Probe for Single Cell Ribosome Imaging and Tracking

Abstract

A B S T R A C T This invention pertains in general to ribosome imaging, in particular ribosome imaging in live cells. In particular, the invention relates to a compound (probe) that selectively binds with eukaryotic ribosomes, such as human ribosomes. Also provided is for a method of preparing the compound and for a method for detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome using the compound.

Description

Title: Probe for Single Cell Ribosome Imaging and Tracking FIELD OF THE INVENTION

[001] This invention pertains in general to ribosome imaging and / or tracking, in particular ribosome imaging and / or tracking in live cells. ln particular, the invention relates to a compound (probe) that selectively binds with eukaryotic ribosomes, such as human ribosomes. Also provided is for a method of preparing the compound and for a method for detecting and / or tracking a eukaryotic, preferably human, 608 ribosomal subunit or ribosome using the compound. BACKGROUND OF THE INVENTION

[002] The background description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[003] Ribosomes are formidable macromolecular machines responsible for protein synthesis and essential components of the cell (1). In eukaryotic cells, ribosomes comprise two subunits and span about 20 nm in diameter.

[004] Ribosomes are extraordinarily large and complex compared to regular enzymes (2). Eukaryotes have 808 ribosomes, each consisting of a small (408) and large (608) subunit. The 408 subunit normally comprises an 188 RNA and 33 proteins whereas the large 608 subunit comprises 58 RNA, 288 RNA, 5.88 RNA subunits and 49 proteins.

[005] Ribosomes have been the focus of scientific investigation for decades due to its critical function, intriguing structure and promise as a cytotoxic drug target (3). In recent years, advances in single-cell technologies have uncovered variability in ribosomal functioning between individual cells (46). In particular, ribosomal dynamics (7) and tracking of ribosome movement in single cells to study translation localization have been of interest (810).

[006] Ribosomes can be studied with a combination of physical techniques, including Xray crystallography (11), electron microscopy (12, 13) and NMR (1416). These techniques have revealed the fascinating structure of ribosomes in spectacular detail.

[007] Biochemical methods such as ribosomal profiling and in vitro assays have uncovered numerous mechanistic insights of ribosome functioning (1720). To study differences in ribosome dynamics between individual cells, techniques based on single-cell sequencing protocols (5, 6) or fluorescence microscopy are being developed. The latter, so far relied on immunostaining (21) or fluorescent in situ hybridization (22) of fixed samples; metabolic labeling of newly synthesized proteins (23, 24); or complex fluorescently tagged protein constructs (7, 25, 26).

[008] However, selective probes for live-cell ribosome imaging are currently lacking, impeding single cell studies of ribosomal movement. Although a few fluorescent dyes that unselectively intercalate with RNA, based on quinolinium (27) and naphtalimide (28) scaffolds, have been developed no fluorescent probes exist that selectively bind to ribosomes.

[009] In light of this, new products, compositions, methods and uses that allow for ribosome imaging and / or tracking, in particular ribosome imaging and / or tracking in live cells would be highly desirable but are not yet readily available.

[010] In particular, there is a clear need in the art for reliable, efficient, and reproducible products, compositions, methods and uses that allow to be used in ribosome imaging and / or tracking in live cells. Furthermore, there is a clear need to new compounds (probes) that selectively binds with eukaryotic ribosomes, such as human ribosomes, and that are suitable for ribosome imaging and tracking, in particular in eukaryotic single cells, including human cells.

[011] Accordingly, the technical problem underlying the present invention can be seen in the provision of such products, compositions, methods and uses for complying with any of the aforementioned needs, or at least providing the public with a useful choice. The technical problem is solved by the embodiments characterized in the claims and herein below. 8UMMARYOFTHE INVENTION

[012] As embodied and broadly described herein, the present invention is directed to the surprising finding that probes may be provided that selectively bind with eukaryotic ribosomes and that allow for ribosome imaging and tracking, in particular in eukaryotic single cells.

[013] According to a first aspect, there is provided for a compound, wherein the compound comprises a cycloheximide moiety and a fluorescent moiety, and wherein the fluorescent moiety is linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety. It was surprisingly found that linking the fluorescent moiety to the C13 position of the cycloheximide moiety, a compound is provided that has high affinity for the eukaryotic ribosome, in particular the 608 subunit of the ribosome, including the 608 subunit of the human ribosome, while at the same time providing the compound with a detectable fluorescent moiety. In addition, it was surprisingly found that the compound according to the invention enables live-cell imaging and tracking over time of ribosomes. With the compound according to the invention has now become possible to visualize ribosomes and ribosome movement in individual live cells, for example, using methods as disclosed herein.

[014] According to another aspect, there is provided for a cell, preferably a live cell, more preferably a human cell, even more preferably a human live cell comprising the compound according to the invention. / nlp

[015] According to another aspect, there is provided for a composition comprising the compound according to the invention. The composition may, for example, further comprise a suitable solution or buffer, for example comprising the compound according to the invention.

[016] According to another aspect, there is provided for a method of preparing the compound according to the invention. The method comprises coupling of the fluorescent moiety to the cycloheximide moiety through the C13 position of the cycloheximide, for example using methods as disclosed herein.

[017] According to yet another aspect, there is provided for a method of detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome, wherein the method comprises contacting the 608 ribosomal subunit or ribosome with a compound according to the invention. By contacting the compound according to the invention with the 608 ribosomal subunit or ribosome the compound is allowed to form a complex with the 608 ribosomal subunit or ribosome which can be detected, for example using methods based on fluorescent detection, including fluorescent microscopy or methods based on flow cytometry, such as methods based on fluorescence-activated cell sorting (FAC8). The methods according to this and other aspects of the invention allow for visualization of ribosomes (and / or the 608 ribosomal subunit(s)) as well as for movement of such ribosomes in individual cells, including human live cells. At the same time the methods according to the invention allow for sorting of cells using, for example flow cytometry, including FACS or FACS-based methods, based on (fluorescent) detection of ribosomes in such (single) cells.

[018] According to another aspect, there is provided for a method of treating a eukaryotic, preferably human, cell, the method comprising contacting the cell with a compound according to the invention, so as to allow the compound to form a complex with a 608 ribosomal subunit or ribosome. In a preferred embodiment, the method according to this and other aspects of the invention inhibits protein synthesis by the cell that is treated with the compound according to the invention. BRIEF DESCRIPTION OF THE DRAWING8

[019] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[020] Figure 1: Concept of cycloheximide probes. A) Structure of cycloheximide and proposed example structures of fluorescent probes with modifications at the C13 position of cycloheximide. B) 8chematic concept of using fluorescent rotor probes to visualize ribosomes using fluorescent microscopy.

[021] Figure 2: 8ynthesis of probes, in vitro activity and fluorescence. A) Probes 1-3 were synthesized starting from cycloheximide, employing a previously published procedure for C13 modification and amide coupling to CCVJ fluorophores with varied linker length (see SI for synthetic scheme and procedures). B) Fluorescent microscopy of HEK 293T / 17 cells incubated with 10 [M probes 1-3. The insets for probe 2 and 3, display the same image with enhanced contrast. 8calebar is 10 um.

[022] Figure 3: Cellular performance of RiboBright. A) Fluorescent microscopy images of HEK 293T / 17 cells incubated with 10 [M RiboBright only and pretreated with cycloheximide or phyllantoside for 30 min. Fluorescence intensity of indicated cross sections is shown. The insets forCHX and phyllantoside display the same image with enhanced contrast 8calebar is 10 um. B) Colocalization between ER (top) and mitochondrial (bottom) specific counterstains with RiboBright determined using the Manders coefficient method. While both ER and mitochondria (M1) colocalize with RiboBright not all of RiboBright (M2) colocolizes with ER and mitochondria.

[023] Figure 4: RiboBright allows tracking of ribosomal movement in single-cells. A) Representative image of HEK293T / 17 cells stained with 10 uM RiboBright for time- lapse imaging (left) and corresponding trajectories obtained by tracking individual fluorescent foci (right). Scale bars are 15 um. B) Example trajectories displaying apparent directionality, normal and confined movement. Scale bars are 1 um. C) MSD curves of the trajectories shown in panel (B), and their associated or values. D) Averaged MSD curves of the trajectories of two biological replicates (dashed and solid line), classified into confined, normal and super diffusive based on their a values. Error bars represent the standard error of the mean. The number of confined tracks are 9854 and 9740, normal-diffusive tracks are 2483 and 2516, and super-diffusive tracks are 654 and 618 for replicate 1 and 2 respectively. The inset represents the percentage of tracks associated with each category for replicate 1. E) Average diffusion coefficient of each type of diffusion. Error bars represent the standard error of the mean (number of biological replicates = 2). The inset shows the diffusion coefficient of the single trajectories of replicate 1. The box plot shows the median and the interquartile range, and the whiskers represent the dispersion of the data.

[024] Figure 5: Representative images of live or fixed HEK293T / 17 cells stained with 10 uM RiboBright (Ex405 / Em525, 100x oil objective, scale bars are 15 um). DESCRIPTION Definitions

[025] A portion of this disclosure contains material that is subject to copyright protection (such as, but not limited to, diagrams, device photographs, or any other aspects of this submission for which copyright protection is or may be available in any jurisdiction.). The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or patent disclosure, as it appears in the Patent Office patent file or records, but otherwise reserves all copyright rights whatsoever.

[026] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein.

[027] For purposes of the present invention, the following terms are defined below.

[028] As used herein, the singular form terms a, an, and the include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a cell includes a combination of two or more cells, and the like. For example, a method for administrating the compound according to the invention includes the administrating of a plurality of the compound (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules).

[029] As used herein, about and approximately", when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of120% or 110%, more preferably 15%, even more preferably 11%, and still more preferably 10.1% from the specified value, as such variations are appropriate to perform the disclosed invention. Unless otherwise clear from context, all numerical values provided herein include numerical values modified by the term about.

[030] As used herein, and / or refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[031] As used herein, "at least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. As used herein, the term "at most" a particular value means that particular value or less. For example, "at most 5" is understood to be the same as "5 or less" i.e., 5, 4, 3, -10, -11, etc.

[032] As used herein, comprising or to comprise is construed as being inclusive and open ended, and not exclusive. 8pecifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components. It also encompasses the more Iimiting to consist of.

[033] As used herein, conventional techniques or methods known to the skilled person refer to a situation wherein the methods of carrying out the conventional techniques used in methods of the invention will be evident to the skilled worker. The practice of conventional techniques in molecular biology, biochemistry, chemistry, cell culture, flow cytometry, microscopy and related fields are well-known to those of skill in the art and are discussed, in various handbooks and literature references.

[034] As used herein, "exemplary" or for example means "serving as an example, instance, or illustration," and should not be construed as excluding other configurations, including those disclosed herein.

