WO2005045427A1

WO2005045427A1 - High-throughput screening of clathrin-independent endocytosis to identify therapeutic molecules

Info

Publication number: WO2005045427A1
Application number: PCT/EP2004/012858
Authority: WO
Inventors: Johannes Ludger; Christophe Lamaze; Alexandre Fradagrada; Agnès SAINT-POL
Original assignee: Institut Curie; Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2003-10-24
Filing date: 2004-10-25
Publication date: 2005-05-19

Abstract

A method for screening inhibitors or stimulators of clathrin-independent endocytosis, wherein the clathrin-dependent endocytosis has previously been inhibited comprising the use of a biomarker molecule such as Shiga toxin, ricin toxin, cholera toxin, Pseudomonas exotoxins and verotoxins.

Description

"High-throughput Screening of Clathrin-Independent Endocytosis to Identify Therapeutic Molecules"

The invention relates to means for controlling clathrin- independent endocytosis. It more particularly relates to a method for screening molecules capable of specifically modulating clathrin -independent endocytosis. Endocytosis at the plasma^' membrane is a key cellular process that is required for the biologica.1 activity of a variety of molecules that are internalized into the cell. It is the pioneering work by Roth and Porter (J.Cell Biol., 20, 313-330, 1964) that identified clathrin-coated pits as specialized plasma membrane domains responsible for the selective recruitment of cargo molecules that are to be internalized into the eukaryotic cell. This seminal ultrastructural characterization was to be followed by 30 years of intense biochemical and molecular studies that established clathrin-dependent endocytosis as a major sorting mechanism responsible for the efficient uptake of various lipids, proteins and other molecules from the cell surface . Along these years, other endocytic pathways that do not use the clathrin coat had also been observed. The lack of identified physiological cargo and selective tracers has often confined the study of these so-called clathrin-independent pathways to the use of non-specific bulk uptake of solutes or exogenous molecules such as bacterial protein toxins. The recent advances in identifying the machinery of clathrin- depe-ndent endocytosis and the design of specific molecular inhibitors of this route have helped to definitely establish the physiological importance of clathrin-independent endocytosis. Thus, recent years have witnessed the resurgence of studies focused on clathrin-independent endocytosis, leading to the identification of physiological markers and the accumulation of evidence suggesting the existence of several clathrin-independent pathways. Today, the existence of these alternate pathways is no longer a matter of controversy and current efforts are aimed at elucidating the molecular machinery of these poorly characterized endocytic processes and the comprehensive description of their physiological role(s) in the cell. ^■ Several classes of cargo molecules use the clathrin- independent endocytosis route: i) Clathrin-independent endocytosis is used by a variety of signaling molecules whose activity and specificity ^■ is dependent on their routing. Examples are the prion ^• protein, interleukin 2 receptor, interferon gamma receptor, chemokine receptor CCR5, eNOS, E-cadherin, metabotropic gluta ate receptor mGluR5, M2 muscarinic acetylcholine receptors, GABA(A) .receptσrs, Urokinase-type .plasminogen activator receptor, autocrine motility factor receptor, and cholecystokinin receptor, ii) Pathogens such as semian virus 40, Chlamydia trachomatis, and Campylobacter jejuni, and protein toxins such as the plant protein toxin ricin and the bacterial toxin cytotoxic necrotizing factor 1 also use clathrin-independent endocytosis to enter into cells and to reach their target molecules, iii) The biogenesis of organells such as synaptic vesicles also depends on clathrin-independent endocytosis. Based on this original observation, the inventors have devised approaches that allows for high-throughput screening of inhibitors or stimulators of clathrin-independent endocytosis, using specific marker molecules. Modulating clathrin-independent endocytosis then leads to the modulation (inhibition or stimulation) of the function of molecules that use this pathway, thereby interfering with pathological manifestations related to their presence and/or dysfunction. Intracellular transport (including endocytosis) is a critical process that is required for the biological activity of a variety of molecules that are internalized into the cell. This process is important for bacterial toxins whose activity depends strictly on reaching intracellular cytosolic targets and signaling molecules whose activity and specificity is dependent on their routing. Membrane trafficking plays also a major- role in . controlling the signaling cascade of mitogenic receptors such as tyrosine kinase receptors that are essential in cell proliferation and tumor growth. The invention thus relates to a method for specifically screening of inhibitors or stimulators of clathrin-independent endocytosis in cells whose clathrin-dependent endocytosis has previously been inhibited. The invention more particularly relates to a - method for screening inhibitors or stimulators of clathrin-independent endocytosis, wherein the clathrin-dependent endocytosis has previously been inhibited. Said method comprises the . use of a biomarker molecule selected in the group comprising Shiga toxin, ricin toxin, cholera toxin, Pseudomonas exotoxins and verotoxins, and fragments (sub-units) thereof. The invention also relates to a method for specifically screening inhibitors or stimulators of clathrin-dependent endocytosis wherein the clathrin-dependent endocytosis has not been previously inhibited. Said method comprises the use ' of a biomarker molecule selected in the group comprising Shiga toxin, cholera toxin, verotoxins. It particularly relates to a method for the screening, of