Jennifer Apodaca

Fall 2006

Gen 875 Class Presentation

Chemical Genetics and Genome Wide RNAi Screening of Cytokinesis Inhibitors and Targets.

Phenotypic screening approach using genome-wide RNAi and large scale chemical genetic screens in cultured Drosophila cells:

• To create an inventory of all genes required for cytokinesis.•Identify small molecules that target their products.

Chemical Genetics

Forward Chemical Genetics

Reverse Chemical Genetics

Biological process(cytokinesis)

Identify small molecules that cause phenotypic

changes

Identify the protein targets

Determine Mechanism

Protein of Interest(actin)

Identify small molecules that inhibit or activate

in vitro

Phenotypic effect

Chemical Genetics: Technical Overview

RNAi in Drosophila

Zamore PD, Ancient pathways programmed by small RNAs. Science 296(5571):1265-9.

Cells that fail cytokinesis acquire two nuclei

• This general phentoype is a unique and irreversible consequence of cytokinesis failure and can therefore be scored by automated fluorescence microsopy.

Parallel Screening Protocols for Cytokinesis in Drosophila Cells

Screen for compounds that cause different phenotypes

Screen for RNAi targets that cause different phenotypes

Compare small molecule and RNAi phenotypes

or

RNAi

Chemical Genetics

Methods

• After fixation, cells were stained with amine reactive TMR-NHS ester to visualize cytoplasm and Hoechst dye to visualize DNA. In the RNAi screened cells , microtubules were visualized by immunofluorescence(monoclonal anti-tubulin)

• Cells were imaged by automated fluroescence microscopy and assay wells containg a high frequency of binucleate cells were identified by automated image analysis and visual inspection.

51,000 small moleculesTaken from compound stocks Dissolved in DMSO 10 mg/ml

RNAi

Chemical Genetics

+

10,000 Kc167 Drosophila cells/well for RNAi screen

Or20,000 Kc167 Drosophila

cells/well for small molecule screen.

19,470 gene specific dsRNA

90% of annotated genome in triplicate

Incubated for 2 days, for completion of at least

one cell cycle

Incubated for 4 days, for depletion and turnover of

targeted gene products

Small Molecule Screening Results

• The 51,000 small molecules included a mixture of commercial “drug-like” molecules, natural product extracts, and natural-product-like libraries. 50 small molecules cytokinesis inhibitors were identified, 25 of the most potent and readily available were selected for further analysis.

OO

SO

OMe

N

N CF3

N

HN

O

OMe

Binucleine 5

Binucleine 6

Binucleine 4

Extract fromIrcinia ramosa(Swinholide A)

F

Cl

N

N

CN

N

N

F3C

NNH

O

NH

O

S

Cl

N

O

HN O

N

Cl Cl

ClO

Cl

Binucleine 1 Binucleine 3Binucleine 2

ON

N

N

N

OMe

MeO

NH

NH

NHS

O

O

MeO

O

OMe

N

N

NH

N

O

N

Binucleine 9Binucleine 8Binucleine 7

O

AcO

O

OH

O

MeO2C

O

OHMeO2C

O

O

O

HO

N

NMeO

MeO

NH2

N

N O

O

Binucleine 12Binucleine 11Binucleine 10

NHN

N

N

CF3

S

N

N

SMe

N CF3

CF3

HO

HN

Binucleine 13 Binucleine 15Binucleine 14

N

N

NH

N

F

OMe

Cl

S

HN

OO

NH2

HO

HO

OHHN

N

N NH

N

N

Binucleine 17Binucleine 16 Binucleine 18

N O

O

HN

NH

F

OMe

Binucleine 20

Binucleine 19

Extract fromClathria sp.2

O

ON

N

N

O

NO N

H

O OO

N

Binucleine 22Binucleine 21

Extract fromCowania mexicana

N

NH

O

O

OO

O

NH

HN

Br

HO

Binucleine 24Cytochalasin D

Binucleine 23

Binucleine 25Jasplakinolide

HN

O

O

HOO O

H H

OH

Retesting of the 25 small molecules

• Initial screen of small molecules was at a nominal concentration of 25 uM. Retesting was performed at 3 different concentrations: 100uM, 30uM and 10uM.

• To determine cross-reactivity with other species, cytokinesis inhibition in HeLa(64%, 16/25 active) and BSC-1 tissue culture cells(52%, 13/25) and growth inhibition in drug-sensitive S. cerevisiae(48%, 12/25) was assayed using all 25 compounts.