[035] As used herein, "in vivo" refers to an event that takes place in a cell, in particular a live cell; "in vitro" refers to an event that takes places outside the cell. Detailed description

[036] The invention is defined herein, and in particular in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention.

[037] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment envisaged herein. Thus, appearances of the phrases in one embodiment or in an embodiment in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments.

[038] Furthermore, while some embodiments described herein include some, but not other features included in other embodiments, combinations of features of different embodiments are also envisaged herein, and form different embodiments, as would be understood by those in the art.

[039] It is contemplated that embodiments described herein in relationship to any method, use, or composition can be implemented with respect to any other method, use or composition described herein. Embodiments discussed in the context of methods, use and / or compositions of the invention may be employed with respect to any other method, use or composition described herein. Thus, an embodiment pertaining to one method, use or composition may be applied to other methods, uses and compositions of the invention as well.

[040] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[041] As embodied and broadly described herein, the present invention is directed to the surprising finding that probes may be provided that selectively bind with eukaryotic ribosomes and that allow for ribosome imaging and tracking, in particular in eukaryotic single cells.

[042] According to a first aspect, there is provided for a compound, wherein the compound comprises a cycloheximide moiety and a fluorescent moiety, and wherein the fluorescent moiety is linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety.

[043] Cycloheximide (4-{(2R)-2-[(18,38,58)-3,5-DimethyI-2-oxocyclohexyl]-2- hydroxyethyl} piperidine-2,6-dione) is a widely-used eukaryotic protein synthesis inhibitor. The compound is a fungicide produced by Streptomyces griseus and blocks the elongation phase of translation by binding to the E-site of the 608 ribosomal subunit to block eEF2-mediated tRNA translocation. The molecular structure of cycloheximide is shown below, with the carbon as position 13 indicated. o HN OH o o . C13 position H "Me

[044] In the compound according to the invention, cycloheximide is coupled with a fluorescent compound through the C13 position of the cycloheximide. In other words, in the compound according to the invention a fluorescent moiety is linked to the cycloheximide moiety via the C13 position shown above.

[045] In an embodiment, the compound according to the invention is a compound represented by Formula I: o HN OH o '? Me o / Formula l wherein: [F] is a fluorescent moiety, X is any atom, preferably N, and / \ / links the fluorescent moiety to the cycloheximide moiety.

[046] In some embodiments, X as shown in Formula l is part of the fluorescent moiety according to the invention. In a preferred embodiment, X as shown in Formula l represents a nitrogen atom (N).

[047] As will be understood by the skilled person, / \ / represents a covalent bond that links the fluorescent moiety to the cycloheximide moiety.

[048] The skilled person will understand that the fluorescent moiety may be linked directly to the carbon at the C13 position, or that the fluorescent moiety is linked indirectly to the carbon at the C13 position (i.e. wherein the fluorescent moiety is linked to the cycloheximide moiety via an intermediate atom or functional group).

[049] Within the context of the current invention the term cycloheximide moiety is to be understood as comprising a cycloheximide group that is connected through the C13 position of the cycloheximide to any other moiety (within the context of the current invention, wherein the other moiety is a moiety that is fluorescent).

[050] In a preferred embodiment of the invention, the cycloheximide moiety in the compound according to the invention retains the biological activity of cycloheximide (despite it now being linked to a fluorescent moiety via the C13 position).

[051] In a more preferred embodiment of the invention, the cycloheximide moiety in the compound according to the invention retains the biological activity of cycloheximide, wherein the biological activity is inhibition of translational elongation and / or inhibition of protein synthesis.

[052] Within the context of the current invention the term fluorescent moiety is to be understood as comprising a fluorescent group that is connected through the C13 position of the cycloheximide. As indicated above, the fluorescent moiety may be linked directly to the carbon at the C13 position of the cycloheximide moiety, or the fluorescent moiety is linked indirectly to the carbon at the C13 position of the cycloheximide moiety (i.e. wherein the fluorescent moiety is linked to the cycloheximide moiety via an intermediate atom or functional group). Within the context of the current invention, the fluorescent moiety in the compound according to the invention retains the activity of the original fluorescent molecule (despite it now being linked to the cycloheximide moiety via the C13 position).

[053] This skilled person understands that the term retain activity and the like, and as used herein, indicates that the moiety in the molecule according to the invention has an activity that is comparable, preferably similar, to the activity of the compound from which the moiety originates (for example, cycloheximide or a fluorescent molecule).

[054] In some embodiments, the compound according to the invention non- covalently binds with the 60 8 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit, i.e. by non-covalent binding.

[055] In some embodiments, the compound according to the invention covalently binds with the 60 8 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit, i.e. by covalent binding, for example by the use of photo-activable groups.

[056] In some embodiments, the compound according to the invention comprises further moieties. In such embodiments, to the compound according to the invention comprising a cycloheximide moiety and a fluorescent moiety, additional moieties are linked. Such additional moieties may be linked to the compound according to the invention through the cycloheximide moiety and / or through the fluorescent moiety. In a preferred embodiment, such compound according to the invention, comprising, in addition to the cycloheximide moiety and the fluorescent moiety, retains the activity of being able to selectively bind with eukaryotic ribosomes and allow for ribosome imaging and tracking, in particular in eukaryotic single cells, as disclosed herein. Examples of such additional moieties include, for example, additional fluorescent groups or functional groups including groups that provide enzymatic and / or biological activity.

[057] In a preferred embodiment, the compound according to the invention does not comprise such additional moieties.

[058] As indicated above, the fluorescent moiety comprised in the compound according to the invention may be linked directly to the carbon at the C13 position of the cycloheximide moiety, or the fluorescent moiety may be linked indirectly to the carbon at the C13 position of the cycloheximide moiety.

[059] In the latter case, the fluorescent moiety may be linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety by means of a spacer. ln such embodiments, the spacer links the fluorescent moiety to the cycloheximide moiety through the C13 position of the cycloheximide moiety.

[060] As will be understood by the skilled person, the spacer (or linker) may first be connected to the cycloheximide moiety, after which the fluorescent moiety is linked to the spacer, thereby linking the fluorescent moiety to the cycloheximide moiety.

[061] As will be understood by the skilled person, the spacer (or linker) may also first be connected to the fluorescent moiety, after which the cycloheximide is linked to the spacer, thereby linking the cycloheximide moiety to the fluorescent moiety.

[062] Within the context of the current invention the term spacer is to be understood as any group that can form, in the compound according to the invention, a link between the cycloheximide according to the invention and the fluorescent moiety of the invention. As will be understood by the skilled person, any (molecular) spacer is allowed as long as the spacer does not negatively affect the activity of the compound according to the invention as disclosed herein (i.e. as long as the compound according to the invention is suitable as a compound that binds with eukaryotic ribosomes and allow for ribosome imaging and tracking, in particular in eukaryotic single cells, as disclosed herein.).

[063] Therefore, in some embodiments according to the invention, there is provided for a compound according to the invention, wherein the fluorescent moiety comprises a spacer, and wherein the fluorescent moiety is linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety via the spacer.

[064] As shown in the examples, a spacer may affect the fluorescent properties of the compound according to the invention. A skilled person is well capable of establishing by routine experimentation - whether a spacer is desirable in the compound according to the invention or not, and if so, what type of spacer may be used.

[065] In some embodiments, it is preferred that the spacing between a particular fluorescent moiety and the cycloheximide is increased. ln such embodiments, a spacer may be used that increases the spacing (distance) between the cycloheximide moiety and the fluorescent moiety.

[066] In some embodiments, it is preferred that the spacing between a particular fluorescent moiety and the cycloheximide is not increased. ln such embodiments, no spacer should be used that increases the spacing (distance) between the cycloheximide moiety and the fluorescent moiety.

[067] The skilled person understands how the spacing between the cycloheximide moiety and the fluorescent moiety in the compound according to the invention may be varied, for example by including spacers with varying length (e.g. chain length), for example as disclosed herein in the examples.

[068] In some embodiments, the spacer comprises a chain length of, for example 0 to 10 (linear, adjacent) atoms, preferably carbon atoms. In some embodiments the spacer comprises a chain length of less than 10 (linear, adjacent) atoms, preferably carbon atoms. ln some embodiments the spacer comprises a chain length of more than 10 (linear, adjacent) atoms, preferably carbon atoms.

[069] In a preferred embodiment, the compound according to the invention does not comprise an (additional) spacer linking the fluorescent moiety to the cycloheximide moiety through the C13 position of the cycloheximide moiety.

[070] In a preferred embodiment, the compound according to the invention comprises an (additional) spacer linking the fluorescent moiety to the cycloheximide moiety through the C13 position of the cycloheximide moiety.

[071] As will be understood by the skilled person, the fluorescent moiety may be linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety via any type of covalent bond (see Formula I above).

[072] However, in a preferred embodiment, the fluorescent moiety is linked to the cycloheximide moiety through the C13 position of the cycloheximide moiety via an amide bond. ln such preferred embodiment, the compound according to the invention is a compound represented by Formula II below (and wherein [F] represent a fluorescent moiety): 0 HN OH O U g Me o / Formula II

[073] In a further preferred embodiment, the compound according to the invention is a compound represented by Formula III below (and wherein [F] represent a fluorescent moiety): 0 HN OH o U 5 Me o / 5 °| H MN Me Formula III

[074] In even more preferred embodiments, the compound according to the invention is a compound selected from Formula IV, Formula V or Formula VI: o N o o El ; Me o . % \ N "Me CN N Formula IV, o N o o tl ; Me o . % \ N / \ / \N Me CN N Formula V, O N o O lj 5 Me CN 0 N / \ / \ "" && \ / \o N Me o Formula VI.

[075] In a preferred embodiment, the compound according to the invention is according to Formula IV. In a preferred embodiment, the compound according to the invention is according to Formula V. In a preferred embodiment, the compound according to the invention is according to Formula VI.

[076] In a more preferred embodiment, the compound according to the invention is according to Formula IV.

[077] As will be understood by the skilled person, the fluorescent moiety in the compound according to the invention is not in any particular way limited and any suitable fluorescent moiety may be used. Therefore, according to the invention any fluorophore may suitably be used as the fluorescent moiety in the compound according to the invention.

[078] However, in some embodiments the fluorescent moiety is a fluorescent moiety that, when comprised in the compound according to the invention, that allows the compound according to the invention to be taken up by eukaryotic cells, such as eukaryotic live cells. The skilled person understands how to select a suitable fluorescent moiety that can be comprised in the compound according to the invention and / or how to determine its suitability using conventional techniques, for examples, as disclosed herein.

[079] Some non-limiting examples of suitable fluorophores and fluorescent moieties include naphthalimides, benzoxadiazoles, nitrobenzoxadiazoles, fluorescein, FTC, Texas Red, phycoerythrin, rhodamine, coumarins, carboxytetramethylrhodamine, DAPI, indopyra dyes, Cascade blue coumarin, NBD, Lucifer Yellow, propidium iodide, porphyrin, Bodipy, CY3, CY5, alexa, and derivatives and analogues thereof.

