Fluoppi technology for visualizing PPIs in living cells;Case studies including 3 updated PPI pairs.

Tomohiro Nakajo1, Ken Inoue2, Chiemi Matsumi2, Taku Watanabe2

1 MBL International Corporation, Woburn, MA,USA 2 Research & Development Division, Medical & Biological Laboratories, Co., Ltd., JAPAN

Correspondence to: Tomohiro Nakajo, email: tomohiro.nakajo@mblintl.com

Abstract ; Undesired compounds such as light quenchers, aggregation prone compounds, protein expression inhibitors or cytotoxic compounds arisen from any assay systems make discovery of true PPI Modulators (PPIMs) a tough subject. In this study, activities of several small molecule PPIMs were visualized and validated by Fluoppi technology without utilizing enzyme reactions or increasing/ decreasing of light intensities for measurement. Fluoppi utilizes a simple output, where the fluorescence signal is either diffuse or present as puncta based on PPI interactions. It is easy and clear to monitor the flow of formation and/or degradation of the puncta within single cells. This allows researchers to be able to quantify many factors, including PPI modulator durations- which can vary from several minutes to hours. Modulations of PPIs such as KRAS(G12C)-Raf, Mcl1-BH3, Bcl2-BH3, BclxL-BH3, p53-MDM2, p53-MDM4 are presented.

Protein X AG (Azami Green)

Tetramer

Protein Y Ash

Oligomer

Principle : Tetramer fluorescent protein (FP-tag) and Assembly helper tag (Ash-tag) were genetically fused to Protein X and Y, respectively (Fig. 1). In these experiments, we used AG (Azami-Green) as FP-tag. To optimize a Fluoppi system, proteins of interest were fused on either N-terminus or C-terminus of each tag. The eight combinations of expression vectors and four negative controls were transfected into cell lines (Fig. 2). Fluorescence puncta were detected using an inverted fluorescence microscope or an Imaging cytometer. Regular filter sets for GFP were used for detecting AG fluorescence. Either transient or stable transfection were used for PPI analysis.

Fig. 1 Fluoppi Tags.AG fluorescent proteins form a tetramer and Ash-tags form an oligomer spontaneously. Fusing protein X or Y to each tags resulted in the formation of apparent single-molecules carrying multivalent Xs or Ys.

Fig. 2. Mechanisms of action.Before induction of PPI, Fluoppi proteins were diffusely distributed throughout the cytosol. Once PPI was induced, they formed clusters using their multivalent arms. As a result, the fluorescence signal was observed as bright and quantifiable puncta in the cells.

Fig. 5 XIAP-SmacThe assay of PPI between BIR3 domain of XIAP and Smac N-terminal peptide (AVPI) was constructed by using the Fluoppi technology. (A) Amino acid substitutions were introduced at the position of 4th isoleucine of Smac peptide. Numbers in brackets represents reported Kd values. Fluorescent punctum were detected even the Kd value was 93 µM. Fractions of puncta positive cells of five mutants were plotted against their Kd values. (B) Two commercially available Smac mimetics were treated into the stable cell line expressing XIAP-SmacFluoppi. Kinetics and potencies were variable depending on the compound. (C) Thirty minutes after addition of AT-406, the puncta were dissociated and AG tagged molecules were distributed throughout the cytosol. Dose dependent puncta dissociations by the compounds were analyzed by Cell Voyager 7000 (Yokogawa electric Corporation), resulted in IC50 value of 9.2 µM.

puncta

Nuclei

PPI signal =Total fluorescence intensity of puncta

Number of nucleus

Fig. 3 Quantification of PPI signalPPI signals were analyzed using image analysis software. Puncta regions were morphologically identified by a spot detection algorithm. The PPI signals were calculated using the equation above.

reversibleCpd.X (25 µM)

Nor

m. p

unct

a in

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Cel

l

AT-406 (μM)

0.0

0.5

1.0

300 350 400 450 500 550 600 650 700

AG ex

AG em

Shikonin

Fig. 9 Fluoppi can identify pseudo-positive resultsTo determine if Shikonin truly inhibited p53-MDM2 interaction, the images from the pilot screening were re-analyzed in detail. Notably, cells treated with Shikoninshowed a dramatic overall decrease in fluorescence intensity (B) compared to DMSO control (A). However, when brightness was enhanced using imaging software on the same acquired image, weaker yet distinct puncta fluorescence was observed (D) compared to the DMSO control (C). Because the fluorescence didn't become dispersed throughout the cell as seen when treated with Nutlin-3 (Fig.4, C, middle), we conclude that Shikonin did not inhibit protein interaction. Further investigation showed that Shikonin has an absorbance spectrum overlapping the AG fluorescence (Fig.9, E, green line). This photo quenching activity explains the reason why Shikonin induced a pseudo-positive result.Theseresults show that in-depth analysis of images taken with Fluoppi can easily identify false positive hits, a unique feature compared to other end-point based assays.