• Additionally, small molecule inhibitors were tested for their affect of actin polumerization. Binucleines 4,6, 24 and 25 inhibited pyrene-actin plumerization in a pure protein assay. Binucleines 24 and 25 are the actin binders cytochalasin D and jasplakinolide, which had been included as controls. Binucleine 4 is a natural product extract from a sponge Ircinia ramosa. DATA NOT SHOWN.

Small Molecule Results: Kc Kc Kc HeLa BSC-1 RDY98

100 ?M 30 ?M 10 ?M 30 ?M 30 ?M 250 ?M 1Binucleine w w w binucleate no no no 2Binucleine m m w diffuse DNA no no no 3Binucleine toxic m w lc toxic yes yes 4Binucleine s s s MT ext yes yes no 5Binucleine m m w binucleate yes yes no 6Binucleine toxic m m binucleate yes yes yes 7Binucleine m m w binucleate yes yes no 8Binucleine m w w lc yes yes yes 9Binucleine m w w MT ext no no no 10Binucleine w m w binucleate no no yes 11Binucleine toxic s m diffuse DNA yes no no 12Binucleine toxic m w binucleate yes yes no 13Binucleine toxic m m binucleate no no yes 14Binucleine toxic m w binucleate yes yes yes 15Binucleine toxic m m lc toxic no no 16Binucleine w w w lc yes yes yes 17Binucleine w w w MT ext yes no no 18Binucleine w w w binucleate yes no no 19Binucleine s s s MT ext yes yes yes 20Binucleine toxic w w lc no no yes 21Binucleine toxic m w lc no no yes 22Binucleine toxic w w lc yes no no 23Binucleine s s s binucleate yes yes no 24Binucleine s s s MT ext yes yes yes 25Binucleine s s s MT ext yes yes yes

Small Molecule Binucleate Phenotype

Genome-Wide RNAi Screening Results.

• Identified dsRNAs corresponding to 214 genes with phenotypes important for cytokinesis. dsRNAs that resulted in binucleate phenotypes in 2/3 replicate screens were summerzied in the final results.

• These genes resulted in either a strong, medium or weak increase in frequency of binucleate cells and represented a diverse range of predicted cell functions.

• 20% of identfied genes have been previously implicated in processes associated with cytokinesis.

• Eleven of the strong phenotypes identified the following genes: Act57B and Act5C, Myosin heavy chain(zipper), Anillin(scraps), a formin(diaphanous), Rho GTPase (Rho1) and its known guanine nucleotide exchange factor (pebble) and GTPase-activating protein (RacGAP50C), a kinesin (pavarotti), Citron kinase (CG10522), Aurora B kinase (ial), and a PRC1 homolog (fascetto).

• Discovered one new gene essential for cytokinesis (CG4454),

• INCENP, required for cytokinesis, was not identified in the screen because of failure in INCENP dsRNA synthesis. However, it was successfully resynthesized for later experiments.

Penetrance of binucleate phenotype for small molecules and RNAi hits (Figure 1).

A. Small molecules:• 24% (6/25) Strong (s)• 44%(11/25) Medium (m)• 32% (8/25) Weak (w).

B. RNAi targeted Genes• 6% (13/214) Strong (s)• 20% (43/214) Medium (m)• 74% (158/214) Weak (w)

Phenotypic classes for small molecules and genes targeted by dsRNAs

Binucleate

Large, diffuse DNABinucleate with low cell count

Binuclate with microtubule extensions

Binucleate

Distribution of phenotypic classes for small molecules and genes targeted by dsRNAs (Figure 1)

C. Phenotypic classes for small molecules

• 40% (10/25) were binucleate (b)

• 8% (2/25) binucleate with large, diffuse DNA (d)

• 28% (7/25) binucleate with low cell count (lc)

• 24% (6/25) binucleate with microtubule extensions (MT)

D. Genes targeted by dsRNAs

• 51% (109/214) (b)

• 2% (5/214) (d)

• 29% (62/214) (lc)

• 12% (25/214) (MT)

• 5% (10/214) were binucleate with low cell count and microtubule extensions

• 1% (3/214) were binucleate with low cell count and large, diffuse DNA

Predicted Functional Annotations of 214 Genes Associated with RNAi Binucleated Phenotypes (Figure 2)

Microtubule-rich extention subphenotypes correlate in both small Molecule and RNAi datasets

Cells exposed to dsRNA targeting Act5C or to Cytochalasin D.

Binucleate with diffuse DNA sub-phenotype

CG4454 RNAi phenotype and localization matches chromosomal passenger proteins

Detailed comparison of Binucleine 2 and Auroroa B complex phenotypes

Time and Concentration dependence of Binucleine2