * 20 * * * 0 WT R201C R201C/F142S R201C/R231C R201C/L266T R201S R201S/F142S R201S/R231C R201S/L266N

Cells carrying the MAS mutation have constitutively active signaling Hormone Adenyly cyclase GPCR α∗ α∗ βγ βγ GTP MAPK ATP cAMP GDP cascade RESPONSE RESPONSE RESPONSE RESPONSE

Choosing a target site F142 F146 E268 R231 F140 L266

Finding molecules that bind at R231

Dose-Dependent Relationships of Drugs 3 and 18 40 30 pmol cAMP / 25 ul lysate pmol cAMP / 25 ul lysate 30 20 20 10 10 18 3 IC 50 : 0.1 µ M IC 50 : 0.8 µ M 0 0 -10 -8 -6 -4 -10 -8 -6 -4 [DRUG], M [DRUG], M

Conclusions:  McCune-Albright syndrome can be Protein homologies modeled at the cellular level using yeast  Several intragenic suppressors of R201H Homologous biochemical activity were identified, these mutations pathways alone do not abolish Gs signaling. Variation and  The intragenic suppressors were able to selection suppress R201C and R201S mutations processes applied to  We have identified two molecules that drug design inhibit Gs-R201H in a dose-dependent Evolution is manner used to heal disease

Pilot studies Site-directed mutagenesis Matthew Koster, MD Kyle Turcic, MD Joshua Mitchell, MD Alison Dufour, MS Laura Ooms, Ph.D. Raquel Tobar-Rubin, PharmD Dahlia Sultan, PharmD Julie Carroll, MD Brittany Swen, DPT Regina Herrera, DDS Drug screens Julie Carroll, MD Yeast library construction & screen Renee Habbal, DDS Daniela Janevska, Ph.D. Evan Jenkins, MD Eraj Din, MD Joe Cruz Jennifer Haick, Ph.D. Matthew Raub Rebecca Alvarez Morgan Schumacher, MD Support and Collaborators • NIH 1R15DE020190-01 • Benedictine University, Scholl Endowment in the Natural Sciences • Dr. Eric Walters, Rosalind Franklin University