[080] Therefore, in some embodiments, the fluorescent moiety comprised in the compound according to the invention is one selected from naphthalimides, nitrobenzoxadiazoles, fluorescein, FTC, Texas Red, phycoerythrin, rhodamine, coumarins, carboxytetramethylrhodamine, DAPI, indopyra dyes, Cascade blue coumarin, NBD, Lucifer Yellow, propidium iodide, porphyrin, Bodipy, CY3, CY5, alexa, and derivatives and analogues thereof.

[081] However, in some embodiments it is preferred that the fluorescent moiety comprised in the compound according to the invention comprises a fluorescent rotor, preferably wherein the fluorescent rotor is 9-(2-Carboxy-2-cyanovinyl) julolidine (CCVJ).

[082] The skilled person is well-aware of fluorescent rotors. Such rotors include, for example, 4- (N, N-dimethylamino)-benzene, -benzylidene and -cinnamylidene derivatives and coumarin-like compounds, acridiziniums and thioflavin-T, and have photophysical characteristics which depend on the environmental parameters (polarity, viscosity, temperature, etc.) and are useful in detection of biomolecular interactions. The fluorescence of molecular rotors contingent upon target binding and that makes them a versatile tool for detecting specific biomolecular interactions (see J. Am. Chem. Soc. 2014, 136, 17, 61596162).

[083] Also provided is for a compound according to any one of the previous claims, wherein the compound is complexed with a eukaryotic, preferably human, 608 ribosomal subunit or with a eukaryotic, preferably human, ribosome (comprising the 608 ribosomal subunit).

[084] Also provided is for cell, preferably a eukaryotic cell, even more preferably a human cell, comprising the compound according to the invention. The cell may be a single cell. The cell may be a mouse cell. The cell may be a rodent cell. The cell may be present in a eukaryotic organism. The cell may be isolated from a eukaryotic organism. Preferably the cell is ex vivo, i.e. outside of a eukaryotic organism. Most preferably, the cell is alive cell.

[085] In another most preferred embodiments, the cell is a fixed cell, for example a permeabilized fixed cells. It is surprisingly found that the compounds according to the invention may suitable be used in not only live (living) cell, but also in cells that have been fixed (by using methods well-known to the skilled person).

[086] Thus, in some embodiments the cell according to the invention is a fixed cell. It was surprisingly found by the inventors that the compound according to the invention provides for suitable fluorescent signals in fixed cells. Preferably the cells are fixed using polymeric formaldehyde (PFA; for example, 1-8% at room temperature) or using methanol (for example 100% methanol, for example at 4°C).

[087] As will be understood, the compounds according to the invention can suitable used in fixed cells, for example for spatial / structural analysis (for example, of the ribosomes or the cells) in different pathological conditions, for analysis of ribosomal density, as this provides insight into how this is regulated in development, aging or disease, for use in pathological studies and / or longitudinal studies for patient, for multiplexed imaging, for analysis of changes with specific biological or pathological applications, for use in retrospective or long-term studies wherein it may not be not feasible with live-cell imaging alone. The ability to use the compounds according tin the invention in fixed tissue of fixed cell is in particular important since imaged based mRNA and protein quantification is currently performed in fixed cells. Therefore, the compound according to the invention, and use thereof in fixed cells provides an additional application where it can be multiplexed with other existing methods. Finally, clinical tissue samples are often fixed. The current invention now surprisingly allows for the compounds according to the invention to be used in such fixed clinical tissue samples, for example as part of clinical studies.

[088] Also provided is for a composition comprising a compound according to the invention. The composition preferably comprises at least one other compound. Such compound is not in particular limited and may, for example, be selected from a solute, a buffer, a stabilizer, a compound to be screened, and the like. Also contemplated are compositions wherein the compound according to the invention is comprised in a package, for example in a container.

[089] Also provided is for a method of preparing a compound according to the invention, wherein the method comprises coupling of the fluorescent moiety to the cycloheximide moiety through the C13 position of the cycloheximide, for example as exemplified in the examples.

[090] In the Examples the synthesis is disclosed for compounds according to the invention, and wherein the fluorescent moiety is based on 9-(2-Carboxy-2-cyanovinyl) julolidine (CCVJ). However, as will be understood by the skilled person, the method of preparing a compound according to the invention may utilize any other suitable fluorescent moiety, including those disclosed herein. Therefore, for this embodiment according to the invention, the Examples are to be understood as exemplifying the method of preparing a compound according to the invention, independent of the fluorescent moiety that is to be linked to the cycloheximide through the C13 position of cycloheximide.

[091] Also provided is for a method of detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome, wherein the method comprises contacting the 608 ribosomal subunit or ribosome with a compound according to the invention, so as to allow the compound to form a complex with the 608 ribosomal subunit or ribosome and detecting the complex.

[092] The skilled person understands how to contact the 608 ribosomal subunit or ribosome with the compound according to the invention, and under what conditions, for example, such as those described herein, so as to allow the compound to form a complex with the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit.

[093] In some embodiments, the eukaryotic, preferably human, 608 ribosomal subunit or ribosome is an isolated eukaryotic, preferably human, 608 ribosomal subunit or ribosome, for example isolated from a cell, preferably human cell.

[094] In other embodiments, the eukaryotic, preferably human, 608 ribosomal subunit or ribosome is an isolated eukaryotic, preferably human, 608 ribosomal subunit or ribosome is comprised in a cell, preferably live cell, preferably live human cell.

[095] Detection of the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit may be by any suitable means. Any methodology that can detect fluorescence may be used in the method according to the invention. Such methodology includes, but is not limited to fluorescent microscopy, time-lapse fluorescent microscopy, and flow cytometry, such as FACS based methods.

[096] The method according to the invention allows detecting of the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit in single cells and over time. Therefore, the method according to the invention allows detection the dynamics of ribosomes in single cells, in particular single live cells. For example, in some embodiments, detection in a live cell may be performed every 100 1000 millisecond, for example every 400 milliseconds for a period of 10 seconds 300 seconds or more, for example for 60 seconds, for example, using time-lapse fluorescent microscopy. Such method allows tracking of ribosomes in single cells, as shown in the Examples herein.

[097] In another embodiment, detection of the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit includes flow cytometry, for example FAC8. The skilled person is well aware of such techniques and how to apply these in the context of the current invention, for example, as described herein. Using flow cytometry, for example FACS, it is possible to determine, for example, total fluorescence in a live cell, and sort cell based thereupon.

[098] Therefore, also provided is for the method of detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome wherein the method is an in vitro method or an in vivo method, and / or wherein the method is for detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome in a cell, preferably wherein the method is for single-cell tracking of a eukaryotic, preferably human, 603 ribosomal subunit or ribosome.

[099] Therefore, also provided is for the method of detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome wherein detecting comprises fluorescent microscopy, time-lapse fluorescent microscopy, and / or flow cytometry.

[100] In another embodiment, detection of the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit with the compound according to the invention allows for the quantifying of ribosome content in individual cells, for example using microscopy or flow cytometry. Therefore, also provided is for a method of quantifying of ribosome content in a cell, the method comprising detecting a eukaryotic, preferably human, 608 ribosomal subunit or ribosome with a compound according to the invention and based thereupon quantify ribosome content of the cell.

[101] As will be understood by the skilled method, the compounds according to the invention also allow for identifying and / or isolating (sub)populations of cells based on the ribosome content of the cells. In one embodiment, the identifying and / or isolating of such (sub)population involves flow cytometry and / or fluorescent microscopy.

[102] Also provided is for a method of treating a eukaryotic, preferably human, cell, wherein the method comprising contacting the cell with a compound according to any one of the previous claims, so as to allow the compound to form a complex with a 608 ribosomal subunit or ribosome. As explained above, the skilled person is well aware on how to perform such method, for example as disclosed herein.

[103] In some embodiments, such method provides for inhibition of protein synthesis in the cell that is treated with the compound according to the invention.

[104] Also provided is for a method of screening for candidate agents that bind to the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit, wherein the method comprises providing a compound according to the invention to a cell and providing a candidate agent to the cell and detecting binding of the compound according to the invention with the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit, wherein in change in binding of the compound according to the invention is indicative for binding of the candidate agent with the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit. ln some embodiment the compound according to the invention and the candidate agent are provided to the cell at the same time. In some embodiments the compound according to the invention is provided to the cell before the candidate agent is provided to the cell. In some embodiments the cell is first provided with the candidate agent before the cell is provided with the compound according to the invention. Therefore, in some embodiment there is provided for a method of screening candidate agents that bind to the 608 ribosomal subunit or ribosome comprising such 60 8 ribosomal subunit, and preferably, that inhibit ribosome function (e.g. inhibit protein synthesis).

[105] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[106] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by references.

[107] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[108] It will be understood that all details, embodiments, and preferences discussed with respect to one aspect of embodiment of the invention is likewise applicable to any other aspect or embodiment of the invention and that there is therefore not need to detail all such details, embodiments, and preferences for all aspect separately.

[109] Having now generally described the invention, the same will be more readily understood through reference to the following examples which is provided by way of illustration and is not intended to be limiting of the present invention. Further aspects and embodiments will be apparent to those skilled in the art. EXAMPLES Example Summary

[110] In this study, we describe the development of fluorescent probes (including one referred to as RiboBright; a compound according to Formula IV above) that selectively binds to the 608 subunit of the eukaryotic, including human, ribosome. The probes are based on the scaffold of the natural product cycloheximide and is modified with a fluorescent moieties, in particular fluorescent rotors at the C13 position of the cycloheximide, using C-H activation.

[111] It was surprisingly found that the probes retain biological activity based on in vitro and in vivo translation assays and selectively interact with the ribosomal E-site as shown by DMS-MaPseq experiments and competition with cycloheximide and phyllantoside.

[112] The compounds according to the invention, including RiboBright, enable visualization of ribosomes using fluorescence microscopy and are compatible with flow cytometry. Furthermore, they facilitate tracking of ribosome movement in live cells.

[113] Time-lapse imaging revealed three distinct ribosomal populations that displayed confined, normal and super diffusion respectively. Given these results, we show that the compounds according to the invention will enable ribosomal studies using fluorescent microscopy and allow for investigating ribosome movement at the single-cell level.

[114] Fluorescent imaging of the eukaryotic ribosome with the compounds according to the invention will help elucidate cellular functioning of this complex macromolecular machine in health and disease. The compounds according to the invention are compatible with live-cell confocal imaging and selectively binds and stains ribosomes. The compounds according to the invention can be applied to time-lapse microscopy to track movement of ribosomes in single cells and with thatwe identify three populations that display distinct motions. The compounds according to the invention, including RiboBright, are user-friendly probes that is easily applied by simple incubation and can reproducibly be visualized with different techniques.