small molecules, siRNA,, antibody, phages, peptides, retroviral insertional elements, said molecules having a modulatory effect on the transport of Shiga toxin or verotoxins. The method of screening of^' the invention comprises detecting the action of said toxins on protein synthesis by infecting cells by an adenovirus coding^' for a reporter molecule or a fluorescent protein. The invention particularly relates to a method wherein said toxins are conjugated to fluorochromes to follow the protein's arrival in different comportments of the cell and detecting any change in the intracellular distribution of the toxin. Regarding Shiga toxin, it is produced by Shigella dysenteriae and by enterohemorrhagic strains of Escherichia coli is composed of two subunits, A and B. The A-subunit has a ribosomal RNA N-glycanase activity . that leads to the inhibition of protein biosynthesis by covalent modification of ribosomal RNA. The cellular entry of the A-subunit depends strictly on its binding to the B-subunit that interacts with the cellular toxin receptor, the- _. glycosphingolipid globotriaosyl ceramide (Gb₃ or CD77). The inventors have shown that after its endocytosis into the early endosome, Shiga toxin or the non-toxic B-subunit alone are transported to the endoplasmic reticulum, via the Golgi apparatus, using the so-called retrograde transport route. The toxicity of Shiga toxin relies on the modification of ribosomes in the cytosol, a location reached by retrotranslocation from the lumen of the endoplasmic reticulum. Thus, since Shiga toxin activity depends on its arrival in the endoplasmic reticulum, any molecule or drug interfering specifically with its transport from the plasma membrane to the^' endoplasmic reticulum will interfere with its toxicity. The inventors have shown for the first time that Shiga toxin entry into cells is not at all affected even if clathrin-dependent endocytosis is specifically and efficiently inhibited using the RNAi technique to down-modulate clathrin heavy chain expression (Figure 1) . Thus, Shiga toxin appears as a perfect tool to screen for modulators of the clathrin- independent endocytosis. The inventors have then established new tools and transport assays to follow clathrin-independent endocytosis of said biomarkers. These assays are used to screen small molecules, siRNAs, antibody phages, peptides, retroviral insertional elements, and others, for their modulatory effects on this^' transport step. Such compounds are blockers of the toxic activity of the Shiga toxin. Therefore, they are of interest in the treatment of the hemolytic and ure ic syndrome, one of the leading causes for childhood mortality in the world. Besides, the screen also identifies molecules interfering with the first step in transport, i.e. the binding of Shiga toxin to its receptor, Gb3 glycolipid, at the plasma membrane. In this case, the screen leads to the identification of therapeutic compounds in pathological situations where Gb3 is involved such as the entry of HIV in AIDS or in lysosomal storage diseases such as the Fabry' s syndrome where alpha- galactosidase deficiency causes an accumulation of Gb₃ leading to renal and cardiac dysfunction. Similar to Shiga toxin, other protein toxins use the retrograde route to transfer their catalytic subunits to the cytosol where they can act on their molecular targets. These toxins are, among others, cholera toxin, and the verotoxins. Inhibitors of the retrograde route neutralize their toxic activity and are therapeutic agents for the treatment of pathologies associated with these toxins, including their use as weapons. .Inhibitors of clathrin-independent endocytosis are also of use in other physiological or pathological situations that involve passage via this pathway, such as those described above, notably in the case of growth factor receptors whose implications in tumor development does not need to be shown anymore. The invention also gives means for inhibiting clathrin- dependent endocytosis and thus to unravel a clathrin- independent uptake route for the biomarkers of the invention that can be specifically inhibited by low temperature. More particularly, clathrin-dependent endocytosis inhibited for example with isRNA, then clathrin-independent endocytosis is inhibited by low temperature incubation. Under clathrin heavy chain RNAi conditions, the only uptake route open for biomarkers selected in the group comprising Shiga toxin, ricin toxin, cholera toxin, Pseudomonas exotoxins and verotoxins is clathrin-independent endocytosis . This route will be totally inhibited if endocytosis is done under low temperature conditions in clathrin heavy chain RNAi cells. Small molecules such as above mentioned will have similar effects and will then be identified. The invention thus also relates to a method comprising inhibition of clathrin-dependent endocytosis, then clathrin- independent endocytosis using, as biomarkers of said second pathway toxins trafficking through the independent endocytosis pathway such as Shiga toxin, ricin toxin, cholera toxin, Pseudomonas exotoxins and verotoxins, fragments thereof. Other characteristics and advantages of the invention will be described in the following examples given for illustrating purposes. It will also be referred to figure 1 to 7, represent,, respectively: -Figure 1: Biochemical analysis of endocytosis under clathrin dysfunction conditions. (A) siRNA efficiently down-modulated CHC expression in HeLa cells, as detected by Western blot analysis. The histogram shows the mean of two determinations. (B-C) HeLa cells were transfected with control plasmid (CTL) or plasmid expressing siRNA against CHC (siCla) . The following experiments were done under the indicated conditions: (B) endocytosis of Tf; (C) endocytosis of STxB. The means of three independent experiments are shown. Note that under conditions of inhibition of clathrin function, as revealed by inhibition of Tf uptake, endocytosis of STxB was very little affected.

