Small molecule screening cellular assay using chemically modified beads

Abstract

A method for screening a DNA-encoded library of chemical structures (2) for activity in a cellular target (11) is provided. The chemical structures (2) of the library and corresponding encoding DNA (4), and optionally chemical probes (7, 8, 9) that react with a response molecule (12), are covalently attached to beads (1). The method includes providing an incubation medium (13) or an aliquot thereof containing the cellular target (11) and one or more beads (1), releasing the chemical structures (2) from the beads (1) in the incubation medium (13) or an aliquot thereof by cleaving the structural linker (3), incubating the released chemical structures (2) and the cellular target (11), and sequencing the encoding DNA present or remaining on the beads (1). Beads (1) suitable for the method are also provided.

Description

[Technical field]

[0001] The present invention relates to screening assays for screening small molecules for potential efficacy in altering the activity of cellular targets of pharmaceutical interest, and means for performing the assays. [Background technology]

[0002] Many targets of pharmaceutical interest are active in a cellular context, and when investigating small molecules that alter the activity of a target, it is beneficial to conduct the investigation in a cellular context, which can involve screening a large diversity of compounds.

[0003] One of the desired responses of the system to small molecules that modulate a target may be the release of molecules from the cell. These molecules may be smaller products of the cell, such as enzymes, proteins, nucleic acids or metabolites. The release of molecules may occur by directed release or by simple leakage. During screening, these released molecules are detectable as a measure of the action of the compound.

[0004] In the pharmaceutical industry, screening of small molecule compounds in cellular assays is generally accomplished by combining the compound and cells in a single vessel (Jones E, Michael S, Sittamparam GS, "Basics of Assay Equipment and Instrumentation for High Throughput Screening", May 1, 2012 (updated April 2, 2016); Sittampalam GS, Coussens NP, Brimacombe K, et al., "Assay Guidance Manual" (Internet), ethesda (MD), Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004). Thus, small molecules can act on cellular molecules outside the cell or in the cell membrane, or enter the cell and perform their action inside the cell. The ability of the compound to modulate the activity of the cell is then determined by monitoring the output of the assay, which can be evaluated. The output of the assay can be, for example, the processing of a substrate by an enzyme. The processing of the substrate can generate a fluorescent signal. Another means is, for example, when the binding of the small molecule to a protein target is monitored, for example, by a change in fluorescence.

[0005] A large number of different small molecules can be interrogated (=screened) for their ability to modulate the activity or binding of a target using microcompartments such as in the form of n-well plates (n is typically 96, 384 or 1536). Commercially available microplate readers are commonly used to measure the activity of each small molecule by measuring the remaining substrate (or appearance of product) in each individual well in the plate. Another known method of dividing such an assay into a large number of microcompartments is the production of aqueous droplets containing the assay reagents in the form of a water-in-oil emulsion. Summary of the Invention [Problem to be solved by the invention]

[0006] The logistics of long-term storage of small molecule plates is challenging when using a microplate reader, especially when dealing with very large numbers of plates. Furthermore, the time required to assess product formation in a microplate increases linearly with the number of small molecules, which becomes problematic when investigating millions of small molecules (using current technology, the assessment of 2 million molecules takes on the order of 10 business days) (Brouzes E., Medkova M., Savenelli N., Marran D., Twardoski M., Hutchison JB, Rothberg JM, Link DR, Perrimon N., Samuels ML, PNAS 106, pp. 14195-14200 ((2009)).

[0007] Such assays require that the structures of chemical compounds found to be useful be easily determined. A solution to this problem is to provide the compounds to be screened in the form of a DNA encoded chemical library (DEL) (Brenner and Lerner, Proc. Natl. Acad. Sci. USA, 89:5381-5383 ((1992)). In a DEL, each compound is pre-tagged with a unique DNA sequence that corresponds to its structure. Thus, it is not necessary to identify the structure of the useful compound itself (this task may or may not be structure dependent), only the corresponding DNA tag needs to be sequenced, a standard procedure that is the same for all useful compounds.

[0008] However, such DEL-based assays are hindered by the problem that compounds associated with DNA tags may not behave the same in the assay as free compounds. WO2018 / 087539 suggests cleaving the compound and its associated DNA tag prior to the assay, but leaving the free compound and its associated DNA tag in "spacial association."

[0009] ACS Chem. Biol. 13, pp. 761-771 (2018) discloses a small molecule assay using silica beads with a quenched fluorescent probe and a small molecule bound to it with a DNA tag. In this assay, the beads are permeated into cellular targets that themselves act as microcompartments. The small molecule is detached from the permeated beads by light. Any small molecule that is lethal to the cellular target will trigger apoptosis of the cellular target in the permeating bead, triggering the release of caspase-3, which removes the quencher from the fluorophore. Such fluorescent cellular targets (and fluorescent microcompartments) are sorted using flow cytometry, and the DNA of any beads contained therein is sequenced to find the corresponding small molecule that triggered apoptosis.

[0010] US5958703A discloses supports with tethers that can be modified with reporter molecules, and related screening procedures. This publication "isolates" the supports with the modified tethers. Thus, this publication does not pool all the supports before isolating the supports with modified tethers.

[0011] The release of compounds in WO2013 / 057188A1 is made so that the compounds "remain inside the solid support / beads" or "each compound is physically located inside its parent solid support" or each compound is made "inside the bead" or allows the "substrate" to be absorbed "inside the support / bead". Therefore, this publication does not release the compounds to be assayed into the incubation medium, but keeps them inside the beads. The "physicochemical or biological system" that this publication targets is therefore a soluble species, not a cellular target. In the latter case, there may be two heterogeneous phases (compound-containing beads and cellular targets) that preclude interactions between them.

[0012] ACS Com. Sci. 19, pp. 524-532 (2017) describes the principle of the water-in-oil assay. Beads with DNA-encoded libraries are encapsulated in droplets and incubated in a test assay. Any droplets (and any positive microcompartments) that show a positive reaction in the incubation assay are first sorted, and beads separated from such sorted droplets are further examined for statistically relevant hits.

[0013] ACS Com. Sci. 21, pp. 425-435(2019) also describes the principle of the water-in-oil droplet assay. Beads with DNA-encoded libraries are encapsulated in droplets and incubated in a screening assay for autotaxin inhibitory activity. This assay uses homogeneously dissolved autotaxin as a target, rather than a cellular target. In this publication, hit droplets are first sorted. Figure 1 shows the "droplet sorting conjugation (5)" with fluorescence detection.

[0014] In these prior art bead-based assays, "positive" microcompartments (microwells, droplets, cells, etc.) are first selected or sorted, then all beads contained in the sorted microcompartments are pooled and their DNA tags are analyzed. However, a "positive" microcompartment may contain more than one bead, of which usually only one bead is a hit bead (i.e., a bead that yields an active small molecule) and the other beads are non-hit beads. The publication identified these non-hit beads as "passenger beads" and derived a mathematical expression for the "false discovery rate" that describes the degree of separation and discovery of such non-hit beads. Confirmation that the separated beads are non-hit beads is only possible in a second test.

[0015] In such a prior art assay, the probability P h can be calculated as explained below.

[0016] Bead population in the microcompartment P b is the Poisson distribution:

[0017]

number

[0018] In equation (1), k m is the number of beads in the microcompartment under consideration (0, 1, 2, ...). mcannot exceed an upper threshold K (which depends on the volume of the beads and the volume of the microcompartment, and on the reproducibility of the volumes). For example, it is not possible for the total volume of all beads contained in a microcompartment to be greater than the volume of the microcompartment itself. If the volume of the microcompartment is variable and infinite, the upper threshold K can tend to infinity. However, real microcompartments such as microwells or droplets are very small and very well reproducible in volume. Therefore, for real microcompartments, K is typically less than 10. Reducing the volume of the microcompartment and K is one of the common ways to lower λ. The average bead population λ of a microcompartment must be smaller than the maximum possible bead population K. λ is the average bead population of a microcompartment and is defined by equation (2).

[0019]

number

[0020] In formula (2), M is the number of all micro-partitions, and the sum spans all M micro-partitions. b is the number of times that a given micro-partition actually has (k m ) the probability of not containing a bead. This type of bead distribution is obtained with all known devices that divide a bead population into microcompartments.

[0021] The k of a given micropartition m k in beads p The probability that a hit bead actually exists is P p is hypergeometrically distributed as shown in equation (4) below.

[0022]

number

[0023] In equation (4), N is the number of individual chemical structures in the DNA-encoded library. r is the so-called "library hit rate" (the fraction of library compounds that are "active" chemical structures in the assay of interest (a number of unknowns close to zero)). ε is the so-called "library equivalence" (the number of beads with the same chemical structure in the library linked to it). k p is the number of hit beads. k m is as defined above. Here, we assume that εN, rεN, and ε(1-r)N are integers or numbers rounded to integers.