[115] To enable live-cell imaging of ribosomes, we here use cycloheximide (CHX) to develop ribosome-selective fluorescent probes (Fig. 1B). By appending a fluorophore to the C13 position of cycloheximide (Fig. 1A) using C-H activation (33, 34), we provide a compound that has high affinity for the 608 subunit of the human ribosome, while rendering it fluorescent.

[116] In vitro translation assays showed that the probes display similar biological activity as CHX. Using DMS-MaPseq we further demonstrated that probe 1 (Formula IV, dubbed RiboBright), and CHX interact with the same ribosomal binding pocket.

[117] Importantly, applying the probe to Human Embryonic Kidney (HEK293T / 17) cells and performing fluorescent microscopy allowed us to visualize ribosomes and ribosome movement in individual live cells. Moreover, co-staining validated localization of the fluorescent signal to the ER, confirming ribosomal staining. Finally, time-lapse imaging revealed three distinct ribosomal populations that exhibit confined, normal and super diffusion respectively. Results and Discussion Design and synthesis of cycloheximide probes

[118] To obtain fluorescent probes that selectively interact with the ribosome and therefore enable live-cell ribosomal imaging we used CHX. This natural product binds the 608 subunit and displays high affinity for the ribosomal exit site (E-site), competing with deacetylated tRNAs(3). Previous studies showed that modifications at the glutarimide, C8 alcohol and C10 ketone (Fig. 1A) diminish binding affinity (35, 36). Recently, an elegant synthetic route was described (33)(34) to modify CHX at the C13 position using C-H activation (Fig. 2A) affording the C13 aminated analogue 2. We therefore chose to append a fluorophore at the C13 position to yield fluorescent probes.

[119] Three probes that differ in spacing between CHX and the fluorophore were prepared, corresponding with the compounds according to Formula IV, Formula V and Formula VI.

[120] The fluorescent rotor CCVJ was chosen as the fluorophore (Fig. 2A). This molecular rotor has been reported to display increased emission when interacting with a biomolecular target (3741), which could provide additional resolution during live- cell imaging. Probes were synthesized by conjugating aminated analogue 2 with carboxylic acid bearing CCVJ derivatives, yielding probes 1-3 (corresponding with compounds according to Formula IV, Formula V and Formula VI) (Fig. 2A). In vitro and cellular fluorescent assessment of probes 1-3.

[121] To assess if probes 1-3 retained CHXs biological activity, in vitro translation assays were performed. Binding of the probes to the ribosome would block translation of a Iuciferase-encoding mRNA, which can be measured spectroscopically. Probes 1- 3 and CHX were incubated at increasing concentrations with the in vitro translation system and the luminescence measured. CHX displayed an IC5o of 0.45 uM. Interestingly, probes 1-3 showed similar activity with ICsos of 0.62 uM, 1.05 uM and 0.49 uM respectively, indicating that the conjugated fluorophore indeed does not substantially affect ribosomal binding.

[122] Probes 1-3 displayed similar fluorescent properties in PBS with emission maxima at 500 nm (Aex: 460 nm). To study the potential increased emission in crowded environments, emission spectra were recorded in glycerol. Emission and quantum yields increased for all probes, indicating that fluorescence is indeed enhanced in restricted environments (42).

[123] Next, we assessed the performance of probes 1-3 in live HEK293T / 17 cells. When cells were incubated with probe 1, defined foci with enhanced fluorescence were observed throughout the cytoplasm (Fig. 2B), irrespective of cellular seeding concentration. Interestingly, significantly weaker signal was observed for probe 2 and 3. This could be explained by the increased spacing between the CHX scaffold and the CCVJ rotor, where the increased rotational freedom when bound to the target corresponds to decreased emission (42). Based on these results, probe 1 was used for further experiments and dubbed RiboBright. RiboBright selectively binds ribosomes in human and mouse cells

[124] To further verify that RiboBright binds ribosomes in cells, a cellular translation assay was performed. HEK293T / 17 cells were pretreated with RiboBright orCHX and subsequently exposed for 30 minutes to the puromycin analogue O-propargyl- puromycin (OPP), allowing for incorporation of OPP into newly synthesized proteins (43). After fixation, the nascently translated polypeptides were fluorescently labelled by click chemistry and imaged. As expected, with increasing concentrations of CHX and RiboBright, the incorporation of OPP into newly synthesized proteins is decreased. This occurred more gradually for CHX than for RiboBright, indicating that RiboBright inhibits protein synthesis and therefore might bind more readily than CHX in live cells.

[125] To determine if RiboBright retained affinity for the same binding pocket as CHX, a DMS-MaPseq experiment was performed (44). To this end, HEK293T / 17 cells were treated with RiboBright orCHX for 30 min and then exposed to 2% DMS. Cells treated with RiboBright or CHX showed strong protection of C4341 (3). The N-3 position of this cytosine forms a hydrogen bond with the C8 alcohol of CHX, protecting it from methylation by DMS (34). These results suggest that RiboBright indeed interacts with the same binding pocket as CHX, allowing selective imaging of ribosomes.

[126] To further verify the selectivity of RiboBright towards the ribosomal E-site, we conducted cellular competition experiments with CHX. HEK293T / 17 cells were pretreated with increasing concentrations of CHX for 30 min and then incubated with RiboBright and imaged immediately. A significant lower signal was observed compared to cells treated with RiboBright only (Fig. 3A), which displayed a concentration-dependent trend verifying that the probe likely occupies the same binding pocket as CHX.

[127] To exclude off-target interactions that CHX and RiboBright could have in common due to their similar structure, we repeated this experiment with phyllantoside (45). This structurally unrelated compound interacts with the same binding pocket as CHX and should therefore also be able to compete with RiboBright. When cells were pretreated with phyllantoside for 30 minutes, only a weak fluorescent signal was observed comparable to CHX pretreatment (Fig. 3A), further validating the selective nature of the probes.

[128] In addition, treatment with actinomycin D, decreased nuclear RiboBright signal consistent with previously reported effects (46). Lastly, RiboBright was further tested in mouse embryonic stem cells as well as by flow cytometry with both adherent (HEK 293T / 17) and suspension (K562, Jurkat) human cell lines. Together these results indicate that RiboBright selectively binds ribosomes in a range of cell types and can be visualized by microscopy and flow cytometry.

[129] Next, organelle specific counterstains were applied to determine the cellular localization of RiboBright. To this end, cells were simultaneously incubated with RiboBright, along with ER and mitochondrial staining respectively. Next, the degree of colocalization between RiboBright and the two respective stains was quantified using the Manders Coefficient. While both mitochondria and ER colocalized strongly with RiboBright (Fig. 3B, left), not all of RiboBright signal colocalized with either of these two stains. Overall, fluorescent signal from the ER staining correlated more strongly with RiboBright signal (mean Pearson r = 0.78) than fluorescent signal from the mitochondria (mean Pearson r = 0.58). This indicates that RiboBright binds to ribosomes in the ER as well as mitochondrial ribosomes or ribosomes in proximity to mitochondria. RiboBright allows for single-cell tracking of ribosomes in live cells

[130] After validating that RiboBright is suitable for imaging ribosomes, we examined if the probe could be used to track ribosomal movement. To this end, cells were incubated with RiboBright and imaged with a frequency of 400 ms for 1 minute, enabling the tracking of individual fluorescent foci (Fig. 4A). Interestingly, different fluorescent foci either displayed rapid movement with an apparent directionality, displayed random movement or remained relatively stationary (Fig. 4B). For each track, the mean squared displacement (MSD) was computed at increasing time lags (Fig. 4C). The dependence of MSD on time, allows quantitative determination of the type of motional behaviour (Brownian or non-Brownian) where nonlinear regression of the MSD data yields an or value per trajectory (Fig. 4C, see respective d) (47). Specifically, a linear relationship (d=1) indicates normal diffusion; confined diffusion resulting from steric barriers, crowding, or binding to cellular structures, is characterized by d<1; and super-diffusion caused by direct motion, typically exhibits d>1. Notably, as previously described (47), some foci transitioned between different behaviors. The Packing coefficient (Pc) can identify these transitions (48), as Pc values inversely scale with the area of confinement. As expected, the instantaneous diffusion coefficient (Di) calculated for the same time windows, decreased during the Brownian-defined periods. While for such transitions d-estimation from MSD is a generalization, the dominant behavior of a track could still be estimated and the average MSD of all tracks showed good reproducibility between replicates. Specifically, 75.9% of tracks were characterized by confined diffusion, 19.1% by normal diffusion, and only 5% exhibited super diffusion (Fig. 4D). The quantified diffusion coefficients also showed very good reproducibility among replicates, both at a population level (Fig. 4E) and at a single-cell level. Average D was ~0.007 um? / sl, ~0.015 umZ / s1 and ~0.029 um? / sl, for the confined, normal and super diffusive trajectories, respectively. This is within a comparable range as previous reported values of untethered (0.047 um? / sl) or tethered (0.0016 um? / sl) mRNAs associated with ribosomes (49). Collectively, these data show that RiboBright can be used to quantify ribosome movement in live cells.

[131] Having confirmed that RiboBright is capable of binding ribosomes in live cells, we tested its ability of binding them in fixed cells. For this purpose, we fixed cells for 15 minutes with 4% PFA (polymeric formaldehyde) at room temperature or 100% methanol at 4°C, followed by two washing steps with PBS 1x, 5 minutes each. After fixation, RiboBright was added to the cells to a final concentration of 10 uM, in PBS 1x. The results are shown in Figure 5, showing comparable fluorescent signals. Conclusion

[132] Taken together, we have developed fluorescent probes for live cell imaging of ribosomes by, in the examples, appending a CCVJ fluorescent rotor to the C13 position of CHX. Using an in vitro translation assay, we show that the probe retains its biological activity as compared to CHX, despite the incorporation of the fluorescent group. Future iterations involve conjugation of other fluorophores.

[133] Cellular assays reveal that the compounds according to the invention selectively interacts with the same binding site as CHX. 3pecifically, using DMS- MaPseq a strong mutational signal was observed for the binding pocket residue C4341, that disappeared in the presence of the probe, suggesting occupancy of the binding site and protection against DMS modification. Using competition experiments with two structurally dissimilar natural products that both bind the E-site of ribosomes CHX and phyllantoside no fluorescence was observed. This suggests that RiboBright is competed out of the binding pocket. Together, these results imply that the probe selectively binds to the E-site of ribosomes, enabling ribosomal imaging.

[134] When applied in time-lapse imaging, three distinct ribosomal populations were apparent. These populations displayed different movements that were classified as confined, normal and super diffusion. Thus, we demonstrate that this setup can be implemented to track ribosomes in live single cells using the compounds according to the invention, including RiboBright.

