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 Gαs

 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

GPCR Hormone

α

GDP

βγ

α

GTP

βγ MAPK cascade

Adenyly cyclase

ATP cAMP

RESPONSE RESPONSE

Cells carrying the MAS mutation have constitutively active signaling

GPCR Hormone

α

GDP

βγ

α

GTP

βγ MAPK cascade

Adenyly cyclase

ATP cAMP

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

α

GDP

βγ

α

GTP

βγ MAPK cascade Temporary cell-cycle arrest Gα-GDP inactivates βγ to restore cell division Normal yeast signaling

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 Gα causes same biochemical defect as in human protein

α*

GTP

βγ

MAPK cascade

Permanent cell-cycle arrest No Gα-GDP inactivation of βγ without a suppressor mutation Yeast with overactive Gα

The MAS mutation activates yeast Gα 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 Gα causes same biochemical defect as in human protein

α*

GTP

βγ

MAPK cascade

Permanent cell-cycle arrest No Gα-GDP inactivation of βγ without a suppressor mutation Yeast with overactive Gα

α **

GTP

α **

GXP

βγ Inactivation of βγ Restoration of cell cycle progression Colony formation

Variation

Randomly mutated library screened for intragenic suppressor mutations of an “MAS allele”

39 Clones remained FOAR

,

plasmids were sequenced

Library: 7800 unique mutants in Gpa1 R297H 55,000 clones screened

424 colonies grew on FOA

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?

GPCR Hormone

α

GDP

βγ

α

GTP

βγ MAPK cascade

Adenyly cyclase

ATP cAMP

RESPONSE RESPONSE

Homology is not identity, but it works sometimes!

W T R 2 1 H F 1 4 2 S I 1 8 2 T Q 2 1 3 S H 2 2 R R 2 3 1 C T 2 4 2 A V 2 5 6 I E 2 5 9 G K 3 R L 2 6 6 T I 3 8 3 T 20 40 60 80 100

* * * * ** **

R201H + Basal cAMP (% forskolin response)

Examining the variations at each site that can suppress the MAS mutation

F142 F140 F146 L266 E268 R231

WT R201H R231C R231S R231A R231E R231I R231K 25 50 75 100

* * * *

R201H + cAMP (% forskolin response)

McCune-Albright Syndrome

 Mosaicism for constitutively

active Gαs

 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

WT R201C R201C/F142S R201C/R231C R201C/L266T R201S R201S/F142S R201S/R231C R201S/L266N 20 40 60 80 100

* * * * * *

Basal cAMP (% forskolin response)

Cells carrying the MAS mutation have constitutively active signaling

GPCR Hormone

α∗

GDP

βγ

α∗

GTP

βγ MAPK cascade

Adenyly cyclase

ATP cAMP

RESPONSE RESPONSE

RESPONSE RESPONSE

Choosing a target site

E268 R231

F142 F140 F146 L266

Finding molecules that bind at R231

Dose-Dependent Relationships of Drugs 3 and 18

• 10
• 8
• 6
• 4

10 20 30

3 IC50: 0.8 µM [DRUG], M pmol cAMP / 25 ul lysate

• 10
• 8
• 6
• 4

10 20 30 40

18

IC50: 0.1 µM

[DRUG], M pmol cAMP / 25 ul lysate

Conclusions:

Protein homologies Homologous biochemical pathways Variation and selection processes applied to drug design Evolution is used to heal disease

 McCune-Albright syndrome can be

modeled at the cellular level using yeast

 Several intragenic suppressors of R201H

activity were identified, these mutations alone do not abolish Gs signaling.

 The intragenic suppressors were able to

suppress R201C and R201S mutations

 We have identified two molecules that

inhibit Gs-R201H in a dose-dependent manner

• NIH 1R15DE020190-01
• Benedictine University, Scholl Endowment in the Natural Sciences
• Dr. Eric Walters, Rosalind Franklin University

Support and Collaborators

Yeast library construction & screen Daniela Janevska, Ph.D. Eraj Din, MD Jennifer Haick, Ph.D. Rebecca Alvarez Site-directed mutagenesis Kyle Turcic, MD Alison Dufour, MS Raquel Tobar-Rubin, PharmD Julie Carroll, MD Pilot studies Matthew Koster, MD Joshua Mitchell, MD Laura Ooms, Ph.D. Dahlia Sultan, PharmD Brittany Swen, DPT Regina Herrera, DDS Drug screens Julie Carroll, MD Renee Habbal, DDS Evan Jenkins, MD Joe Cruz Matthew Raub Morgan Schumacher, MD