[0024] If a given micro-partition has k m Contains beads of exactly k p The probability P that a bead in a cell becomes a hit bead after incubation with the cell target (these events are independent of each other) is given by the following equation (5):

[0025]

number

[0026] In the prior art process, k associated with the micro-partitions p An incubated microcompartment was recognized as “positive” if k was greater than 0 (and therefore contained at least one hit bead). Such “positive” microcompartments also had a k greater than 0. m The amount of all beads (hit beads and non-hit beads) obtained from all such "positive" microcompartments B tot is expressed by the following equation (6).

[0027]

number

[0028] In equation (6), k p The sum of is over all possible numbers of hit beads in the microplot, and the total number of beads in the microplot, k mThe range is up to k m The sum of k is up to an upper threshold K, which is the maximum number of beads that a microcompartment can contain. In equation (6), we use k m Or another number k m ' are mutually exclusive events, given a number k m A given microcompartment with beads is divided into a number k p Or another number k p 's hit beads are also assumed to be mutually exclusive events.

[0029] The amount of hit beads obtained from all such "positive" microcompartments B h is defined by equation (7).

[0030]

number

[0031] In equation (7), all symbols and legends are as above. Equation (7) involves the same assumptions regarding mutual exclusivity as described above for equation (6).

[0032] The above P h is defined by equation (8).

[0033]

number

[0034] k in the numerator of Eq. (8) p The corresponding k in the denominator m Because this P is smaller h is less than 1. P h becomes smaller as λ increases because the λ polynomial in the denominator of (8) grows faster with λ than the λ polynomial in the numerator, and because, as mentioned above, k p is the corresponding k mHowever, Ph is largest when λ=0. That is, these prior art processes have the lowest degree of false discovery of non-hit beads at a practically unrealizable average bead population λ in a microcompartment. A practically realizable average bead population has λ greater than 0 (there should be microcompartments containing one or more beads).

[0035] Thus, the prior art processes are not simultaneously optimal for both minimizing false discovery and analysis of non-hit beads and for a practically achievable microcompartment bead population, i.e., one is traded off against the other.

[0036] The present invention is intended to solve the above problems. [Means for solving the problem]

[0037] The present invention is as follows. 1. A method for screening a DNA-encoded library of chemical structures for activity in a cellular target, comprising: the cellular target is known to release or alter the release of a response molecule when contacted with an active chemical structure; The chemical structures of the library and their corresponding encoding DNA, and optionally chemical probes that react with the response molecule, are covalently attached to the beads; Each bead is a) a plurality of instances of a single chemical structure from said library; b) a plurality of instances of a DNA sequence encoding said chemical structure; having each of said instances of said chemical structure is covalently attached to said bead via a structural linker; the structural connector is severable at a severable structural connector portion; the DNA sequence is covalently attached to the bead via a tag linker; the tag linker comprises a cleavable tag linker moiety and is cleavable by a cleavage agent; c) the releasable structural linker moiety is not releasable under reaction conditions that cleave the releasable tag linker moiety, and vice versa; The tag linker and / or the detachable tag linker site and / or the DNA sequence may be detachable by the response molecule; When the DNA sequence and / or the cleavable tag linker site and / or the tag linker are cleavable by the response molecule, the bead preferably lacks a response molecule-sensitive chemical probe, The method comprises the following steps (i), (ii) and (iii): (i) It has one of the following steps (ia) or (1-b): (ia) for each distinct chemical structure and each distinct cellular target to be assayed, providing an incubation medium comprising said cellular target and one or more of said beads coupled to said distinct chemical structure; Releasing the chemical structures from the beads in the incubation medium by cleaving the structural link at the cleavable structural link site; incubating the cell targets and the released chemical structures in the incubation medium; or (ib) providing a single incubation medium containing the cellular targets and all of the beads coupled to all of the chemical structures of the library; partitioning a plurality of aliquots having one or more beads from the incubation medium; releasing the chemical structures from the beads in each of the aliquots by cleaving the structural link at the cleavable structural link site; incubating the cell targets and the released chemical structures in an aliquot of the incubation medium; having (ii) if the encoding DNA sequence and / or the detachable tag linker site and / or the tag linker are detachable by a response molecule, (ii-a-1) the incubation medium or the aliquot is monitored for release of the coding DNA sequence or fragments thereof from the beads; if said release is detected, separating all of the beads from all of said incubation medium or all of said aliquots and pooling all of said separated beads; (ii-a-2) the cleavable tag linker sites on the pooled beads are cleaved by the cleavage agent to release the encoding DNA sequence or a fragment thereof; (ii-a-3) the released coding DNA sequences or fragments thereof are amplified and sequenced to identify the complete coding DNA sequence in the coding DNA library; (ii-a-4) the remaining complete DNA sequences contained in the encoding DNA library that were not identified in step (ii-a-3) are linked to corresponding chemical structures in the encoding DNA library; Alternatively, when the beads carry the response molecule sensitive chemical probes, (ii-b-1) the incubation medium or an aliquot thereof is monitored for a reaction of the probe with the response molecule or a change in said reaction; If said reaction or change therein is detected, separating and pooling all of the beads from all of said incubation media or all of said aliquots; (ii-b-2) beads that show a reaction or a change in the reaction of the probe are extracted from the pool; (ii-b-3) the cleavable tag linker site on the beads extracted from the pool is cleaved by the cleavage agent to release the encoding DNA sequence covalently attached to the extracted beads; (ii-b-4) the released DNA sequences are amplified and sequenced; (ii-b-5) the DNA sequences sequenced in step (ii-b-3) are associated with corresponding chemical structures in the encoded DNA library; (iii) The chemical structure associated in step (ii-a-4) or (ii-b-5) is selected as a further active chemical structure.

[0038] Preferred embodiments of the method are subject to the dependent claims. [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 is a schematic representation of a bead suitable for processing according to the invention and which forms part of the invention; [Diagram 2] FIG. 2 is a schematic diagram of another bead suitable for treatment according to the present invention. [Diagram 3] FIG. 2 is a schematic diagram of the incubation medium containing the beads and cell targets. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] In contrast to the prior art process, the process according to the present invention separates and pools all beads (whether hit or non-hit) from all incubation media or aliquots thereof from all assay compartments (whether "positive" or not). Hit beads are then identified among the pooled beads. In the process according to the present invention, the P of the selected beads is his individual and independent of the average bead population λ present in the microcompartment. In the process according to the invention, it is not a problem that more beads have to be identified based on the probe reaction or based on DNA sequence determination. For the former, there are automated sorting tools (e.g. FACS in the case of fluorescent probes) that perform very powerful sorting, and for the latter, there are automated next generation sequencing (NGS). Furthermore, there is the possibility to test only a part of the bead pool for hit beads. Such a part of the bead pool may be a. A representative part of it (e.g. the number of beads, which is the number of chemical structures present in the library) is multiplied by the "library equivalent" ε defined above, where ε is typically between 1 and 100, preferably between 5 and 50.

[0041] One advantage of first pooling all beads and then selecting the hit beads is explained in the following four paragraphs.

[0042] In the prior art process described above, the hit microcompartments (e.g., hit droplets) are first sorted. Therefore, all microcompartments (those containing beads and those not containing beads) must be tested for activity by the sorter. h The number of micropartitions M that must be tested for activity in the prior art process if it is expected to obtain hit beads of priorart uses the above formula (7).

[0043]

number

[0044] The method of the present invention first separates and pools all beads (hit and non-hit beads) from all microcompartments (incubation medium or aliquots thereof), and then selects the hit beads. The number of microcompartments required for separating and pooling beads is M inventionThe number of separated and pooled beads (hit and non-hit beads) is λM invention where λ is the average bead population of the microcompartment, calculated according to formula (2) above. Then, the number of hit beads rλM is determined by sorting the hit beads from the pool. invention where r is the "library hit rate" as described above. This number of hit beads is used for comparison with the prior art process described above, B h Therefore, the following equation (10) is obtained.