[135] The compounds according to the invention, including RiboBright are useful for studying ribosomal functioning in live cells and will enable investigations into ribosome movements at the single cell level. Material and Methods

[136] Biochemical Methods

[137] Quantum Yield Determination.

[138] The quantum yield of probe 1 (RiboBright) was determined following the method of Mchedlov-Petrossyan and coworkers (1) using aminofluorescein as a reference and the following equation: 1 _ Em(1)x <p<2> <p( ) t

[139] cp(1): Probe 1 quantum yield

[140] Em(1): Probe 1 fluorescence at 460 nm excitation and 500 nm emission for water and 460 nm excitation and 510 nm in glycerol

[141] cp(2): 5-Aminofluorescein quantum yield from literature

[142] cp(water) = 0.008

[143] cp(glycerol) = 0.1

[144] Em(2): 5-Aminofluorescein fluorescence at 460 nm excitation and 500 nm emission and 460 nm excitation and 510 nm in glycerol.

[145] In vitro translation assays.

[146] The in vitro translation assays were performed using Flexi Rabbit Reticulocyte Lysate System, purchased from Promega Corporation. The assays were performed according to manufacturers instructions in a total reaction mixture of 10 uL, containing 7 uL Flexi Rabbit Reticulocyte Lysate, 0.1 uL Amino Acid Mixture Minus Leucine, 0.1 uL Amino Acid Mixture Minus Methionine, 0.28 uL Potassium Chloride (2.5 M), 0.2 uL Milli-Q water, 0.2 uL Luciferase Control RNA (1 mg / mL), 1 uL Luciferase Assay Reagent and 1.12 uL control medium (in H2O with 1.5% DMSO). The reaction mixtures were incubated at 30 °C for 4 hours while luminescence was being measured in real time. Each assay was performed in triplicates and the luminescence signal was measured using BioTek Synergy H1 Plate Reader.

[147] Cell culture.

[148] HEK293T / 17 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher) supplemented with 4.5 g / L D-Glucose, L-glutamine, Sodium Pyruvate, 10% (v / v) fetal bovine serum (FB8, ThermoFisher) and antibiotic solution (50 U / mL Penicillin and 50 ug / mL Streptomycin). mESC-E14 (mESCs) were obtained from Hendrik Markss group at Radboud University (2). Cells were cultured in plates precoated with 0.1% gelatin (Sigma Aldrich, 48723-500G) and maintained undifferentiated in high glucose-Dulbeccos Modified Eagles Medium (GibcoT'V', 41965039) supplemented with 15% fetal bovine serum (GibcoT'V', A3840002), 2 mM L- glutamine (GibcoT'V', A2916801), 1 mM sodium pyruvate (GibcoT'V', 11360039), 1% penicillinstreptomycin (GibcoT'V', 15140122), 0.1 mM beta-mercaptoethanol (GibcoTM 31350010) and 500 U / mL recombinant leukemia inhibitory factor (LIF; Millipore, ESG1107) at 37 °C in 5% CO2. Jurkat and K562 obtained from Leor Weinbergers group (Gladstone Institutes, UCSF) cells were grown in suspension in RPMI 1640 medium (ThermoFisher) supplemented with 2 mM L-Glutamine, 10% (v / v) fetal bovine serum (FB8, ThermoFisher) and antibiotic solution (50 U / mL Penicillin and 50 ug / mL Streptomycin). Cells were passaged every 2-3 days, using 0.05% Trypsin / EDTA (GibcoT'V') for the adherent cell lines. All cell lines were incubated at 37°C, in a humified 5% CO2 atmosphere, until reaching a confluency of 70-80%.

[149] DMS-MaPseq.

[150] HEK293T / 17 cells were seeded at a concentration of 1x105 cells / mL in a 6-well plate two days prior treatment. At the moment of treatment, the culture medium was replaced with warm complete DMEM supplemented with 10 [M probe, 10 uM CHX or an equivalent volume of DMSO. Cells were incubated with the respective molecule for 30 minutes at 37°C. A final concentration of2% ofDMS was added directly to the wells and cells were incubated for exactly 4 minutes at 37°C. Medium was discarded and cells were washed with fresh Wash Buffer (60% v / v PBS 1x, 40% v / v ß- mercaptoethanol). Cells were collected, washed once with PBS 1x and centrifuged at 500 g for 4 minutes at RT. The cell pelletwas lysed using TRlzol reagent and incubated at RT for 5 minutes. Subsequently, 0.5 volume of chloroform was added to the cell Iysate. Samples were centrifuged for 15 minutes, at 12000 g at 4°C. The aqueous phase was transferred to clean tubes. RNA was then purified using the Zymo® Research Clean & Concentrator-5 kit, as per manufacturers instructions. RNA was eluted in 15 uL nuclease-free water (NF water). Total RNA quality was assessed by capillary electrophoresis (Bioanalyzer, Agilent RNA 6000 Nano Kit).

[151] The probed RNA was reverse transcribed using TGIRT-IIITM Reverse transcriptase. Briefly, to 5 uL of probed RNA, 1 uL of 5X Reverse Transcription buffer (250 mM Tris-HCI pH 8.3; 375 mM KCI; 15 mM MgCI2), 0.5 uL of random hexamers (10 uM) and 0.5 uL of dNTPs (10 mM) was added. The mixture was incubated for 8 min at 98 °C, to simultaneously fragment and denature the RNA, and immediately transferred to ice. Sample was supplemented with 0.25 uL of SUPERaseIn (20 U / uL), 0.25 uL DTT (0.1 M) and 0.25 uL TGIRT-IIITM Reverse transcriptase (200 U / L). Reverse transcription was carried out at typical conditions (25 °C 10 min, 57 °C 2 hours, hold 4 oC). Then, 1 uL of ice-cold proteinase K (1 ug / uL) was added to each sample, to degrade the reverse-transcriptase. Reaction was carried out at 37 °C for 20 minutes. Proteinase K was deactivated by adding 1 uL proteinase inhibitor cocktail (Sigma Aldrich, cat. P186; 1:2 dilution in water). Second strand synthesis was carried out using the NEBNext® Ultra IITM Non-Directional RNA Second Strand Synthesis kit (New England Biolabs, cat. E6111). Whilston ice, 2 uL of NEBNext® UltrallTM Second Strand Synthesis reaction buffer, 1 uL of NEBNext® Ultra IITM second strand synthesis enzyme mix and 11 uL of nuclease-free water were added to the sample. The reaction was carried out as per manufacturer instructions. The resulting DNA was cleaned by adding 72 uL NucleoMagTM NGS Clean-up and Size Select beads (Macherey- Nagel), eluting in 12.5uL NF water. The eluted dsDNA was then used as input for the KAPA Hyperprep kit with Library amplification from Roche (cat. No. 07962363001). Sequencing was carried out on an Illumina NextSeq 2000 system with a P2 100 cycle flow cell.

[152] All sequencing data was analyzed using the RNA-framework.(3) Briefly reads were aligned to Human Ribsomal RNA recovered from the RNA central Database (Accession codes: UR80000726FAB and UR8000075EC78): rf-map -mp "-very- sensitive-local" -cmn 0 -ctn -cq5 20 - b2 -ow bi. Briefly -mp enables soft-clipping during alignment. Commands -ctn and -ctm remove reads with or trims ambiguously called bases. -cq5 trims all bases with phred quality lower than 20 on the 5 end. Mutations were counted with: rf-count -m -es -ni -nd -na. This counts the mutations, whilst ignoring all insertions, deletions and ambiguously mapped segments and ensuring that nucleotides neighboring the mutation are above 20 in quality. Mutation rates were calculated using rf- norm, ignoring G and T bases and setting minimum coverage to 400: rf-norm -nm 1 -n 400 -rb AC -ni-sm 4. Mutation rate was calculated as reported by Rouskin and coworkers (4) and normalized with 2-8% normalization.

[153] Translation blocking assay.

[154] The retained ability of the probe to block translation was tested using Click-iTTM Plus OPP Alexa FluorTM 647 Protein Synthesis Assay Kit. HEK293T / 17 cells were seeded at a concentration of 3.5x105 cells / mL in a ibiTreat 8-well #1.5 polymer coverslips (Ibidi) previously coated with Poly-L-Lysine 0.01% (Sigma-Aldrich). The day after, medium was replaced with warm complete DMEM containing the probes orCHX at the final desired concentration, or an equivalent volume of DMSO. After 30 minutes of incubation at 37°C, medium was replaced with warm complete DMEM containing 20 [M CIick-iT® OPP reagent and the same drug used in the pre-incubation step. Cells were incubated for 30 minutes at 37°C, then fixed using PBS in 4% formaldehyde and permeabilized with 0.5% Triton X-100. Fresh Click-iT® OPP Reaction Buffer Additive was prepared by diluting the 10X solution 1:10 in deionized water. A CIick-iT® Plus OPP reaction cocktail was prepared by mixing CIick-iT®OPP Reaction Buffer, Copper Protectant, Alexa Fluor® picolyl azide and Click-iT® OPP Reaction Buffer Additive. Cells were washed once with PBS, the reaction cocktail was added to each well and incubated for 30 minutes at room temperature, protected from light. The solution was removed and cells were rinsed once with of Click-iT® Reaction Rinse Buffer. The HCS NuclearMaskTM Blue Stain was diluted 1:2000 in PBS to obtain a 1X HCS NuclearMaskTM Blue Stain working solution, which was added to the wells, and these were incubated for 30 minutes in the dark. Samples were washed twice with PBS and were ready for imaging.

[155] Probes and ER / mitochondria / staining forimage acquisition.

[156] Cells were seeded at a concentration of 3.5x105 cells / mL in an ibiTreat 8-well #1.5 polymer coverslips (Ibidi) previously coated with Poly-L-Lysine 0.01% (Sigma- Aldrich) for HEK293T / 17 cells, or 0.1% gelatin (Sigma Aldrich, 48723-500G) for mESCs. The day after seeding, culture medium was replaced with warm complete DMEM containing the probes at the final desired concentration, or an equivalent volume of DMSO for the negative controls. For the competition assay cells were pre- treated for 30 minutes with CHX (Sigma Aldrich) or Phyllanthoside (Biosynth). The probe was added directly to the wells at a final concentration of 10 uM, and images were acquired exactly one minute after addition. For ER and mitochondrial staining, cells were incubated for 30 minutes at 37°C respectively in Hank's Balanced Salt Solution (Thermofisher) containing 1 uM ER-tracker (Thermofisher), or complete medium containing 200 nM MitoTracker (Thermofisher). After incubation, the staining solution was exchanged with complete medium containing probe at a final concentration of 10 uM. For the Actinomycin D (Fisher Scientific) treatment, cells were treated for 0, 3, 6 and 10h with 8 uM Actinomycin D or with an equivalent volume of DMSO as control. The probe was finally added directly to the wells at a final concentration of 10 uM, and samples were immediately imaged.

