Small Molecule Screening Cellular Assay Using Decorated Beads

Abstract

A method for screening the activity of a DNA-encoded chemical structure (2) library in a cellular target (11), wherein the chemical structure (2) of the library, the corresponding encoding DNA (4), and optionally a chemical probe (7 / 8 / 9) sensitive to a response molecule (12) are covalently linked to beads (1); the method comprises providing an incubation medium (13) or an aliquot thereof containing the cellular target (11) and one or more beads (1) as defined above, releasing the chemical structure (2) from the beads (1) in the incubation medium (13) or an aliquot thereof by cleaving a structural linker (3) and incubating the released chemical structure (2) with the cellular target (11); and sequencing the encoding DNA present on or retained by the beads (1). Also provided are beads (1) suitable for the method.

Description

Technical Field

[0001] The present invention relates to a screening assay for small molecules having potential efficacy in altering the activity of a cellular target of pharmaceutical interest, and methods for performing the assay. Background Art

[0002] Most targets of pharmaceutical interest are active in a cellular environment. When investigating small molecules that alter target activity, it can be beneficial to do so in a cellular setting, including when screening large numbers of different compounds.

[0003] A desired system response to a target-modulating small molecule might be the release of molecules from the cell. These molecules could be enzymes, proteins, nucleic acids, or smaller cellular products such as metabolites. Release can be achieved through targeted release or simple leakage. During screening, these released molecules are detected as a measure of compound action.

[0004] Screening small molecule compounds in cell-based assays in the pharmaceutical industry is typically accomplished by combining the compound and the cell in a container (Jones E, Michael S, Sittampalam GS. Basics of Assay Equipment and Instrumentation for High Throughput Screening. 2012 May 1 [Updated 2016 Apr 2]. In: Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004). Thus, the small molecule can act on cellular molecules outside the cell or in the cell membrane, or it can enter the cell and perform its action inside the cell. The ability of the compound to modulate cellular activity is then determined by monitoring feasible analytical readouts. Such analytical readouts can be, for example, the processing of a substrate by an enzyme. The processing of the substrate can produce a fluorescent signal. Another approach is, for example, to monitor the binding of the small molecule to a protein target by changing fluorescence.

[0005] By using microcompartments, for example in the format of n-well plates (where n is typically 96, 384 or 1536), a large number of different small molecules can be investigated (= screened) for their ability to modulate target activity or binding. Commercially available microplate readers are typically used to measure the activity of each small molecule by measuring the remaining substrate (or product appearance) in each individual well of the plate. Another known method for dividing such an assay into multiple microcompartments is to generate aqueous droplets containing the assay reagent in the form of a water-in-oil emulsion.

[0006] When using a microplate reader, the logistics of long-term storage of small molecule plates is challenging, especially when processing a large number of plates. In addition, the time required for product formation in the assessment microplate increases linearly with the number of small molecules, which is problematic when studying millions of small molecules (using current technology, assessing 2 million molecules requires approximately 10 working days) (Brouzes E., Medkova M., Savenelli N., Marran D., Twardowski M., Hutchison JB, Rothberg JM, Link DR, Perrimon N., Samuels. ML, PNAS 106, pp. 14195-14200 (2009)).

[0007] Any such analysis requires that the structures of the compounds to be found useful be readily determined. One solution to this problem is to provide the compounds to be screened in the form of DNA-encoded chemical libraries (DELs) (Brenner and Lerner, Proc. Natl. Acad. Sci. USA. 89:5381-5383 (1992)). In a DEL, each compound is pre-linked (tagged) with a unique DNA sequence corresponding to its structure. Thus, there is no need to identify the structure of the useful compound itself (a task that may be structure-dependent or even impossible), only to sequence the corresponding DNA tags, a standard procedure that is the same for all useful compounds.

[0008] However, such DEL-based assays lag behind the problem that compounds linked to DNA tags may behave differently in the assay than the free compound. In WO 2018 / 087539, it was proposed to cleave the compound and its linked DNA tag before analysis, but to keep the free compound and its linked DNA tag "spatially associated" with each other.

[0009] ACS Chem.Biol.13,pp.761-771(2018) discloses a small molecule analysis using silica beads with quenched fluorophore probes and small molecules attached thereto and having DNA tags. In this analysis, beads penetrate into cell targets, thereby themselves acting as microcompartments. Small molecules are cut off from the penetrated beads by light. Any small molecule that is lethal to the cell target will cause its apoptosis, release caspase-3, and in the beads that are still infiltrated, the quencher is removed from the fluorophore. These now fluorescent cell targets (thus now fluorescent microcompartments) are sorted out using flow cytometry, and the DNA of any beads contained therein is then sequenced to find the corresponding small molecules that cause cell apoptosis.