[0045]

number

[0046] Comparing equations (9) and (10), in order to obtain the desired number of hit beads Bh under the following conditions, the number of micro-partitions M required in the process according to the present invention is invention is the number of micro-partitions required in the prior art process, M priorart will be smaller than

[0047]

number

[0048] Assuming that the upper threshold K is 1, the above inequality gives us the following: e -λ < 1 This formula does not depend on N, ε, or r, and is satisfied for any λ>0. As already mentioned, λ must be smaller than K. If K=1, then the useful range for λ is 0<λ<1, in which case the process according to the invention will only require a small number of hit beads B h This is more efficient than prior art processes in terms of the number of micro-cells required to obtain

[0049] Thus, a preferred embodiment of the process according to the invention is as follows. 1a) In step (1-a), the incubation medium has an upper threshold K with a value of 1 for the number of beads that may be contained therein. That is, the incubation medium is not allowed to contain more than one bead. Alternatively, 1b) In step (1-b), each aliquot from the incubation medium has an upper threshold K with a value of 1 for the number of beads that may be contained therein. That is, each aliquot is not allowed to contain more than one bead. Furthermore, 2) the average bead population λ is defined as the following formula (2) and is greater than 0 and less than 1.0.

[0050]

number

[0051] In equation (2), (k m ) i is an integer indicating the number of beads in the i-th incubation medium or the number of beads in the i-th aliquot of incubation medium. M is the number of incubation mediums or the number of aliquots in incubation medium. The sum is over all incubation mediums or all aliquots in incubation medium.

[0052] The above requirement of K=1 is easily realized by rigid microcompartments such as microwells with a suitably small volume, since such rigidity enforces such k. For soft microcompartments such as droplets, droplets with beads may be readily generated with a volume small enough to ensure K=1. Alternatively or additionally, the droplets may be further reduced in volume by a "droplet splitter" as used in ACS Comb. Sci. 21, pp. 425-435 ((2019)).

[0053] With reference to Figures 1, 2 and 3, a solution to the above problem is provided by associating small molecules in the form of chemical structures 2 to beads 1 by linking them to beads 1 via a cleavable (or severable or cleavable) structure linker 3. The structure linker 3 comprises a cleavable structure linker site 3a. Furthermore, the bead 1 comprises a DNA barcode 4 linked to the bead 1 via a tag linker 5. The tag linker 5 comprises a cleavable tag linker site 5a (Figure 1), which can be cleaved by a cleaving agent. In the screening method according to the invention, the cleavable tag linker site 5a is used after incubation of the cellular targets and after separation and pooling of the beads, to cleave all DNA barcodes 4 from the beads 1, thereby allowing their sequencing and correlation with the corresponding chemical structures in the library. In the embodiment of FIG. 1, it is assumed that the tag connector 5 and / or the cleavable tag connector site 5a and / or the coding DNA 4 are cleavable by the response molecule 12 generated during incubation, and thus the bead 1 is devoid of (then unnecessary) probes. Alternatively, if neither the tag connector 5, the cleavable tag connector site 5a nor the coding DNA 4 are cleavable by the response molecule 12, as shown in the embodiment of FIG. 2, the bead 1 further comprises chemical probes 7, 8, 9 linked to the bead 1 via a spacer 10. Here, as shown in FIG. 2, the chemical probes 7, 8, 9 consist of a fluorophore 7 and a quencher 8 of the fluorophore 7, and the fluorophore 7 and the quencher 8 are preferably linked together on a spacer 9 that is cleavable to the response molecule. In FIG. 3, the cellular target 11 is bound or co-encapsulated in the incubation medium 13 or an aliquot thereof together with the bead 1 in the test compartment. Also, the chemical structure 2 is released from the bead 1 by cleaving the cleavable structure connector site 3a.Either the reaction of the chemical probe to the response molecule (FIG. 2) or the persistence of the tag linker in an uncleaved form (neither shown in FIG. 3, but implicitly assumed since it is linked to the bead 1) records the presence and / or activity of the response molecule 12 released (or released at different levels) by the cellular target 11 in the culture medium 13 or an aliquot thereof. Although FIG. 3 shows only one bead 1 in the compartment, which is a preferred embodiment of the invention, there may be multiple beads, each with the same chemical structure. Similarly, there may be multiple cellular targets 11 instead of the single cellular target 11 shown.

[0054] The screening process of the present invention uses beads modified with the chemical structures of the library, the corresponding DNA barcodes, and optionally probes for the response molecules. For the purposes of the present invention, any type of granular, solid or gel-like material can be used as a "bead" if the material: a) the substance is inert (particularly in aqueous media) to the incubation medium in which the screening procedure of the present invention is carried out (wherein "inert" means that the substance does not react with the incubation medium and is substantially insoluble therein); and b) the material has a particle surface that contains chemically reactive moieties that covalently attach the chemical structures, chemical probes and DNA barcodes to the surface of the beads via their respective linkers.

[0055] In the case where the chemically reactive moieties are predominantly hydroxyl, the bulk of the beads may be an inorganic particulate material (e.g., silica or alumina). In another preferred embodiment, the bulk of the beads is an organic polymer (particularly polystyrene), which may be surface-modified by the introduction of chemically reactive moieties. There are many such organic bead materials commercially available with modified surfaces (e.g., beads sold under the trade name Tenagel® by Rapp Polymere). These beads may typically contain a surface of chemically reactive moieties in the range of 0.1 to 0.5 mmol / g of beads. The beads are preferably approximately spherical and have an average diameter in the range of 50 μm to 500 μm. These beads, even those with surfaces modified with chemically reactive moieties, are conventional.

[0056] The term "chemical structure" as used herein means a small molecule either in free form or its derivatives bound to beads, whereby from the context in which the term is used it will be clear that one of two meanings applies.

[0057] The screening process of the present invention first detaches the chemical structures from the beads, so that the free DNA-tagged chemical structures penetrate into the cellular target. Once in the cellular target, the chemical structures can interact, react, interfere, promote or inhibit any system present in the cellular target. Such systems may be, for example, any kind of system or receptor involved in cell differentiation, transcription, translation, respiration, membrane structure, and mitosis.

[0058] The structural linker linking the chemical structure to the bead may be any organic divalent group attached to the bead surface on one side and to the chemical structure on the other side. The structural linker comprises a cleavable structural linker moiety, allowing the chemical structure to be released from the bead in the incubation medium. The cleavable structural linker moiety is preferably located in close proximity to the chemical structure, such that, upon cleavage, the chemical structure contains few remaining residues from the structural linker.

[0059] Preferred examples of such cleavable structural linker moieties are set forth in Table 1 below (* indicates the valence that is preferably linked to the remainder of the structural linker, ** indicates the valence that is preferably linked directly to the chemical structure).

[0060] [Table 1]

[0061] These cleavable structural linker moieties themselves are conventional and there are many examples in the literature.

[0062] The beads used in the screening process according to the invention first have a library of chemical structures covalently attached to the surface of the beads via a cleavable structural linker. The chemical structures can be any chemical compound found by nature to be produced by synthetic means or by (modified) biological activity such as transcription. The molecular weight of the chemical structures can range, for example, from a few hundred Daltons to a few thousand or tens of thousands of Daltons.

[0063] Preferred examples of chemical structures that can form part of the screening library are any pharma- ceutically acceptable chemical compounds that satisfy all of the following a) to d): a) The chemical compound contains up to five hydrogen atoms, which are capable of participating in hydrogen bonds, derived from hydroxy (donating one such hydrogen atom) and primary amino groups (donating two such hydrogen atoms) and secondary amino groups (donating one such hydrogen atom). b) The chemical compound contains up to 10 oxygen and nitrogen atoms that can act as hydrogen bond acceptors (moieties that contain lone pairs of electrons that can participate in hydrogen bonding). c) The molecular weight of the chemical compound is a maximum of 500 Daltons. d) The negative common logarithm of the octanol / water partition coefficient (-log(Cn-octanol / Cwater) is at most +5, and preferably in the range of -0.4 to +5. Here, Cn-octanol is the molar concentration of the compound in n-octanol. Cwater is the molar concentration of the compound in water. The n-octanol solution of the compound and the aqueous solution of the compound are in contact with each other and are in thermodynamic equilibrium at 25°C. Compounds that satisfy the above conditions are usually defined as the "rule of five compounds".

[0064] Preferably, the screening process of the present invention may rely on known libraries of chemical structures as candidates to be linked to beads. A review of known libraries is shown in Table 1 of J. Med. Che. 59, pp. 6629-6644 (2016). For use as chemical structures in instant processes employing beads, as shown in Table 1 of this publication, the DNA tag directly linked to the chemical structure via an amino group is replaced by a cleavable structural linker and a bead linked thereto (details are described herein). Furthermore, the split and pool method described herein is used to simultaneously build segments and building blocks of chemical structures and associated DNA tag segments.

[0065] A "plurality" of such chemical structures are linked to the beads. The plurality must be large enough that the number is sufficient or the concentration is high enough in the incubation medium to ensure that the chemical structures, once released from a single bead, will detectably release the response molecule from the cellular target. The "plurality" is limited by the number of reactive sites present on the surface of the beads. If all reactive sites of a bead are linked to chemical structures but no detectable release of the response molecule from the cellular target can be obtained, the chemical structure is presumed to be ineffective for its intended purpose and can be removed from the assay. Furthermore, the "plurality" of chemical structures can range from 0.001 to 0.01 molar equivalents based on the surface of the bead bearing the chemically reactive moieties.