A B

C D

Nor

mal

Enha

nced

E

Wavelength (nm)

Z’ factor = 0.74IC50 = 6.3 μM

Nutlin-3

Shikonin

Inhi

bitio

n (%

)

A commercially available chemical library containing 640 chemical compounds were screened for their inhibitory potential against p53-MDM2 interaction by using the Fluoppi assay. Compounds were added to the cells in triplicate at 2 μg/mL and 10 μg/mL. 60 minutes after adding compounds, cells were

fixed and the fluorescence signals were observed using x10 objective lens on an IN Cell Analyzer 2200. Most of the test compounds did not show inhibition as Nutlin-3 did, however one compound (Shikonin) was considered to be a hit from this plot screening.

DMSO Shikonin (10 μg/mL)

Fig. 8 HTS

Low High

ABT-737 ABT-263 ABT-1995 hours / 1.2 μM

Bcl-x

LBc

l-2

Fig. 7 Inhibition of Bcl2 family interactions.(A) Fluoppi assay plasmid pairs Ash-BclxL and AG-BH3, or Ash-Bcl2 and AG-BH3, were transiently transfected into HEK293 cells by electroporation. Five hours after addition of each compound, cells were imaged by IN Cell Analyzer 1000 (GE Healthcare). The puncta intensities in each concentration of the compounds are displayed as heat maps. Fluorescence images of the cells treated with 1.2 µMcompounds are represented to show the specificities of each compounds. Dose response curves are plotted on the right. Both BclxL and Bcl2 are trucated form generated by removing their mitochondrial localization signal sequences. The BH3 domain of BAX was used for this assay (QDASTKKLSECLKRIGDELDS). All the compounds were purchased from a commercial supplier. (B) Ash-Mcl1 was transiently transfected with AG-BH3 (Green) or MR-BH3 (Red) into HEK293 cells. The same BH3 sequences as (A) were used in this assay. Commercially available A-1210477 was treated for 30 minutes to the cells resulted in dissemination of both colors of punctum. MR; Monti Red fluorescent protein.

Conc

. of e

ach

com

poun

d Co

nc. o

f eac

h co

mpo

und

ABT-737 (0.2 µM)ABT-263 (0.5 µM)ABT-199 (2.5 µM)

EC50

ABT-737 (1.2 µM)ABT-263 (5.3 µM)ABT-199 (0.2 µM)

EC50

puncta intensity / cell

Nor

m. p

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a in

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Cel

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orm

. pun

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inte

nsity

/ C

ell

(μM)

(μM)

0

1

-9-6-3 0 3 6 9121518

add Nutlin-3

wash

recovery

A

- 9 - 6 7 (min)

B

Fig. 4 Nature of punctum.Puncta formed by p53-MDM2 interaction were analyzed. (A) Two punctum were fused and relaxed into a large sphere within a few minutes. Aspect ratios (major/minor axis ratio) were plotted in the chart. (B) A small region inside the puncta was bleached and then, recoveries of fluorescence intensity were plotted. (C) After treatment with Nutlin-3 (20 µM), the compound was washed out. Afterwards, formation of puncta started again. Fluorescence intensities of punctum were quantified. Approximately 90 % of puncta intensities were recovered. These results show the high flexibility and liquidity nature of the puncta, thus it's more likely a phase-separated droplet than an aggregated solid structure.

Nor

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A

0

50

100

0.1 1 10 100

Frac

tion

of p

unct

a (+

) cel

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)

Kd (µM)

Smac (AVPI)(0.45 µM)

I4T(2.1)

I4S(4.4)

I4D(7.3)

I4A(14)

I4E(93)

-1 5 90 (min)

-1 5 90 (min)

+ AT-406 (50 µM)

+ LCL-161 (50 µM)

+ ARS-853 (33.3μM)

B

Smac (AVPI)(0.45 µM)

AVPE (I4E)(93 µM)

Mcl

1(G

reen

)

Mcl

1 (R

ed)

KRAS(G12C)-cRaf

ERK2-ERK2(ERK2 homodimer)

PPI (-) PPI (+)

-9 0 9

C

Time (min)

C 100 µM0.05 µM+ AT-406

A

47 (min)

p53-MDM4B+ RO-5963 (25 μM)

20 (h)

C

Fig.6 Various PPIs and inhibitors.(A) PPI between KRAS (G12C) and cRaf (RBD) was visualized by transient transfection of HEK293 cells with Fluoppi assay plasmid pair. Treatment with commercially available ARS-853 for 47 minutes resulted in dissemination of punctum which were formed beneath the plasma membrane. (B) p53-MDM4 interaction was visualized and inhibited by treating with RO-5963. After twenty hours, punctum were disseminated. (C) Modification of Fluoppi enabled us to visualize protein homodimerization (homoFluoppi). ERK2 homodimer was visualized after addition of EGF for 5minutes in Cos-7 cells. Commercially available ERK2 homodimerization inhibitor (DEL22379), as well as MEK inhibitor (U0126) were treated before induction by EGF, resulted in suppression of puncta formation. Fractions of numbers of puncta positive cells were represented in the chart.

+ A-1210477 (25 μM) 30 (min)

+ A-1210477 (25 μM) 30 (min)

A

B

Reference: Watanabe T, Seki T, Fukano T, Sakaue-Sawano A, Karasawa S, Kubota M, Kurokawa H, Inoue K, Akatsuka J, Miyawaki A. (2017) Genetic visualization of protein interactions harnessing liquid phase transitions. Sci. Rep. 7, 46380.