[157] Single image acquisition and image analysis.

[158] Live and fixed cells were imaged with an Andor spinning disk confocal with FRAP-PA equipped with an Andor iXon 897 EMCCD camera, using a 60x / 1.40 NA, and a 100x / 1.40 NA oil objective. The probes were excited using a 488 nm laser at 25- 40% power with an exposure time of 300 ms. For the translation blocking assay, NuclearMaskTM Blue Stain and OPP signal, indicating translation, were excited using a 405 nm laser at 12% power and a 647 nm laser at 17% power respectively, with 300 ms of exposure time. For each x,y position, a z-stack of 18 steps of 1.15 pm was acquired. For the ER and mitochondrial staining, samples were excited using a 561 laser at 12% and 23% power respectively, and 300 ms of exposure time. Cell masks were obtained using the probe or nuclear signal in the deep-learning method Cellpose 2D (5). The pre-trained models from the notebookjointly developed by the Jacquemet (https: / / cellmig.org / ) and Henriques (https: / / henriqueslab.github.io / ) laboratories were used for this purpose (freely available on GitHub: GitHub - HenriquesLab / ZeroCostDL4Mic: ZeroCostDL4Mic: A Google Colab based no-cost toolbox to explore Deep-Learning in Microscopy). The obtained masks were used to calculate single-cells intensity using in-house Python scripts. Manders and Pearsons coefficients of single cells were calculated from thresholded images using in-house Python programs.

[159] Time lapse acquisition and tracking analysis.

[160] Time lapses of HEK293T / 17 cells stained with 10 uM probe were acquired with an NL5+ line-scanning confocal on an Olympus lX83 microscope equipped with a Prime BSI Express sCMOS camera, using a 100x / 1.40 NA oil objective, in a temperature and CO2 controlled chamber. Samples were excited with a 405 nm laser at 25% power with emission collected at 525 nm, and with 200 ms of exposure time. Images were captured at intervals of 400 ms over a duration of 1 minute. Fiji (6)was used for background subtraction and filtering. Fluorescent signals were tracked in Trackmate (TrackMate (imagej.net)) (7) using the Nearest-neighbor tracker. Tracks consisting of fewer than 20 timepoints were omitted from the analysis. For each track the MSD was computed using the equation (1): 1 Ntl 2 . . , MSD(AtT)=Ê ; (xx,) (1) + (Ym _Yi)2 where At is the time interval between two successive frames, T represents the number of lag times, N is the total number of timepoints, and (xi,yi) denote the spatial coordinates at time i. The time dependence of the M8D, described by the equation (2) was used to classify each track into the different types of diffusional behaviour: MSD(t) = 4DAtr (2) where D denotes the diffusion coefficient, d represents the anomalous diffusion exponent

[161] Employing nonlinear curve fitting, or was estimated from the MSD data. Tracks were categorized based on their a values as follows: a s 0.8 denoted confined diffusion, or 2 1.2 signified superdiffusion, and values falling in between were classified as normal diffusion. The diffusion coefficient (D) was computed for each MSD value of the same track using the equation (3): MSD D =m (3)

[162] Subsequently, the average diffusion coefficient across all MSD values of the same track was calculated. For the single cell analysis, cell masks were obtained as described above, and the tracks were assigned to each mask based on their coordinates. The packing coefficient (Pc) at each time point i was calculated as described by Renner et al. (8) using the following equation (4): i+n1 Pci =Z (X Xi)2+2(Yi+1 Yi)2 (4) Si i=0 where (xi,yi) are the spatial coordinates at time i, n is the length of the time window (n=10), and Si is the surface area of the convex hull of the trajectory segment between time points i and i+n.

[163] The value Si was calculated using the ConvexHull function in 8ciPy, Python (9). The corresponding instantaneous diffusion coefficients (Dinst) were calculated on the same time window, using the following equations (5, 6): i+n1 Squared Displacement = Z (Xi+1 xi)2 + (yi+1 yi)2 (5) i=0 D- : Squared Displacement (6) 4Atn

[164] Flow cytometry.

[165] HEK293T / 17, Jurkat and K562 cells were collected and diluted to a concentration of 1x106 cells / mL in phenol red free media, containing 10 uM probe. Cells were incubated in a pre-heated water bath at 37°C for 30 minutes, and gently agitated every 15 minutes. After incubation, flow cytometry analysis of the probe signal was performed in a BD FACSCaIibur Flow cytometer (Ex488 / Em525). Data analysis was performed with BD Flowjo.

[166] General Synthesis

[167] Temperature are given in degrees Celsius (oC). Unless otherwise stated, reactions were carried out at room temperature (18-25 oC). Progress of reactions were determined using thin-layer chromatography (TLC) silica gel-coated plates (Merck 60 F254) using the described eluent. Column chromatography was performed with VWR chemicals silica gel (0.040-0.063 mm, pore size 60 A). The separation was visualized with UV light and / or by potassium permanganate or ninhydrin staining. Low resolution electron-spray ionisation mass spectra (MS ESI) were recorded on a Thermo Finnigan LCQ Advantage Max Ion Trap mass spectrometer. NMR spectra were recorded in the indicated solvent at 25 °C on a JEOL 500 MHz spectrometer equipped with a Royal HFX probe. Chemical shifts are displayed in parts per million (ppm) with respect to TMS (6 = 0.00 ppm), CDCI3 (6 = 7.26 ppm) or other indicated deuterated solvent as internal reference for 1H NMR; and CDCI3 (6 = 77.16 ppm) or other indicated deuterated solvent as internal reference for 13C NMR. Coupling constants are reported in hertz (Hz) as J values. The multiplicity is described by the number of coupling constants, the number of peaks, and the pattern in the signal (s=singlet, d=doublet, t=triplet, q=quartet, dd= doublet of doublets, dt=doublet of triplets, ddd=doublet of doublet of doublets, m=multiplet, br=broad). The peak assignments in 1H and 13C spectra are based on 2D COSY, HSQC and HMBC spectra. Each stated 1H NMR, 13C NMR and Mass spectra result is depicted in the section SPECTRA. QO OSNH, Ft î F O 0 ms HN o o HN o O c 1? Me TMSOTf o ï Me 4 DCM. -78 toO°C thspb. PIDA Me Me MgO. i-PrOAc, 12h 3(42° / -:) CHX.1 0 ms HN O O O H Me O Pyridine HN OI'LIO M . _* ? e F que ACN:H;O_70°C. 12h 0 Os _ HsN "Me @ ó 0 F @ @TFA c73%; 2 5 (56%) Scheme !: Synthesis ofaminatcd cycloheximide analogue 2.

[168] 4-((R)-2-((1S,38,5S)-3,5-dimethyl-2-oxocyclohexyl)-2- ((trimethy / silyl)oxy)ethyl)piperidine-2,6-dione (3) O TMS\ HN O H o Me O ? fie

[169] Compound 3 was synthesized according to a literature protocol (10). In brief, cycloheximide (4.0 9, 14.2 mmol, 1.0 eq.) was dissolved in CH2CI2 (71 mL) and the solution was cooled at 78 °C. 2,6-lutidine (3.4 mL, 29.80 mmol, 2.1 eq.) was added dropwise followed by TMSOTf (5.4 mL, 29.80 mmol, 2.1 eq.). The reaction mixture was warmed to 0°C and stirred for 2 hours. Then, it was quenched with saturated aqueous NaHCO3 solution and diluted with EtOAc and water. The layers were separated, and the organic layer was washed with 0.1 M aqueous HCI solution and brine. It was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (heptane / EtOAc, 4:1 to 0:1) to give the product as a white solid (2.1 g, 42% yield). 1H NMR (500 MHz, chloroform-d) 6 7.84 (s, 1H), 4.23 (ddd, J = 8.9, 6.1, 2.8 Hz, 1H), 2.83 (dd, J =16.8, 3.5 Hz, 1H), 2.71 2.61 (m, 1H), 2.56 (dp, J = 12.7, 6.3 Hz, 1H), 2.44 (dt, J = 12.3, 5.7 Hz, 1H), 2.37 2.13 (m, 4H), 1.98 (ddd, J = 12.6, 5.5, 2.7 Hz, 1H), 1.89 (ddt, J = 12.7, 5.6, 2.7 Hz, 1H), 1.71 (td, J = 13.2, 4.8 Hz, 1H), 1.43 (ddd, J = 13.7, 8.9, 2.8 Hz, 1H), 1.24 (d, J = 7.2 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.11 (s, 9H).1H NMR in accordance with literature (10).

[170] 2, 6-difluoropheny / ((1R,38,5S)-3-((R)-2-(2,6-dioxopiperidin-4-yl)- 1- ((trimethylsilyl)oxy)ethyl)- 1,5-dimethyl-4-oxocyclohexyl)sulfamate (5). O TMS / S\\ o O ôt F

[171] Compound 5 was synthesized according to a literature protocol (10). In brief, MgO (912 mg, 22.6 mmol, 4.0 eq.) and molecular sieves (200 mg per 1 mmol of 3) were added in a Schlenk tube and they were flamedried. Compound 4 (1.5 g, 7.35 mmol, 1.3 eq.), 2-phenylisobutyric acid (464.7 mg, 2.83 mmol, 0.5 equiv), Rh2(esp)2 (43 mg, 0.057 mmol, 0.01 eq.), and 3(2.0 g, 5.66 mmol, 1.0 eq.) were then added and suspended in i-PrOAc (11.3 mL) at room temperature. After 5 minutes, PIDA (4 mg, 11.32 mmol, 2.0 eq.) was added. The reaction was stirred overnight at

[172] room temperature, filtered through a pad of celite, and purified by column chromatography (heptane / EtOAc, 1:1) to give the product as a white solid (1.77 g, 56% yield). 1H NMR (500 MHz, chloroform-d) 6 7.89 (s, 1H), 7.28 7.19 (m, 1H), 7.01 (t, J = 8.4 Hz, 2H), 5.15 (s, 1H), 4.37 (dt, J = 8.5, 3.9 Hz, 1H), 2.80 (ddd, J = 17.0, 4.1, 1.9 Hz, 1H), 2.72 2.65 (m, 1H), 2.50 (dp, J = 12.7, 6.2 Hz, 1H), 2.43 2.32 (m, 5H), 2.10 (t, J = 12.4 Hz, 1H), 1.89 (t, J = 13.2 Hz, 1H), 1.82 (s, 3H), 1.41 (ddd, J = 13.1, 8.9, 3.5 Hz, 1H), 1.07 (d, J = 6.4 Hz, 3H), 0.11 (s, 9H). 1H NMR in accordance with literature (10).