[0066] The beads used in the process of the present invention secondly have an encoding DNA covalently linked to the bead surface via a tag linker. As mentioned above, the tag linker may be any suitable organic bivalent linker, provided that the tag linker comprises a cleavable tag linker site, which is capable of releasing the DNA barcode from the bead after incubation of the beads with the cellular target, separation and pooling of the beads. Examples of cleavable tag linker sites and associated cleavage agents that allow their isolation and their pooling can be as described above for the cleavable structural linker sites and associated cleavage agents, and are preferably nucleotide sequences. More preferably, the cleavable tag linker site is a nucleotide sequence that can be cleaved by a restriction endonuclease as a cleavage agent, in which case the nucleotide sequence comprises a recognition site for that restriction endonuclease. Numerous examples of suitable restriction endonucleases and their associated recognition sites are known in the literature. For example, the second column of Table 2 of WO2010 / 94036A1 discloses a classification of restriction endonucleases and their associated recognition and cleavage sites. The most preferred restriction endonuclease as cleavage agent is Stu1.

[0067] In the case of a cleavable tag linker site that is cleavable by a restriction endonuclease, it is further preferred that the tag linker also comprises a PEG divalent spacer having a length of 5-10 ethylene glycol units (preferably 8 ethylene glycol units) and located immediately adjacent to the cleavable tag linker site, which may be advantageous for cleavage of the recognition site by the restriction endonuclease.

[0068] The cleavable structural linker site is preferably placed in close proximity to the encoding DNA so that, upon cleavage, the encoding DNA contains few residues remaining from the structural linker. Preferably, the tag linker itself is constructed by so-called "click chemistry" reactions.

[0069] The above preferred variations of tag linkers, cleavable tag linker sites and DNA barcodes may be accompanied by a PEG spacer that forms part of the tag linker and may be located in close proximity to the cleavable tag linker site, and are preferably constructed according to the following synthetic scheme:

[0070] [ka]

[0071] In this scheme, Fmoc-PEG derivatives are commercially available, for example, from JenKem (USA), Abbexa (UK) or Iris Biotech (Germany). Here, n is 5-10, preferably 8. m is 0 or 1 and k is 0-2. Preferably, m and k are 0, or m is 0 and k is greater than 0. C6 amino-terminal deoxythymidine (dT) derivatives are commercially available, for example, from GeneLink (USA). The Fmoc-protected amine moiety can then be deprotected and the free amine can be linked to a DNA tag linker already linked to a bead, or the free amine can be directly linked to a bead with carboxyl surface functionality, in which case the PEG linker itself will form the DNA tag linker. One or two oligos as X and / or Y can act as headpiece DNAs that can be successively linked in the split and pool method described herein to further encode DNA segments. When both X and Y are DNA oligos, they can then be used to construct a DNA tag in the form of a hairpin DNA. A cleavable tag linker site can be formed, for example, by converting the double bond near a deoxythymidine moiety to a vicinal diol using alkaline hydrogen peroxide, which can then become a tag linker site cleavable by NaI04 (see Table 1 above). Alternatively, oligos as X and / or Y may contain restriction endonuclease recognition sites as cleavable tag linker sites, as described above.

[0072] For the processes of the invention and beads of the invention, the cleavable structural linker moiety and the cleavable tag linker moiety are "orthigonal" in reactivity, i.e., the cleavable structural linker moiety is not cleavable under reaction conditions that cleave the cleavable tag linker moiety, and vice versa.

[0073] The tag linker and / or the cleavable tag linker site and / or the encoding DNA may be cleavable by a response molecule generated during incubation, in which case no chemical probe is required, and the cleavable tag linker site itself may be cleavable first by a cleaving agent and optionally further by a response molecule, where the cleaving agent and the response molecule may be different or may be the same, preferably different.

[0074] The coding DNA itself is preferably a hairpin DNA. This allows the construction of a complete double-stranded DNA from only a single DNA strand, since it itself is double-stranded. Therefore, only one coding DNA sequence needs to be constructed. The coding DNA may contain further pre- and / or post-sequences, in addition to the actual coding sequence characteristic of the encoded chemical structure. These sequences may be necessary or useful when linking the coding DNA to the bead and / or when cleaving the tag linker at the cleavable tag linker site. These further pre- and / or post-sequences are typically the same for all coding DNA instances.

[0075] A bead bears a "plurality" of such encoding DNA instances, which plurality must be large enough in number to allow the encoding DNA instances from a single bead to be amplified (e.g., by PCR) and subsequently sequenced. Additionally, the "multiple" encoding DNA instances can range from 0.001 to 0.01 molar equivalents based on the above surface of the bead having chemically reactive moieties.

[0076] The beads used in the present invention may comprise a chemical probe that reacts with the response molecule and is linked to the bead. The chemical probe may be a substrate for the released response molecule. This substrate may be an enzyme, protein or molecule such that the bead fluoresces (or ceases to fluoresce) after incubation with the released substance and appropriate additives. Alternatively (or in addition), the released enzyme may degrade or alter the DNA barcode attached to the bead so that detection by amplification or hybridization is modulated. The probe may comprise a chemical indicator that includes a reporter molecule. The chemical indicator may be, for example, colorimetric (i.e., the result of a colored reaction product between the response molecule and the probe that absorbs light in the visible range), fluorescent (e.g., based on an enzyme that converts the response molecule and the probe into a reaction product that fluoresces when excited by light of a particular wavelength) and / or luminescent (e.g., based on bioluminescence, chemiluminescence and / or photoluminescence). Preferably, the probe reacts by fluorescence when in contact with the response molecule, and is therefore a combination of a fluorophore or a quencher associated with a fluorophore. The response molecule cleaves the quencher from the fluorophore. However, the chemical probe is only present if the bead does not contain a tag linker that is cleavable by the response molecule.

[0077] The spacers linking the probes to the beads may be as described above for structural and / or tag linkers.

[0078] The cellular target is preferably a prokaryotic or eukaryotic cell. More preferred examples of prokaryotic cells are gram-positive bacteria, gram-positive coccus, gram-negative coccus and gram-negative bacteria. More preferred examples of eukaryotic cells are fungal cells and animal cells, especially human cells. The cellular target comprises a protein, receptor or other species that is susceptible to interaction with chemical structures that penetrate into the cellular medium from the outside of the cellular target. Following the interaction, it is also possible to initiate or increase the production of a response molecule, or to decrease or stop the production of a response molecule.

[0079] The beads used in the screening process of the present invention are synthesized by attaching the coding DNA directly to the tag linker and attaching the chemical structure to the linker and to the bead according to known chemistry.

[0080] Alternatively, the coding DNA and chemical structures may be synthesized directly and stepwise on the beads, for example by split and pool synthesis. In this variation, a "scaffold" (a chemical core structure common to all small molecules of the library to be tested) is first linked to the beads via a cleavable linker as described above, and a tag linker without coding DNA is also attached to the beads. The scaffold is typically a ring-containing structure that includes a set of diversity sites, typically 1-3 diversity sites. Each such diversity site is a functional group to which a variety of different substituents can be linked in one synthetic route. The reactivity of the diversity sites is "orthogonal" when multiple diversity sites are present. That is, one diversity site can react under its particular reaction conditions without affecting the other diversity sites under its particular reaction conditions. The other diversity site is only reactive under reaction conditions different from the one diversity site. The bead batch containing the scaffold thus introduced is divided into many sub-batches as there are first substituents in the chemical library, and each sub-batch is modified with one of such first substituents, and simultaneously or subsequently, the associated first coding DNA increment is added to the tag linker. The sub-batches are then repooled into a single batch. The subdivision into a number of sub-batches equal to the number of further substituents of the library, the ligation of the further substituents and the further associated coding DNA increments to the DNA tag already bound to the beads by using a conventional ligase such as T4 ligase, and the repooling are repeated. The repetition is carried out as many times as possible, as long as there are further substituents to be linked to the scaffold. That is, until all diversity sites in the scaffold are modified with the respective substituents of the chemical library.

[0081] Examples of diversity sites for scaffolds follow Table 2 below (* indicates possible attachment points of the diversity site to the scaffold).

[0082] [Table 2]

[0083] The diversity sites and reactions described above are known to be compatible with DNA moieties present in the same molecule or reaction medium and therefore can be modified in the presence of the encoding DNA (see Satz, A. L., Cai, J., Chen, Y., Goodnow, R., Gruber, F., Kowalczyk, A., Petersen, A., Naderi-Oboodi, G., Orzechowsky, L., Strebel, Q., Bioconjugate Chemistry 26, pp. 1623 ff. (2015)).