[0010] US 5,958,703 A discloses a vector with a tether that can be modified with a reporter molecule, and related screening methods. The disclosure "isolates" vectors with modified tethers. Therefore, the disclosure does not compile all vectors before isolating the vectors with the modified tethers.

[0011] WO 2013 / 057188 ​​A1 releases compounds so that they are “retained within the solid support / beads,” or “each compound is physically located within its parent solid support,” or “released within the beads,” or even allows the “substrate” to be “absorbed into” the support / beads. Thus, the disclosure does not release the compounds to be analyzed into the incubation medium, but rather retains them within the beads. Therefore, the “physicochemical or biological system” targeted by the disclosure is a soluble substance, not a cellular target: in the latter case, there would be two heterogeneous phases (the compound-containing beads and the cellular target), which would hinder interaction between them.

[0012] ACS Comb. Sci. 19, pp. 524-532 (2017) describes the principle of an oil-in-water droplet-based assay. Beads encoding a DNA library are encapsulated in the droplets and incubated in a test assay. Any droplets that show a positive reaction in the incubation assay (and therefore any positive microcompartments) are first sorted, and beads isolated from these sorted droplets are then further examined to see if they are statistically relevant hits.

[0013] ACS Comb. Sci. 21, pp. 425-435 (2019) also describes an oil-in-water droplet-based assay. Beads encoding a DNA library are encapsulated in droplets and incubated in a screening assay for autotaxin inhibitory activity. This assay does not use cellular targets, but rather homogenously dissolved autotaxin as a target. This publication also first sorts out the hit droplets: Figure 1 A "droplet sorting junction (5)" using fluorescence detection is shown.

[0014] In these prior art bead-based assays, a "positive" microcompartment (such as a microwell, droplet, cell, or other) is first selected or sorted, and then all beads contained in the sorted microcompartments are pooled together and analyzed for their DNA tags. However, a "positive" microcompartment may contain more than one bead, of which typically only one is a hit bead (i.e., a bead that provides an active small molecule), while the others are non-hit beads. The aforementioned disclosure designates these missed beads as "passenger beads" and derives a mathematical expression for the "false discovery rate" to describe the degree of separation and discovery of such missed beads. Only in a second round of testing can a separated bead be confirmed to be a missed bead.

[0015] As described below, the probability that a bead isolated in such prior art analysis is a hit, P h .

[0016] Bead clusters in microcompartments—P b is Poisson distributed:

[0017]

[0018] where k m is the number of beads in the microcompartment in question (hence 0, 1, 2, etc.). m An upper threshold value K cannot be exceeded, which depends on the volume of the beads, the volume of the microcompartment, and the reproducibility of this volume. For example, the total volume of all beads contained in a microcompartment cannot be greater than the volume of the microcompartment itself. If the volume of the microcompartment is variable and can reach infinity, then K can also reach infinity. However, real microcompartments, such as microwells or droplets, are very small and have very good reproducibility in volume. So for real microcompartments, K is usually less than 10. Reducing the volume of the microcompartment and thus reducing K is also a common way to reduce λ: the average bead population λ of a microcompartment must be smaller than its maximum possible bead population K. λ is the average bead population of a microcompartment, defined as

[0019]

[0020] Where M is the number of all microcompartments, and the sum covers all M microcompartments. b is the actual content of a given microcompartment (k m ) beads. This type of bead distribution is obtained by splitting the bead population into microcompartments using all known devices.

[0021] The k in a given microcompartment m The bead has exactly k p The probability of hitting the bead P p is a hypergeometric distribution:

[0022]

[0023] where 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 in question; a number close to zero but unknown), ε is the so-called “library equivalent” (the number of beads with the same library chemical structure attached to them), and k p is the number of hit beads, k m As defined above, the assumption here is that εN, rεN, and ε(1-r)N are integers or are rounded to integers.

[0024] A given microcompartment contains k m bead and where exactly k p The probability P that a bead is a hit bead after incubation with the cellular target (these events are independent of each other) is:

[0025]

[0026] If its associated k p If k is greater than 0 (and therefore contains at least one hit bead), the prior art method identifies the incubated microcompartment as "positive". The k of such a "positive" microcompartment m is also greater than 0. The amount B of all beads (hits and misses) obtained from all such "positive" microcompartments tot Therefore

[0027] where k p The sum of covers the number of all possible hit beads in the microcompartment, which is at most the total number of beads k in it. m , and k m The sum of is at most a threshold K, which is the maximum number of beads a microcompartment can contain. Formula (6) assumes that a given microcompartment contains a number k m or another number k m ' beads are mutually exclusive events, and one has a given number k m A given microcompartment of beads contains a number k p or another number k p 'The hitting beads are also mutually exclusive events.