[173] (1R,38,5S)-3-((R)-2-(2,6dioxopiperidin-4-yl)- 1-((trimethylsilyl)oxy) ethyl)- 1,5-dimethyl-4-oxocyc / ohexan-1-aminium (2). O O O own / '9 H3N ~"Me © eTFA

[174] Compound 2 was synthesized according to a literature protocol.(10) ln brief, Compound 5 (1.57 g, 2.80 mmol, 1.0 eq.) was dissolved in ACN / H20, 2 / 1 mixture (28 mL) and pyridine (4.5 mL, 56.1 mmol, 20.0 eq.) was added. The reaction was stirred at 70 °C overnight. It was then concentrated and purified by reversed-phase column chromatography (5% to 50% CH3CN in water, 0.1% TFA) to give the product as a white solid (841 mg, 73% yield). 1H NMR (500 MHz, deuterium oxide) 6 4.23 (ddd, J = 10.2, 4.3, 3.0 Hz, 1H), 2.94 2.55 (m, 4H), 2.50 2.33 (m, 4H), 2.22 (dddd, J = 22.4, 12.7, 5.8, 3.7 Hz, 2H), 1.93 (t, J =13.2 Hz, 1H), 1.79 1.68 (m, 1H), 1.67 (s, 3H), 1.53 (ddd, J = 14.4, 10.2, 4.5 Hz, 1H), 1.45 1.39 (m, 1H), 0.97 (d, J = 6.4 Hz, 3H). 1H NMR in accordance with literature (10).

[175] 2, 6difluorophenyl sulfamate (4). C? F OSNHZ CEOH as]? .ch HCOOH F\î / F + C. N ACN DMA F 4(60%) Scheme 2: Synthesis ofcompound 4.

[176] Compound 4 was synthesized according to a literature protocol (11). In brief, chlorosulfonyl isocyantate (6.0 mL, 69.24 mmol, 3.0 eq.) was cooled on ice and formic acid (2.2 mL, 57.70 mmol, 2.5 eq.) was added dropwise. The mixture was dissolved in CH3CN (43.3 mL) and it was stirred on ice for 30 minutes. It was then warmed to room temperature, stirred for 4h and cooled again at 0 °C. In a separate flask 2,6- difluorophenol (3.0 g, 23.08 mmol, 1.0 eq.) was dissolved in dimethylacetamide (37.5 mL) and itwas added to the reaction mixture. Itwas then warmed to room temperature and stirred for 1 hour. The reaction was quenched with 50 mL water, extracted with Et2O (3x) and the combined organic layers were washed with water (5x), saturated aqueous NaHCO3 solution and brine. It was dried over Mg804 and recrystallized from CH2CI2 to get the product as a white solid (2.9 g, 60% yield). 1H NMR (500 MHz, chloroform-d) 6 7.25 (dq, J = 8.5, 5.0 Hz, 1H), 7.08 6.99 (m, 2H), 5.17 (s, 2H). 1H NMR in accordance with literature (10).

[177] (E)-2-cyano-3-(2, 3, 6, 7-tetrahydro- 1H,5H-pyrido[3, 2, 1-ij]quinolin-9-yl)acrylic acid (CCVJ, 6) & cyanoacetlcacid,& && piperldme N CN NHSDCC CN o o ACN / COOH DMF / 035 o O 7 CCVJ. wen / ', Scheme 3: Synthesis ofcompound 7.

[178] Compound 6 was synthesized according to a literature protocol (12). In brief, a flask was put under Ar flow and julolidenecarboxaldehyde (1.0 g, 4.96 mmol, 1.0 eq.) and cyanoacetic acid (843 mg, 9.92 mmol, 2.0 eq.) were added and dissolved in CH3CN (14.2 mL). Piperidine (0.5 mL, 4.96 mmol, 1.0 eq.) was added and the reaction was put under reflux, under Ar for 2h. Itwas then concentrated under reduced pressure and the resulting red oil was suspended in 1M aqueous HCI solution. The orange precipitate was collected with vacuum filtration. Then, it was purified by column chromatography (CH2CI2 / MeOH, 90:10) to afford the product as a brown solid (640 mg, 48% yield). 1H NMR (500 MHz, dimethyl sulfoxide-d6) 6 7.87 (s, 1H), 7.51 (s, 2H), 3.33 (t, J = 5.8 Hz, 4H), 2.67 (t, J = 6.2 Hz, 4H), 1.91 1.82 (m, 4H). 1H NMR in accordance with literature (12).

[179] 2,5-dioxopyrro / idin- 1-y / (E)-2-cyano-3-(2,3, 6, 7-tetrahydro- 1H,5prrido[3,2, 1- ij]quinolin-9-yl)acrylate (7)

[180] Compound 6 (790 mg, 2.95 mmol, 1.0 eq.), N-Hydroxysuccinimide (509 mg, 4.42 mmol, 1.5 eq.) and N,N'-Dicyclohexylcarbodiimide (912 mg, 4.42 mmol, 1.5 eq.) were dissolved in DMF (26.8 mL) and the reaction was stirred overnight at room temperature. Then, the white precipitate was filtered off and the filtrate was diluted with EtOAc, washed with 1M aqueous HCI solution and brine (3x). It was dried over MgSO4 and concentrated under reduced pressure to give the product as an orange solid. The compound was carried on to the next step without further purification. 1H NMR (500 MHz, chloroform-d) 6 7.94 (s, 1H), 7.54 (s, 2H), 3.39 3.33 (m, 4H), 2.85 (s, 4H), 2.74 (t, J = 6.3 Hz, 4H), 2.01 1.95 (m, 4H). 13C NMR (126 MHz, chloroform- d) 6 169.16, 161.18, 156.81, 149.40, 121.22, 118.67, 116.84, 84.26, 50.53, 34.00, 25.70, 25.01, 20.98.

[181] (E)-2-cyano-N-((1R,3S,5S)-3-((R)-2-(2,6-dioxopiperidin-4-yl)-1-hydroxyethyl)- 1,5-dimethyl-4-oxocyclohexyl)-3-(2, 3, 6, 7-tetrahydro- 1H,5H-pyrido[3,2, 1-i)]quinolin-9- yl)acrylamide (Probe 1, RiboBright) o O HN O O o £th 0% +® Owl / le / 035 DMF. 12h \ Me Haw. oo WH 2 7 Probe1l10%l Scheme 4: Synthesis ofprobe ] (RiboBright).

[182] Compound 2 (30 mg, 0.101 mmol, 1.0 eq.) was dissolved in DMF (1.0 mL). Et3N (0.04 mL, 0.300 mmol, 3.0 eq.) and 7 (45 mg, 0.120 mmol, 1.2 eq.) were added and the reaction was stirred at room temperature overnight. It was then diluted with CH2CI2 and washed with saturated aqueous NaHCO3 solution and brine and dried over Mg804. It was then purified by column chromatography (CH2CI2 / MeOH from 98:2 to 90:10) to give the product as an orange solid (6 mg, 10% yield). Probe 1 was obtained as a mixture ofthe E and Z isomers. 1H NMR (500 MHz, chloroform-d) 6 7.92 (s, 1H), 7.88 (d, J = 6.2 Hz, 1H), 7.39 (s, 2H), 7.16 (s, 1H), 7.05 (s, 0H), 6.08 (s, 1H), 5.90 (s, 0H), 4.24 (d, J = 10.9 Hz, 2H), 3.32 3.21 (m, 6H), 2.83 2.64 (m,8H), 2.59 (dp, J = 11.5, 5.8 Hz, 1H), 2.49 2.34 (m, 5H), 2.33 2.25 (m, 3H), 2.22 2.10 (m, 2H),1.93 1.78 (m, 6H), 1.72 1.60 (m, 13H), 1.35 1.16 (m, 18H), 1.03 (dd, J = 6.4, 3.0 Hz, 4H), 0.89 0.77 (m, 7H). 13C NMR (126 MHz, chloroform-d) 6 213.93 (d, J = 21.7 Hz), 172.04 (d, J = 24.1 Hz), 162.65, 152.41, 151.12,147.32,131.62, 131.18, 120.85, 120.44, 119.66, 118.55, 99.62, 93.45, 66.37 (d, J = 7.4 Hz),53.55 (d, J = 10.2 Hz), 50.79, 50.07, 45.50 (d, J = 21.5 Hz), 41.07 (d, J = 7.6 Hz), 38.56, 37.94, 37.33, 36.57, 31.53, 27.84 27.54 (m), 23.07, 22.81, 21.28 (d, J = 12.5 Hz), 14.08, 1.09.MS (ESI) (m / z) calculated for C31H38N405 [M+H]+: 547.67, found: 547.38.

[183] ​​(E)-3-(2-cyano-3-(2,3, 6, 7-tetrahydro- 1H,5H-pyrido[3,2, 1-ij]quinoline-9- yl)acrylamide)propanoic acid (8a) 0 / ON H7N R OH / VR OH 2 © @TrA Oob a.,mer. 12h 0 7(1)W and ââlâïîàîâèlîîì THEIR \ ONARîNïe a: R= "VE .ProbeZ) 3222:2222: Scheme 5: Synthesis ofprobc 2 and 3.

[184] ß-Alanine (10 mg, 0.114 mmol, 1.0 eq.) was dissolved in dry N,N- Dimethylformamide (1.1 mL). Et3N (0.05 mL, 0.342 mmol, 3.0 eq.) was added, followed by compound 6 (50 mg, 0.137 mmol, 1.2 eq.). The reaction was stirred at room temperature overnight. It was then diluted with CH2CI2 and washed with saturated aqueous NH4CI solution, water and brine. It was dried over MgSO4 and purified by column chromatography (CH2CI2 / MeOH, 90:10) to give the product as an orange solid (16 mg, 41% yield). 1H NMR (500 MHz, dimethyl sulfoxide-d6) 6 7.97 (t, J = 5.8 Hz, 1H), 7.81 (s, 1H), 7.42 (s, 2H), 3.38 (ddd, J = 15.2, 7.7, 3.5 Hz, 4H), 2.64 (dt, J = 29.6, 6.3 Hz, 5H), 2.46 (td, J = 7.1, 4.4 Hz, 2H), 1.91 1.81 (m, 5H). 13C NMR (126 MHz, dimethyl sulfoxide -d6) 6 162.89, 150.88, 147.20, 130.77, 120.96, 119.09, 118.13, 94.94, 55.44, 49.85, 36.38, 34.29, 31.23, 27.61, 21.39, 21.18. MS (ESI) (m / z) calculated for C19H21N303 [M+H]+: 340.16, found: 340.06.