[0084] In the process according to the invention, the incubation of the beads and the cell targets in the incubation medium can be achieved by the following variants: Mutant a): providing an incubation medium; and combining in the incubation medium a plurality of beads including a plurality of instances of a single defined chemical structure and associated encoding DNA and cellular targets; Incubation is carried out in a single incubation medium (this alternative method requires one incubation medium for each chemical structure and each type of cellular target), where "incubation medium" is understood to be a medium volume small enough to be contained in a microcompartment (e.g., a microwell or a droplet in oil or microjetted or ink-ejected), but large enough to contain at least one bead.

[0085] Mutant b): An incubation medium is provided, all beads containing multiple instances of all chemical structures in the library and the cellular target are allowed to combine in the incubation medium, and multiple aliquots are divided from the incubation medium (this alternative method requires one incubation medium for every chemical structure and each type of cellular target). It is understood that an "aliquot of an incubation medium" is a small volume portion separated from a macroscopically sized incubation medium, and does not necessarily represent a defined fraction of the volume of the macroscopic incubation medium, but is small enough to be contained in a microcompartment (e.g., a microwell or a droplet in oil or microjetted or ink jetted) but large enough to contain at least one bead.

[0086] Having more than one bead in one aliquot, where the beads are of different types, can give ambiguous results in prior art assays. However, the process according to the invention is not detrimental to the method of the invention, since it is possible to first separate all the beads (hit and non-hit) and partition the hit beads from the non-hit beads. There can always be many other aliquots containing individual beads of one of these different types. Typically, there are dozens of aliquots containing one bead of a given type, either alone (the prevailing situation) or in combination with one or more beads of other types. The aliquots may be present in sample compartments, which can take any form, including wells of a microplate, microfabricated nanowalls, aqueous droplets in oil, or compartments made of lipid bilayers. Such sample compartments are themselves conventional. The aliquots can be prepared by co-spotting the cell targets and beads in an incubation medium via mechanical micropotting. In this case, the sample compartments can be simply defined as aliquot droplets separated from each other by a spatial distance. Alternatively, aliquots may be generated from the bulk incubation medium containing the cell targets, beads and any additives in the form of aqueous droplets, such as a water-in-oil emulsion, where each monodisperse droplet preferably contains only one bead.

[0087] Only one bead is sufficient to perform the assay of the invention in each incubation medium or aliquot thereof. Therefore, embodiments with a mean bead population λ of about 1 (preferably between 0.2 and 0.9), where λ is defined and calculated as described in the introduction, are preferred.

[0088] Preferably, the cellular targets can be added to the incubation medium before dividing the incubation medium into said aliquots. If the cleavage conditions for cleaving the chemical structures from the beads are harmful to the cellular targets, it may be preferable to add the cellular targets to the already divided aliquots after cleaving the chemical structures from the beads contained in the aliquot (and, optionally, after inactivating any remnants of the harmful cleavage chemicals).

[0089] However, cleavage of the chemical structures from the beads must be performed after division into aliquots to maintain confinement of the cleaved chemical structures and the encoding DNA tags bound to the beads.

[0090] The incubation medium itself is usually an aqueous medium, typically containing further adjuvants and / or nutrients (including enzymes and potential modulators for the cleaved chemical structure) required to perform the assay and / or to maintain the viability of the cellular target. The incubation is typically performed under conditions that allow the viability of the cellular target to be maintained (e.g., at or near room temperature, physiological pH and salt concentration) for a period of time until a change in the detachability or detachment of the response molecule is observed in the incubation medium or in one or more aliquots. The incubation is also performed by reaction of the probe attached to the bead, or by the appearance of cleaved DNA in the incubation medium, or by other techniques (e.g., GC-MS) suitable for detecting the presence or amount of the response molecule in the incubation medium. In the process of the present invention, it is preferred or essential that the reaction of the response molecule with the probe, or, in some cases, any cleavage of the tag linker and / or the cleavable tag linker site, and / or any cleavage of the encoding DNA by the response molecule, is kinetically irreversible. That is, in the case of reversible reactions, reacted probes may be able to revert to unreacted probes, or, in the case of cleaved tag linkers and / or cleaved tag linker sites and / or cleaved encoding DNA, these will rebind by simple re-establishment of thermodynamic equilibrium once the response molecule is separated from the bead as described above.

[0091] Following incubation, there are two alternatives for pooling the beads, isolating the hit beads, characterizing the encoding DNA, and correlating it with the chemical structure of the library.

[0092] Alternative a (where the tag linker and / or the cleavable tag linker site and / or the DNA sequence is cleavable by the response molecule): Once any released coding DNA or fragments thereof are detected in the incubation medium or in any aliquot thereof, or in multiple aliquots, the beads are separated from all the incubation medium or pooled from all the aliquots. This pooling process reduces the P from the average bead population λ that was present in the incubation medium or in that aliquot. hThis eliminates the dependency on (discussed in the introduction). This separation also serves to separate the beads from any remaining response molecules, which is not possible using the methods described in the prior art. Separation of the beads from the aliquot allows the assay to be terminated at the desired time point and then analyzed in the pooled state (i.e., PCR amplification and sequencing). Such follow-up analysis is essentially impossible using the methods described in the prior art. In the absence of such separation, the response molecules may continue to interact with the pooled beads, resulting in further cleavage of the DNA linkers attached to all the pooled beads. This would make it impossible to identify the beads that have active chemical structures attached to them. In this alternative a), the DNA of all the separated beads is released from the beads by cleaving the cleavable tag linker site. All the released DNA material is then analyzed and sequenced. In most cases, there will be instances of the full length of the released coding DNA (e.g., if the response molecule did not cleave either the tag linker, the cleavable tag linker site, or the coding DNA during incubation). These full-length instances of released coding DNA were therefore released from beads carrying chemical structures that were inactive during incubation. Additionally, there may be fragments of released coding DNA (if the response molecule cleaves the coding DNA itself during incubation) and coding of DNA sequences that are not entirely present in the released DNA material (if the response molecule cleaves the tag linker or cleavable tag linker site during incubation). These released fragments of coding DNA and any full-length instances encoding DNA that are not entirely present in the released DNA material are from beads carrying chemical structures that were active during incubation. Correlations with active chemical structures in the library are therefore made from fragments of coding DNA present in the released DNA material and full-length instances encoding DNA that are not entirely present in the released DNA material.

[0093] The degree of removal of the coding DNA during incubation of the screening batch of beads may be based on a comparison with a control batch of beads. The control batch is simply DNA sequenced without incubation. Alternatively, the control batch is optionally incubated under the same conditions as the screening batch (except that the cellular target is not present) before DNA sequencing. Both the screen and the control are performed using the same total number of beads. More preferably, the number of beads in the screening batch and in the control batch is 20-100 times the number of chemical structures in the assayed chemical library. Thus, ε, as defined in the introduction, ranges from 20 to 100. Here, a chemical structure is determined to be a possible hit if at least 5 beads with a given chemical structure are found in the control batch (i.e., if a completely corresponding DNA tag can be found in at least these 5 beads) and if less than 2 beads carrying the chemical structure are found in the screening batch (i.e., if a completely corresponding DNA tag can be found in only less than 2 beads). Here, if substantially all beads carrying a given chemical structure are found in the control batch (i.e., a fully corresponding DNA tag is found in a number of beads substantially corresponding to the number of beads employed for each chemical structure), and if substantially no beads carrying the chemical structure are found in the screening batch (i.e., a fully corresponding DNA tag is substantially not found in the beads of the screening batch), then the chemical structure may be determined to be a definite hit.

[0094] Alternative b (where the beads have chemical probes covalently bound to the response molecule): Once incubation in the incubation medium or aliquot thereof has progressed sufficiently, all beads are separated from the incubation medium or aliquot thereof and separated from any remaining response molecules, as described above, and then pooled. This pooling process removes the P from the average bead population λ that was present in the incubation medium or aliquot thereof. h This separation also serves to separate the beads from the rest of the response molecules. In the absence of such a separation, the response molecules may continue to interact with the probes of further pooled beads, making it impossible to identify the beads with the active chemical structures bound to them. In addition, such a separation is not possible using the methods described in the prior art. The separation of the beads from the aliquot allows the assay to be terminated at the desired time point and then optimizes the sorting of the collected beads, allowing the selection to be achieved at a later time point. In this alternative b), the beads with reacted probes are separated from the beads with unreacted probes. If the reacted probes are fluorescent and the unreacted probes are not (or vice versa), such a sorting can be performed using a commercially available fluorescence-activated cell sorter (it is possible to sort 100 million beads in a few hours, which is 100 times faster than the one described in the prior art). The use of a commercially available fluorescence-activated cell sorter is not possible using the methods described in the prior art, and its high throughput is required to interrogate numerically large chemical libraries. In this alternative b), the DNA of all separated beads with reacted probes is released from the beads by cleaving the cleavable tag linker site. All released DNA material is then analyzed and sequenced. Only full-length instances of the released coding DNA will be present. Correlations with active chemical structures in the library are therefore made from all full-length instances of the coding DNA present in the released DNA material.