- Figure 2: STx is transported to the endoplasmic reticulum, where it retrotranslocates to the cytosol and inhibits protein biosynthesis. As a . consequence, the expression of LacZ reporter gene (-BFA, square) is inhibited. In the presence of BFA, the inhibitory action of STx on protein biosynthesis is prevented (+BFA, circles) . n=5 experiments

- Figure 3: Results of the screening with the Curie Institute's small molecule library. Among 4560 tested drugs, 150 protect the cell from VTl toxicty at a similar or higher level than BFA does. In BFA treated cells (drug 1), the LacZ residual activity is 42,7% relatively to control cells without toxin. Colum 2 (« drug2 ») represents residual activity (7,2%) in control cells treated with 2μL DMSO. - Figure 4 : Duplicate of the 10 first-tested drugs

- Figure 5: In HeLa cells, STxB is transported from the plasma membrane to the endoplasmic reticulum through the early endosome et the Golgi apparatus. The B-subunit is internalized at low temperature into early endosomes , (A) where it colocalized with the transferrin receptor (B) . Cells are then warmed up at 37°C .for 30 min (C-D) or 4 hrs (E-F) . After- 30 min, the B-subunit is detected in^' a perinuclear localization (C) where it is colocalized with -a trans-golgi -marker (D) .

After 4 hours, the B-subunit is present in reticular structures (E) , colocalized with ER marker (F) .

- Figure 6: HeLa Cells are transfected with an adeno.virus coding for the GFP protein (A) . The^' retrotranslocation of STx in the cytosol leads to inhibition of protein synthesis, and the reporter gene cannot be expressed (B, arrows) . In the presence of BFA, the inhibitory action of STx on protein biosynthesis is prevented and expression of reporter gene is restored (C) . Figure 7: which illustrate the inhibitor of clathrin- dependent endocytosis to unravel the clathrin-independent uptake route for Shiga Toxin.

Examples

1. -A novel cell-based assay for the measurement of clathrin-independent endocytosis using the measurement ' of Shiga toxin action on cytosolic ribosomes

Principle: Shiga toxin is endocytosed by clathrin-independent endocytosis after binding to its receptor, Gb3. The toxin eventually reaches the cytosol where it inhibits protein biosynthesis by ribosomal inactivation. Any molecule that interferes with clathrin-independent endocytosis prevents the toxin' s arrival in the cytosol and therefore its inhibitory action on protein synthesis. This principle was used to screen for molecules that restore a normal level of protein synthesis, i.e. prevent the inhibitory action of Shiga toxin on protein synthesis, through a block of clathrin-independent endocytosis.