[185] (E)-3-(2-(2-cyano-3-(2,3, 6, 7-tetrahydro- 1H,5prrido[3, 2, 1-ij]quinolin-9- yl)acrylamido)ethoxy) propanoic acid (8b)

[186] Amino-PEG1-C2-Acid (61 mg, 0.456 mmol, 1.0 eq.) was dissolved in dry DMF (4.6 mL). Et3N (0.2 mL, 1.37 mmol, 3.0 eq.) was added followed by compound 6 (200 mg, 0.547 mmol, 1.2 eq.). The reaction was stirred at room temperature overnight. Then, it was diluted with CH2CI2 and washed with saturated aqueous NH4CI solution and brine. It was dried over Mg804 and purified by column chromatography (CH2CI2 / MeOH, 9:2 to 94:6) to give the product as an orange solid (100 mg, 57% yield). 1H NMR (500 MHz, chloroform-d) 6 7.35 (d, J = 2.9 Hz, 2H), 6.66 (t, J = 5.2 Hz, 1H), 3.68 (td, J = 6.5, 2.7 Hz, 2H), 3.52 (tt, J = 8.4, 4.3 Hz, 5H), 3.23 (dd, J = 7.6, 3.9 Hz, 4H), 2.65 (td, J = 6.6, 3.0 Hz, 5H), 2.53 (td, J = 6.3, 3.1 Hz, 2H), 1.87 (q, J = 6.1 Hz, 5H). 13C NMR (126 MHz, chloroform-d)ö 173.68, 152.40, 131.05, 120.73, 118.59, 93.48, 69.31, 66.31, 50.09, 39.88, 34.76, 27.62, 21.20. MS (ESI) (m / z) calculated for C21H25N304 [M+H]+: 384.18, found: 384.28.

[187] (E)-2-cyano-N-(3-(((1R,3S,5S)-3-((R)-2-(2,6-dioxopiperidin-4-yl)-1- hydroxyethyl)- 1,5-dimethyl-4-oxocyclohexyl)amino)-3-oxopropyl)-3-(2, 3, 6, 7- tetrahydro-1H,5H-pyrido[3, 2, 1-i)]quinolin-9-yl)acrylamide (Probe 2) O O ? Me O O \ du Me N CN

[188] Compound 2 (55 mg, 0.134 mmol, 1.0 eq.), compound 8a (50 mg, 0.147 mmol, 1.1eq.), HATU (56 mg, 0.147 mmol, 1.1 eq.) and Et3N (0.1 mL, 0.215 mmol, 5.0 eq.) were dissolved in CH2CI2 (1.3 mL) and the reaction was stirred at room temperature for 2h. It was then diluted with CH2CI2 and washed with saturated aqueous NaHCO3 solution and brine. It was dried over Mg804 and purified by column chromatography (CH2CI2 / MeOH 98:2 to 90:10) to give the product as an orange solid (16 mg, 19% yield). Probe 2 was obtained as a mixture of the E and Z isomers. 1H NMR (500 MHz, chloroform-d) 6 8.03 (s, 1H), 7.96 (s, 1H), 7.42 (s, 2H), 6.75 6.65 (m, 1H), 5.86 (s, 1H), 4.17 (dd, J = 9.3, 4.0 Hz, 1H), 3.63 (tq, J = 23.3, 6.1 Hz, 2H), 3.32 3.28 (m, 2H), 3.31 3.23 (m, 2H), 2.90 2.83 (m, 1H), 2.80 2.63 (m, 6H), 2.54 (ddd, J = 9.2, 6.6, 3.6 Hz, 1H), 2.46 2.35 (m, 2H), 2.33 (dd, J =14.9, 11.8 Hz, 1H), 2.32 2.23 (m, 1H), 2.05 (t, J = 13.1 Hz, 1H), 1.96 (d, J = 6.0 Hz, 1H), 1.97 1.90 (m, 3H), 1.92 1.79 (m, 1H), 1.70 1.57 (m, 5H), 1.26 (s, 1H), 1.28 1.20 (m, 1H), 1.00 (dd, J = 6.4, 4.1 Hz, 2H). 13C NMR (126 MHz, chloroform-d) 6 213.95, 173.40, 172.38, 172.18, 170.64, 163.29, 152.55, 152.46, 147.38, 132.43, 131.24, 131.20, 120.89, 120.84, 120.41, 119.28, 118.52, 93.29, 92.89, 66.54, 53.51, 53.25, 53.12, 50.95, 50.86, 50.20, 50.12, 45.49, 41.07, 38.56, 38.33, 38.23, 37.35, 37.11, 37.09, 36.65, 36.27, 35.33, 27.85, 27.80, 27.70, 27.68, 24.62, 23.21, 23.11, 21.33, 21.26, 21.23, 14.12, 14.10.MS (ESI) (m / z) calculated for C34H43N506 [M+H]+: 618.32, found: 618.33.

[189] (E)-2-cyano-N-(2-(3-(((1R,3S,5S)-3-((R)-2-(2,6-dioxopiperidin-4-yl)-1- hydroxyethyl)- 1,5-dimethyl-4-oxocyc / ohexyl)amino)-3-oxopropoxy)ethyl)-3-(2,3, 6, 7- tetrahydro- 1H,5prrido[3,2, 1-i)]quinolin-9-yl)acrylamide (Probe 3) O ; Me N O CN H O @Nwo / QKN "Me O H

[190] Compound 2 (60 mg, 0.146 mmol, 1.0 eq.), compound 8b (62 mg, 0.161 mmol, 1.1 eq.), HATU (61 mg, 0.161 mmol, 1.1 eq.) and Et3N (0.1 mL, 0.730 mmol, 5.0 eq.) were dissolved in CH2CI2 (1.5 mL) and the reaction was stirred at room temperature for 2h. It was then diluted with CH2CI2 and washed with saturated NaHCO3 solution and brine. It was dried over Mg804 and purified by column chromatography (CH2CI2 / MeOH 94:6 to 90:10) to give the product as an orange solid (15 mg, 15% yield). Probe 3 was obtained as a mixture of the E and Z isomers. 1H NMR (500 MHz, chloroform-d) 6 7.99 (s, 2H), 7.95 (s, 2H), 7.42 (s, 1H), 7.34 (d, J = 9.0 Hz, 1H), 7.10 (s, 0H), 6.58 (d, J = 5.5 Hz, 2H), 6.12 (s, 2H), 4.19 (d, J =11.1 Hz, 2H), 3.77 3.64 (m, 5H), 3.59 (tq, J = 6.7, 3.2 Hz, 11H), 3.28 (dt, J = 15.8, 5.8 Hz, 13H), 2.99 2.92 (m, 3H), 2.80 2.66 (m, 19H), 2.56 2.44 (m, 3H), 2.45 2.20 (m, 20H), 2.12 1.99 (m, 2H), 1.99 1.90 (m, 13H), 1.84 (t, J = 13.0 Hz, 2H), 1.68 (d, J = 3.7 Hz,25H), 1.50 1.34 (m, 2H), 1.24 (s, 6H), 1.18 1.08 (m, 2H), 0.99 (d, J = 6.3 Hz, 7H), 0.89 0.77 (m, 1H). 13C NMR (126 MHz,chloroform-d)6213.95,172.32,172.13,170.91,162.89, 152.79, 147.39, 132.83, 131.25, 120.89, 120.38, 119.75, 118.53, 92.75, 69.62, 69.40, 67.29, 67.11, 66.39, 66.20, 53.51, 52.78, 50.87, 50.20, 50.14, 45.34, 41.06, 40.96, 40.02, 38.55, 38.35, 38.22, 37.33, 37.28, 29.78, 27.82, 27.68, 23.25, 23.11, 21.34, 21.23, 14.18. MS (ESI) (m / z) calculated for C36H47N507 [M+H]+: 662.35, found: 662.18.

[191] Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

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Claims

1. A compound, in which the compound consists of a cycloheximide part and a fluorescent part, and in which the fluorescent group is coupled to the cycloheximide portion via the C13 position of the cycloheximide portion.

2. The connection according to one of the preceding claims, in which the cycloheximide portion is linked to the via an amide bond fluorescent part.

3. The connection according to one of the preceding claims, whereby the fluorescent part consists of one of the following groups: naphthalamides, nitrobenzoxadiazoles, fluorescein, FTC, Texas Red, phycoerythrin, rhodamine, coumarins, carboxytetramethylrhodamine, DAPI, indopyra dyes, Cascade blue coumarin, NBD, Lucifer Geel, propidium iodide, porphyrin, Bodipy, CY3, CY5, alexa and derivatives and analogues thereof.

4. The connection according to one of the preceding claims in which the fluorescent part contains a fluorescent rotor, preferably in which the fluorescent rotor 9-(2-Carboxy-2-cyanovinyl)julolidine (CCVJ), a is naphthalimide or a nitrobenzoxadiazole, preferably CCVJ.

5. The connection according to one of the preceding claims in which the connection a connection is according to formula !: O HN OH o tl Ë Me O : - / \ / X Me Formula l in which: [F] is a fluorescent part, X is an arbitrary atom, preferably N, connect the fluorescent part to the cycloheximide part.

6. The connection according to one of the preceding claims, in which the connection a connection is according to formula Il: il HN OH O H Îll Me 0% ? - / \ / lMe Formula II 7. The connection according to one of the preceding claims, in which the connection a connection according to formula III is: HN OH o 5th | M o? % °| Me i Formula Ill 8. The connection according to one of the preceding claims, in which the connection one of the formulas IV, V, or VI is, preferably one of formula IV: 0 No 0 H ? Me o . O \ N "Me N N Formula IV, 0 No H ? Me 0 . O o \ N / \ / \N Me N N Formula V, O No H ? Me NO CN o N A __ / \ / \0 N Me O Formula VI.

9. A connection pursuant to one of the preceding claims, in which the connection is complexed with a eukaryotic, preferably human, 608 ribosomal subunit or with a eukaryotic, preferably human, ribosome.

10. A cell containing a connection according to one of the preceding conclusions.

11. A compound containing a connection according to one of the preceding conclusions.

12. A method for preparing a compound according to one of the preceding conclusions 1 - 9, where the method the linking of the fluorescent part to the cycloheximide part via the C13 position of the cycloheximide includes.

13. A method for detecting a eukaryote, preferably human, 608 ribosomal subunit or ribosome, whereby the method includes (a) bringing the 608 ribosomal subunit or the 608 into contact ribosome immediate connection according to one of the preceding conclusions 1 to and with 9, so that the connection forms a complex with the 608 ribosomal subunit or the 608 ribosome, (b) detection of the complex formed in step a).

14. The method referred to in claim 13, where the method is an in vitro method or is an in vivo method, and / or in which the method is for detecting of a eukaryote, preferably human, 608 ribosomal subunit or ribosome in a cell, preferably where the method is for single-cell tracking of a eukaryote, preferably human, 608 ribosomal subunit or ribosome.

15. The method according to one of the preceding conclusions 13 - 14 in which the detect fluorescence microscopy, time-lapse fluorescence microscopy and / or includes flow cytometry.

16. A method for treating a eukaryotic, preferably human, cell, where the method consists of bringing the cell into contact with a connection according to one of the preceding claims 1 - 9, so that the connection can form a complex with a 608 ribosomal subunit or 5 ribosome, and can preferably inhibit protein synthesis by the cell.

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