[0095] In alternative b), the number of beads used for each chemical structure of the library is preferably in the range of 5 to 10. ε, as defined in the introduction, is then in the range of 5 to 10.

[0096] In either of the above separation alternatives a) or b), the incubation reaction may be terminated, if necessary, before or during the first step in the separation of the beads, for example by destruction or inactivation of the auxiliary and / or response molecules. Such destruction or inactivation may be carried out, for example, by a denaturing agent such as ethanol, or by heating, evaporation, precipitation, change in pH, oxidative destruction or salting out.

[0097] In either of the above separation alternatives a) or b), the pooling is either from the entire used incubation medium (incubation variant a above) or from all used aliquots thereof (incubation variant b above).

[0098] In either of the above separation alternatives a) or b), any released coding DNA or released fragments thereof are preferably amplified by standard techniques (such as PCR) and sequenced using standard protocols. Next generation high throughput sequencing can result in 200 million sequence reads per sequencing lane. In this way, a 10-fold increase in the number of samples interrogated has little impact on processing time, and thus 10 million small molecules can be interrogated as easily as 1 million molecules. Thus, the process according to the invention is scalable in terms of both logistics and processing time.

[0099] The screening method of the present invention can be useful, for example, in the following screening.

[0100] a) Lethality of the chemical structures of the compound library against a pathogenic cellular target. In this case, the response molecule may be a protein that indicates death or apoptosis of the cellular target, or it may be a degradation product from the cellular target itself. The onset of release of the response molecule during incubation of the cellular target indicates lethality.

[0101] b) Beneficial effects of the chemical structures of the compound library on the cellular target. In this case, the responsive molecule may be any compound known to be released at a certain level by a healthy cellular target, or any compound known to be released at a higher (or lower) level on a defective cellular target. A decrease (or increase) in the release of the responsive molecule during incubation of the cellular target indicates a benefit.

[0102] c) Suitability of the chemical structure as a prodrug capable of penetrating the cellular target, in cases where the drug itself is not capable of penetrating the cellular target.

[0103] A first preferred application for the treatment according to the invention is the secreted embryonal alkaline phosphatase (SEAP) reporter assay. This assay uses engineered reporter cells that indirectly report internal activation by chemical structures upon enhanced expression of SEAP secreted from cellular targets into the incubation medium or aliquots thereof. Such engineered reporter cells, on the other hand, can be custom designed according to known techniques. Published examples include, for example, the Huh7.5-EG(_4B ​​5A) SEAP cell line (Pan KL., Lee JC., Sung HW., Chang, TY., Hsu, JTA, Antimicrob Agents. Chemother 53(11) pp. 4825-4834 (2009)) (secretes SEAP upon infection with Hepatitis C virus), and the 293e / CRE-SEAP cell line (Durocher D., Perret S., Thibaudeau E., Gaumond MH., Kamen A., Stocco R., Abamovitz M., Anal. Biochem. 284, pp. 316-326(2000)) (secretes SEAP upon activation of a G protein-coupled receptor that is an important target for many known drugs). Such engineered cells may be commercially available. Thus, examples are the cell line HEK-Blue® IFN-a / b, which expresses and secretes SEAP upon stimulation with human IFN-a or IFN-b, and the TFIP1-Blue® ISG cell line marketed by InvivoGen, which expresses and secretes SEAP upon activation of Stimulator of Interferon Genes (STING), the activation of which is important in the treatment of cancer and infectious diseases. All these cell lines can be used in the SEAP assay of the present invention as well.

[0104] The chemical structures used as candidates in the SEAP assay of the present invention and linked to beads may, on the one hand, be compounds of the "rule of 5" type described above. A second suitable category is any compound that can be synthesized using known DNA-compatible chemistry. A third suitable category of candidate chemical structures is macrocyclic peptides. Libraries of cyclic peptides are disclosed, for example, in ACS Chem. Biol. 13, pp. 53-59 (2017). For use as chemical structures in alternative methods and beads, as shown in this publication, the DNA tag directly linked to the cyclic peptide via the amino group is replaced by a cleavable structural linker and a bead linked thereto (details are described herein). Further exemplary subsets of candidate chemical structures may, for example, follow one of the following libraries shown in Table 3 below.

[0105] [Table 3]

[0106] Diversity site X that appears in all examples in Table 3 1 , X 2 are independent of each other and may be, for example, -O-, -NH-, -(CH2)-, natural or unnatural amino acids (preferably linked to the respective carbonyl scaffolds at their amino groups and to the respective amino scaffolds at their carboxyl groups). The diversity sites X1, X2 may also be dipeptides or tripeptides consisting of natural amino acids (preferably linked to the respective carbonyl scaffolds via their N-terminus and to the amino scaffolds via their C-terminus).

[0107] Attachment of these chemical structures across a cleavable structural linker may be accomplished similarly to that outlined under Table 1.

[0108] The probes linked to the beads in the SEAP assay of the invention may on the one hand be organic, in particular aromatic or polyaromatic, hydroxyl-containing fluorophores or chromophores, the hydroxyl being converted to a phosphate that is susceptible to cleavage by the released SEAP. Upon cleavage by the SEAP released into the incubation medium or aliquot, the phosphate group is hydrolyzed, thus allowing the fluorescence of the fluorophore or the coloring or color change of the chromophore. Known examples of such phosphate-quenched fluorophores are 1-oxo-3',6'-diphosphono-spiro[isobenzofuran-3,9'-xanthene]-5-carboxylic acid and 1-oxo-3',6'-bis(phosphonooxymethoxy)spiro[isobenzofuran-3,9'-xanthene]-5-carboxylic acid. These phosphorylated chromophores or fluorophores preferably contain a carboxylic acid group that allows them to be bound to the beads by amide group formation using a suitable amino-terminated linker already attached to the beads. The reactive fluorescence is easily detectable in the pooled beads as in the corresponding homogeneous prior art assays. The reactive coloring or reactive color change of the bead-bound chromophores can be measured, for example, by reflectance of the pooled beads instead of standard spectroscopy as used in the corresponding homogeneous prior art assays.

[0109] A second preferred application for the screening method of the present invention is the screening of antibiotics. Here, the cellular target would be a pathological bacterium that releases a nuclease as a response molecule upon cell death. In this second application, the beads preferably contain multiple instances with a single chemical structure and multiple instances of an encoding DNA sequence covalently attached to the beads via a tag linker that contains a moiety cleavable by said nuclease. Alternatively, the beads lack other chemical moieties that are sensitive, cleavable and / or reactive to the response molecule. That is, the beads may consist of a bead core, a linked chemical structure, and a linked encoding DNA, but lack others. Such beads themselves are an object of the present invention. Here, any of multiple possible antibiotic candidates may be linked to an equal number of beads (or an equal number of bead batches) in one step, and at the same time, a corresponding encoding DNA tag may be attached to each such bead (or to the beads in each bead batch). Alternatively, the beads may be constructed stepwise using a suitable scaffold that has a high probability of generating an antibiotically active chemical structure when modified with further substituents. Here, such a scaffold may be, for example, a cyclic oligopeptide comprising lysine, asparagine and / or serine units providing, for example, hydroxy and / or amino as diversity sites for attaching further substituents.

[0110] A third preferred application for the screening method of the present invention is the screening of small molecules for possible anti-cancer drugs. Effective anti-cancer drugs can induce apoptosis of target cancer cells. Such apoptosis is accompanied by the release of caspases. In this third preferred application, the beads of the present invention preferably have a moiety represented by the following formula as a probe. The beads are similar to the microparticle-based system described by Yozwiak CE, Hirschhorn, T., and Stockwell, BR in ACS Chem. Biol. 2018, 13, 761-771. [ka]

[0111] Here, "spacer" is a divalent residue. R 1 is the fluorophore and R 2 is a quencher that acts by fluorescence resonance energy transfer (FRET), or R 1 The peptide sequence in the document is SEQ ID NO: 3. This probe is cleaved at the rightmost D amino acid of the DEVD sequence to release caspase-3, which is R 2 This removes the quencher as a positive signal and allows fluorescence by R1. The fluorescent beads can be sorted using a commercially available fluorescence activated cell sorter (FACS). The positively classified beads are subjected to DNA amplification and sequencing to determine that the small molecule was originally attached to these beads and caused apoptosis and caspase release. Preferably, in the above formula, R1 and R2 are selected according to one of the rows in the following table:

[0112] [Table 4]

[0113] The invention will now be illustrated by the following non-limiting examples.