Analysis of protein biosynthesis: The biosynthesis of proteins is followed by infecting cells by an adenovirus coding for a reporter molecule, such as galactosidase, luciferase, green fluorescent protein (GFP) , and others. Other reporter gene- expressing viruses can be used, such as retroviruses, vaccinia virus, and others. Alternatively, stable cell lines with inducible reporter gene expression are ^' used. In the case of galactosidase-expressing adenovirus, the protocol is as follows: DO

HeLa cells, or other cells such as Vero cells, A431 cells, are grown at 37 °C in 96-wells plate containing 50 μL DMEM w/o phenol red/ 1%FCS / 5mM sodium pyruvate / 4 mM glutamin /

0.01% Streptomycin-Penicillin ("culture medium"), at a confluency of 2.10⁴ cells/well.

Dl

25 μL of culture medium containing the drug to be 'tested are added to each well. This step and the following are executed by a robot, such as BioRobot3000 from Quiagen. As a positive control, brefeldin A (BFA) at 15 μg/mL (3x) is added in 25 μl of culture medium. The rAdv5 virus dilution is prepared in culture medium from a stock at 2.7.10¹¹ infectious particles (IP)/mL. 3 dilutions were tested: 90, 180, 360 IP/cell. For each of them,^' different concentrations of Shiga toxin (stock 165 ng/μL) are prepared : 0.5, 1, 4 ng/mL in medium already containing -the virus. The conditions are summarized below :

rAd 5 90 180 360 90 180 360 (part/cell)

Stx (ng/mL) 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.5 1 0 0.. 4 4 4 4 4 4

BFA (5μg/mL) - +

50 μL of culture medium containing virus/Shiga toxin are added to the cells (see table for the conditions) . Incubate 16h at

37°C / 5%C0₂.

D2 _. '

125 μL of lysis buffer "(0.1 M sodium phosphate / 45 mM βmercapto-ethanol / 1 mM MgCl₂ / 0.2% Tx-100) are added per well, followed by 20 min incubation at room temperature. 25 μL O-Nitrophenyl-beta-D-Galactopyranoside (ONPG) at 3.5 mg/mL in 0.1 M sodium phosphate are added. Incubate until yellow coloration appears (5 min at RT) . The reaction is stopped with 50 μL Na₂C0₃ 2M and the absorbance is read at 420 nm. Typical results are presented in Figure 2. 2. Use of high throughput screening for inhibitors of clathrin-independent endocytosis DO

HeLa cells, or other cells such as ,Vero cells, A431 cells, are grown at 37 °C in 96-wells plate containing 200 μL . DMEM w/o phenol red/l%FCS/5mM sodium pyruvate/4 mM glutamin/0.01% Streptomycin-Penicillin ("culture medium") , at a confluency of 2.10⁴ cells/well.

Dl ,

2 μL of the drug to be tested are added to each well at a final concentration of 0.1 mM. This step and all the following are executed by a robot, such as BioRobot3000 from Quiagen. As a positive control, brefeldin A (BFA) at 50 μg/mL (lOOx) is added in 2 μl of DMSO.

Adenovirus expressing beta-galactosidase (rAdv5) dilutions are prepared in culture medium from a stock at 2.7.10¹¹ infectious particles (IP)/mL. We use a dilution of 45 IP/cell. Shiga toxin (stock 165 ng/μL) is prepared at 4 ng/mL in medium already containing the virus.