[0114] Example 1: Assay of Fluoroquinolone Activity Against Bacteria

[0115] The test is performed with fluoroquinolones, such as ciprofloxacin or levofloxacin, that are capable of inducing oxidative stress in bacteria, which can cause DNA damage and therefore the release of endonucleases (such as BapE DNA endonuclease) from oxidatively stressed bacteria.

[0116] Step 1: Preparation of barcode DNA and attachment of the corresponding attachment to beads (protocol for each individual barcode DNA corresponding to one fluoroquinolone to be analyzed)

[0117] As described in the general description of application section, each headpiece DNA containing a unique nucleotide sequence for each fluoroquinolone "tag" is provided and functionalized with 8 PEG chains and primary amines. To this solution of functionalized headpiece DNA (1 mM in H20) is added Hunigs' base (5 equivalents) and ((1R,8S,9S)-bicyclo[6.10]non-4-yn-9-ylmethyl N-succinimidyl carbonate (3 equivalents) (supplied as a 0.2 M solution in NMP) at 0°C. Upon completion of the reaction, as judged by ESI-TOF mass spectrometry, it is purified according to the reported precipitation procedure. The resulting functionalized DNA headpiece (in DNA-BCN below) is resuspended in water at a concentration of 1 mM and used without further purification.

[0118] Tetagel beads functionalized with azidopentanoic acid (loaded at 0.29 mmol / g) are divided into a number of batches corresponding to the number of fluoroquinolones to be assayed. The beads in each batch are washed briefly with PBS, then with water, and finally with CH3CN. Each of the DNA-BCNs obtained from the previous step (0.004 equivalents) is dissolved in a 1:1 mixture of PBS:CH3CN (alternatively, 10% pyridine in water may be used) and added to one of the batches with azide-modified beads. Strain promoted alkyne azide cycloaddition (SPAAC) is then carried out at 45°C in an incubator for about 24 hours. The DNA-BCN functionalized bead batches are washed with CH3CN, PBS, and finally with molecular biology grade water and kept at 0°C in an ice bath until further use.

[0119] Step 2: Attaching the fluoroquinolone to the beads

[0120] All DNA-BCN functionalized bead batches obtained from step 1 are converted from the remaining azide moieties to primary amine moieties using triphenylphosphine in DCM and H20 at room temperature.

[0121] Each batch of beads thus obtained is reacted with the respective fluoroquinolone antibiotic according to the following protocol: the beads undergo standard peptide coupling and are functionalized with an acid-labile Rink linker, followed by functionalization with a PEG8 linker and two lysine residues. Each batch of beads (1.0 equiv., 0.0096 mmol) is treated with a preactivation solution of 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (secondary alcohol photolabile linker; 4.0 equiv., 38.4 μmol), HATU (3.8 equiv., 36.5 μmol), and Hunig's base (6.0 equiv., 57.6 μmol) in 100 μl NMP at room temperature for 2-12 hours. The beads are then carefully washed and an analytical sample is checked by LCMS upon acid-mediated cleavage. A solution of fluoroquinolone antibiotic (10.0 equiv., 48.0 μmol), DIAD (15.0 equiv., 72.0 μmol), triphenylphosphine (15.0 equiv., 72.0 μmol) is prepared in 150 μL of anhydrous THF and a small amount of NMP to aid in dissolving the fluoroquinolone, and Mitsunobu complex formation is carried out at 0°C. The so-prepared solution is poured onto the beads (1.0 equiv., 0.0048 mmol). The reaction is first carried out at 0°C and then at room temperature for 12 hours to replace the hydroxyl of the photocleavable linker already bound to the beads with the carboxylate from the fluoroquinolone. The beads are then carefully washed and an analytical sample is checked by LCMS upon acid-mediated cleavage. All batches of beads are finally pooled to obtain a single pool, each containing beads modified with a single unique fluoroquinolone / DNA-BCN combination.

[0122] Step 3: Encapsulation of the beads and bacterial cells and assay (protocol for each bacterial strain to be tested)

[0123] Using a flow-focusing microfluidic chip, aqueous droplets are generated in oil, resulting in an emulsion of monodisperse droplets. The aqueous phase contains cells of the bacterial strain of interest, beads prepared as described above that are fully functionalized and barcoded, and additives, such that each droplet contains zero, one or more beads and zero, one or more, or up to tens of thousands of bacterial cells. The droplets are collected in a microscopic reaction tube. The droplets with the beads are exposed to UV light for several minutes to release the fluoroquinolone from all the beads by photolytic cleavage of the photolabile linker described above. The droplets are incubated for several hours. The droplets are then optionally heat inactivated (depending on the desired assay reaction). Alternatively, the emulsion is broken by organic solvent, which also stops the detection reaction. The beads are collected on a frit. The filtrate is amplified by PCR and sequenced to test for the presence of any headpiece DNA that may be cleaved by the bacterial endonuclease. The presence indicates that the fluoroquinolone associated with the headpiece DNA is active against that bacterium.

[0124] Example 2: Sequential construction of bead-linked encoding DNA, allowing for split and pool synthesis of linked libraries of chemical structures and beads containing linked encoding DNA.

[0125] To a solution of the headpiece DNA oligomer functionalized with 8 PEG chains and a primary amine (1 mM in H20) and the primary amine described in the general description of the application, Hunigs' base (5 eq.) and (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl n-succinimidyl carbonate (3 eq.) (supplied as a 0.2 M solution in NMP) are added at 0 °C. Upon completion of the reaction, as judged by ESI-TOF mass spectrometry, the reaction is purified according to the reported precipitation procedure. The resulting functionalized DNA headpiece (DNA-BCN) is resuspended in water at a concentration of 1 mM and used without further purification.

[0126] Tetagel beads functionalized with azidopentanoic acid (loaded at 0.29 mmol / g) are washed briefly with PBS, then with water, and finally with CH3CN. Each of the DNA-BCNs obtained from the previous step (0.004 equivalents) is dissolved in a 1:1 mixture of PBS:CH3CN (alternatively, 10% pyridine in water may be used) and added to the Tentagel beads. Strain-promoted alkyne azide cycloaddition (SPAAC) is then carried out in an incubator at 45°C for about 24 hours to obtain beads with the first headpiece DNA oligomer ligated.

[0127] The beads from the previous step are washed with CH3CN, then with PBS, and finally with molecular biology grade water. The beads are kept at 0°C in an ice bath while the ligation mixture is prepared. For the DNA loading from the previous step, the additional labeled DNA to be ligated to the existing DNA tag (1.2 equivalents) is diluted in DNAse free water, 10X T4 ligation buffer is added, and the mixture is kept on ice. The ligation final volume is 150 μL for 10-15 mgs of beads. T4 DNA ligase is then added and the mixture is poured onto the beads. The reaction is then carried out at room temperature for 5-16 hours.

[0128] The DNA tagging steps described above are performed as many times as there are variable substituents to be linked to the fixed scaffold in the split-and-pool synthesis.

[0129] Example 3: Bead Sorting

[0130] This process is applicable, for example, to the beads used in the assay of the present invention, which contain a fluorophore and its associated quencher as a probe, the quencher being removed by the response molecule. After the incubation is completed, the beads are separated from the incubation medium and its response molecule, and then the separated beads (and both fluorescent and non-fluorescent probes) are pooled. The pooled beads are sorted using a commercially available fluorescence-activated cell sorter (FACS) by gating on the desired properties of the beads (fluorescent or not, and potentially other gates for quality control). The positively classified beads were subjected to DNA amplification and sequencing.

[0131] Example 4: Bead-based secreted embryonic alkaline phosphatase (SEAP) reporter assay

[0132] Step 1: Preparation of quenched fluorescein-labeled beads

[0133] A quenched fluorescein derivative (e.g., 1-oxo-3',6'-diphosphono-spiro[isobenzofuran-3,9'-xanthene]-5-carboxylic acid or 1-oxo-3',6'-bis(phosphonooxymethoxy)spiro[isobenzofuran-3,9'-xanthene]-5-carboxylic acid) is incubated with single-stranded DNA (modified with 5'-amino C6) and a water-compatible acylating reagent (e.g., DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride)) in an aqueous buffer. The encoded DNA library beads with bead-bound DNA barcodes contain single-stranded DNA ends whose sequences are complementary to those of the fluorescein single-stranded DNA conjugates. Upon incubation with the pool of library beads, the fluorescein single-stranded DNA complex anneals with its complement on the beads to form a stable double-stranded DNA duplex.

[0134] Alternatively, the fluorescein single-stranded DNA conjugate can be annealed with another single-stranded DNA oligomer to form a duplex with an overhang. In this case, the coded DNA library bead with the bead-bound DNA barcode is terminated with a short overhang of 2-4 bases, which is complementary to the overhang of the fluorescein-bound double-stranded DNA. The double-stranded DNA oligomer and the barcode are then covalently linked using ligase as previously described (see Clark et al.). Using any of the above methods, the library beads can be labeled with any probe as desired.

[0135] Step 2: Encapsulation of beads and eukaryotic cells (THP1-Dual®KI-hSTING-S154, InvivoGen) and assay

[0136] Using a flow-focusing microfluidic chip, aqueous droplets are generated in oil, resulting in an emulsion of monodisperse droplets. The aqueous phase contains the desired eukaryotic cells, beads (additionally labeled with phosphorylated fluorescein) prepared as described above that are fully functionalized and barcoded, and additives, such that each droplet contains zero, one or more beads and zero, one or more, or up to several hundred eukaryotic cells. Note that the additives can include 2'3'-cGAMP if the cells are THP1-Dual®KI-hSTING-S154. In addition, a positive control (known irreversible small molecule inhibitor H-151, InvivoGen) may be used as an additive to provide a baseline for subsequent statistical analysis and bead sorting. The droplets are collected in a microscopic reaction tube. The droplets with the beads are exposed to UV light for several minutes to release the library members from all the beads by photolytic cleavage of the photolytic linker described above. The droplets are incubated for several hours. The droplets are then heat inactivated. Alternatively, the emulsion is broken by organic solvent, which also stops the detection reaction. The beads are collected on a frit and pooled. Also, weakly fluorescent hit beads are sorted from the pool by FACS. The weakly fluorescent beads are then amplified by PCR and sequenced. Weak fluorescence indicates that the released small molecule associated with the barcode DNA has successfully inhibited the activation of STING, thereby preventing the release of SEAP.

Claims

1. A method for screening a DNA-encoded library of chemical structures (2) for activity in a cellular target (11), comprising: The cellular target (11) is known to release or alter the release of a response molecule (12) when contacted with an active chemical structure; The chemical structures (2) of the library and their corresponding encoding DNA sequences (4) are covalently attached to beads (1); Each bead (1) is a) multiple forms of a single chemical structure (2) in said library; b) a plurality of forms of an encoding DNA sequence (4) that encodes said chemical structure (2); having Each of said forms of said chemical structure (2) is covalently attached to said bead (1) via a structural linker (3); The structural connector (3) is detachable at a detachable structural connector portion (3a); The coding DNA sequence (4) is covalently attached to the bead (1) via a tag linker (5), The tag linker (5) includes a detachable tag linker portion (5a) and is detachable by a cleavage agent (6); the cleavable structural linker moiety (3 a) is not cleavable under reaction conditions that cleave the cleavable tag linker moiety (5 a), and vice versa; the tag linker (5) and / or the detachable tag linker portion (5a) and / or the coding DNA sequence (4) are detachable by the response molecule (12); when a chemical probe (7, 8, 9) which reacts with the response molecule (12) is covalently bound to the bead (1) or when the coding DNA sequence (4) and / or the detachable tag linker site (5a) and / or the tag linker (5) are detached by the response molecule (12), The method comprises the following steps (ib-1), (ib-2), (ii), and (iii): (ib-1) providing a single incubation medium (13) for mixing the cellular targets (11) and all the beads (1) linked to all the chemical structures (2) of the library; partitioning a plurality of aliquots from the incubation medium in the form of droplets large enough to contain at least one bead and having zero, one or more beads (1); having The average bead population λ of the partitioned aliquot is between 0.2 and 0.9, where λ is defined by the following equation (2): [0010] In formula (2), (k m ) i is an integer indicating the number of beads in the i th aliquot, M is the number of all partitioned aliquots, (ib-2) releasing the chemical structures (2) from the beads (1) in each of the aliquots by cleaving the structural connectors (3) at the cleavable structural connector sites (3a); incubating the cellular targets (11) and the released chemical structures (2) in the aliquot of the incubation medium (13); having (ii) when the coding DNA sequence (4) and / or the detachable tag linker portion (5a) and / or the tag linker (5) are detachable by the response molecule (12), (ii-a-1) the aliquots of the incubation medium (13) are monitored for release of the coding DNA sequence (4) or fragments thereof from the beads (1), and if said release is detected, separating all beads (1) from all the aliquots of the incubation medium (13) and pooling all the separated beads (1); (ii-a-2) the cleavable tag linker sites (5a) on the pooled beads (1) are cleaved by the cleavage agent (6) to release the encoding DNA sequence (4) or a fragment thereof; (ii-a-3) the released coding DNA sequence (4) or a fragment thereof is amplified and sequenced to identify the complete DNA sequence in the DNA coding library; (ii-a-4) the complete coding DNA sequence (4) contained in the DNA-encoded library that was not identified in step (ii-a-3) and the fragments of the coding DNA sequence (4) identified in step (ii-a-3) are associated with corresponding chemical structures (2) in the DNA-encoded library; Alternatively, when the bead (1) has the response molecule sensitive chemical probe (7, 8, 9), (ii-b-1) an aliquot of the incubation medium (13) is monitored for a reaction of the probes (7, 8, 9) to the response molecule (12) or a change in said reaction, and if said reaction or change is detected, separating and pooling all beads (1) from all of the aliquots of the incubation medium (13); (ii-b-2) beads (1) that have shown a reaction or a change in the reaction of the probe are extracted from the pool; (ii-b-3) the cleavable tag linker site (5a) on the beads (1) extracted from the pool is cleaved by the cleavage agent (6) to release the coding DNA sequence (4) covalently attached to the extracted beads (1); (ii-b-4) the released coding DNA sequence (4) is amplified and sequenced; (ii-b-5) the encoding DNA sequence (4) sequenced in step (ii-b-3) is associated with a corresponding chemical structure (2) in the DNA-encoded library; (iii) the chemical structure (2) associated in step (ii-a-4) or (ii-b-5) is selected as the further active chemical structure; A method comprising:

2. 10. The method of claim 1 , the tag linker (5) and / or the detachable tag linker portion (5a) and / or the coding DNA sequence (4) are detachable by the response molecule (12); The steps (ii-a-1), (ii-a-2), (ii-a-3), and (ii-a-4) are carried out. A method comprising:

3. 3. The method of claim 2, The cellular target (11) is a prokaryotic cell known to undergo cell death upon contact with an active chemical structure, releasing a nuclease as the response molecule (12); The tag linker (5) and / or the detachable tag linker portion (5a) and / or the coding DNA sequence (4) are detachable by the nuclease. A method comprising:

4. 10. The method of claim 1 , The bead (1) comprises chemical probes (7, 8, 9) which react with the response molecule (12) and are covalently bound to the bead (1), The steps (ii-b-1), (ii-b-2), (ii-b-3), (ii-b-4), and (ii-b-5) are carried out. A method comprising:

5. 5. The method of claim 4, The cellular target (11) is a eukaryotic cell that has been engineered such that when a desired cellular target or pathway is contacted with an appropriate chemical structure (2), the response molecule (12) is secreted embryonic alkaline phosphatase (SEAP); The chemical probe reacts with the secreted embryonic alkaline phosphatase A method comprising:

6. The method according to claim 4 or 5, The chemical probe (7, 8, 9) is a combination of a fluorophore (7) and a quencher (8) that acts by fluorescence resonance energy transfer (FRET) or contact quenching to the fluorophore (7), The fluorophore (7) and the quencher (8) are bound to each other via a spacer (9) that is detachable by the response molecule (12); In the step (ii-b-1), the incubation medium (13) or an aliquot thereof is monitored for cleavage of the spacer (9) by the response molecule (12) accompanied by the fluorescence of the fluorophore (7). A method comprising:

7. The method according to any one of claims 1 to 6, The cleavable tag linker site (5a) is a nucleotide sequence that is cleavable by a restriction endonuclease as the cleavage agent (6). A method comprising:

8. 8. The method of claim 7, The tag linker (5) is -0-(CH 2 -CH 2 -0) n -, and having a bivalent spacer (5b) located immediately adjacent to said detachable tag linker portion (5a), Here, n is an integer from 5 to 10. A method comprising:

9. The method according to any one of claims 1 to 8, Each of the beads (1) comprises the multiple forms of the chemical structures (2) attached to the bead (1) via structural connectors (3) that are cleavable by UV light. A method comprising:

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