20 μL of culture medium containing virus/Shiga toxin are added to the cells, following incubation for 16 hours at 37°C/5% C0₂. D2 11 μL of lysis buffer (0.1 M sodium phosphate/450 mM βmercapto- ethanol/10 mM MgCl₂/2% Tx-100) are added per well, followed by 20 min incubation at room temperature. 50 μL O-Nitrophenyl-beta-D-Galactopyranoside (ONPG) at 1,75 mg/mL in 0.1 M sodium phosphate are added, followed by incubation until yellow coloration appears (15 min at RT) . The reaction is stopped with 50 μL Na₂C0₃ lM/4mM EDTA and the absorbance is read at 405 nm. Typical results are presented in Figure 3 and Figure 4. 3. Use of high- throughput immunofluorescence analysis of retrograde transport In another embedding, trafficking of Shiga toxin will be analyzed by immunofluorescence using high-throughput image processing. For example, the In Cell analyzer is a recently developed high-throughput screening platform with three incorporated high-speed, cooled CCD cameras that simultaneously image red, blue, and green emissions from fluorochromes . Subcellular compartment analysis, i.e. endosomes, nucleus, Golgi apparatus, is made possible as the system generates information on a cell-by-cell basis. The inventors' approach exploits Shiga toxin B-subunit conjugated to fluorochromes, such as Cy3, FITC, TRITC, to follow the protein' s arrival in different compartments of the cell: the endosome, the Golgi apparatus, and the endoplasmic reticulum (Figure 5). As reference points, the. Golgi ^• apparatus, the nucleus, or other compartments are labeled with appropriate probes. Any drug interfering with clathrin- independent endocytosis of Shiga toxin, thus with trafficking of Shiga toxin itself and its activity, results in changes in the intracellular distribution of the toxin, which is detected on a cell-to-cell basis. In this case, cells were infected with a virus, such as adenovirus, coding for the GFP protein or- another fluorescent protein. Alternatively, stable cell lines with inducible fuorescent protein expression are used. The arrival of Shiga toxin in the cytosolic leads to protein synthesis inhibition. As a result, no GFP fluorescence is detectable. Any drug interfering with the trafficking of Shiga toxin will result in protection of protein biosynthesis and the appearance of GFP fluorescence in the cells (Figure 6) . This approach, as the ones described above, presents the advantage that a positive signal is linked to inhibition of transport. False positive results are avoided that result from cell death or other unspecific drug action. Other protein biosynthesis inhibiting toxins, such as cholera toxin, verotoxins, were also used in this assay. ^"4. Inhibition 1) of clathrin-dependent endocytocytosis, and 2) of clathrin-independent endocytosis Methods : The experiments were done as described in Saint-Pol et al (2004) Dev. Cell^' 61525-534. Briefly, Shiga toxin B- subunit (STxB) was biotinylated using NHS-SS-Biotin (Pierce) . Serum starved cells were detached from plates with 2mM EDTA in PBS and incubated in presence of 1 μM biotin-STxB for 30 minutes on ice. After washing, cells were incubated at 30 °C for the indicated times (1.5 x 10⁵ cells per data point) Subsequent treatment with lOOmM of the non-membrane permeable reducing agent sodium 2-mercaptoethanesulfonic acid (MESNA) on ice for 20 min led to cleavage of biotin on cell surface exposed STxB. After washing, excess MESNA was quenched with 150 mM iodoacetamide for 20 min and after centrifugation, cells were lysed in blocking buffer (10 mM Tris, pH 7.4, 1 mM ^'EDTA, 50 mM NaCl, 0.2% BSA, 0.1% SDS Triton X-100) before loading on ELISA plates coated with anti-StxB antibody (13C4). After an overnight incubation, biotinylated STxB were detected using streptavidin-HRP (Roche) . RESULTS: the inhibition of clathrin dependent endocytosis by treatment of cells with anti-clathrin heavy chain (CHC) RNAi does not affect the endocytic uptake^' of Shiga toxin, when the experiment is done at 37 °C (Fig. 7; compare control (CTRL) 37 to RNAi (CHC) 37) .

Claims

1. A method for screening inhibitors or stimulators of clathrin-independent endocytosis, wherein the clathrin- dependent endocytosis has previously been inhibited.

2. The method of claim 1, comprising the use of a biomarker molecule selected in the group comprising Shiga toxin, ricin toxin, cholera toxin, Pseudomonas exotoxins and verotoxins, and fragments (sub-units) thereof.

3. A method for the screening of inhibitors or stimulators of clathrin independent endocytosis comprising using a marker molecule selected in the group comprising Shiga toxin, cholera toxin and verotoxins.

4. The method according to anyone of claims 1 to 3 for screening of small molecules, siRNA, antibody, phages, peptides, retroviral insertional elements, said molecules having a modulatory effect on the transport of said toxins, particularly of Shiga toxin.

5. The method according to anyone of claims 1 to 4, comprising detecting the action of said toxins on protein synthesis by infecting cells by an adenovirus coding for a reporter molecule or a fluorescent protein.

6. The method according to anyone of claims 1 to 5, wherein said toxins are conjugated to fluorochromes to follow the protein's arrival in different comportments of the cell and detecting any .change in the intracellular distribution of the toxin.