[0028] The amount of hit beads obtained from all such "positive" microcompartments is B h for

[0029] All symbols and explanations are as above. Formula (7) makes the same assumption about mutual exclusion as the above formula (6).

[0030] Then the above P h yes

[0031]

[0032] The P h is less than 1, because k in the numerator p Less than the corresponding k in the denominator m .P h It gets smaller as λ increases because the λ polynomial in the denominator increases faster than the λ polynomial in the numerator, which is also because k p Less than the corresponding k m However, P h The value reaches a maximum at λ = 0. That is, these prior art methods have the lowest degree of false discovery of missed beads in a microcompartment with an average bead population λ that is not actually feasible. A actually feasible average bead population has a λ greater than 0 (there should be a microcompartment containing one or more beads therein).

[0033] Therefore, there is no optimal solution for minimizing false discoveries, analysis of miss beads, and analysis of viable microcompartment bead populations in the prior art methods; other improved methods are necessary.

[0034] The present invention seeks to overcome the aforementioned problems. Summary of the Invention

[0035] The present invention provides:

[0036] 1. A method for screening a library of DNA-encoded chemical structures that are active in a cellular target; wherein the cellular target is known to release a response molecule or alter the release of a response molecule when contacted with the active chemical structure; wherein the chemical structures of the library, the corresponding encoding DNA, and optionally a chemical probe sensitive to the response molecule are covalently attached to beads, wherein each bead comprises

[0037] a) multiple instances of a single chemical structure of the library, each instance covalently attached to the bead via a structural linker that is cleavable at a cleavable structural linker site; and

[0038] b) multiple instances of a DNA sequence encoding the chemical structure, each DNA sequence covalently linked to the bead via a tag linker, the tag linker comprising a cleavable tag linker site and cleavable by a cleavage agent;

[0039] c) under the reaction conditions that cleave the cleavable tag linker site, the cleavable structure linker site is not cleavable, and vice versa;

[0040] and the tag adapter and / or the cleavable tag adapter site and / or the DNA sequence can optionally be cleaved by the response molecule; provided that, if the encoding DNA sequence and / or the cleavable tag adapter site and / or the tag adapter can be cleaved by the response molecule, then the beads preferably do not contain a chemical probe sensitive to the response molecule;

[0041] The method comprises the following steps:

[0042] (i) Either

[0043] (ia) providing, for each individual chemical structure and each individual cellular target to be analyzed, an incubation medium comprising said cellular target and one or more beads as defined above, said beads having that individual chemical structure attached thereto, releasing said chemical structure from the beads in the incubation medium by cleaving the structure linker at a cleavable structure linker site, and incubating said cellular target and said released chemical structure in the incubation medium;

[0044] or

[0045] (ib) providing a single incubation medium comprising a cellular target and all beads as defined above, having all chemical structures of the library attached thereto, separating aliquots thereof comprising one or more beads from the incubation medium, releasing the chemical structure from the beads contained in each aliquot by cleaving the structure linker at a cleavable structure linker site, and incubating said cellular target and said released chemical structure in aliquots of the incubation medium;

[0046] (ii) either, if the encoding DNA sequence and / or the cleavable tag adapter site and / or the tag adapter is cleavable by the response molecule, then:

[0047] (ii-a-1) monitoring the incubation medium or aliquots thereof for release of any encoding DNA sequence or fragment thereof from any beads; and if so, separating all beads from all incubation medium or all aliquots thereof and pooling all separated beads;

[0048] (ii-a-2) the cleavable tag linker sites in the pooled beads are cleaved by a cleavage agent to release any encoding DNA sequence or fragment thereof;

[0049] (ii-a-3) amplifying and sequencing the released coding DNA sequences or fragments thereof to identify therein any complete DNA sequence of the DNA encoding library; and

[0050] (ii-a-4) correlating the remaining portions of the complete DNA sequence of the DNA-encoded library that were not identified in step (ii-a-3) with the corresponding chemical structures of the DNA-encoded library;

[0051] Alternatively, if the beads contain a chemical probe sensitive to the response molecule:

[0052] (ii-b-1) monitoring the incubation medium or aliquot thereof for any reaction or change in reaction of any probe with the response molecule and, if so, separating all beads from all incubation medium or aliquots thereof and pooling them;

[0053] (ii-b-2) extracting beads that exhibit the probe response or a change in the probe response from the pool;

[0054] (ii-b-3) cleaving the cleavable tag linker site in the beads extracted from the pool with a cleavage agent to release any DNA sequence covalently attached to the separated beads;

[0055] (ii-b-4) amplifying and sequencing the released DNA sequence; and

[0056] (ii-b-5) correlating any DNA sequences sequenced in step (ii-b-3) with the corresponding chemical structures of the DNA-encoded library;

[0057] and

[0058] (iii) selecting any chemical structure as linked in step (ii-a-4) or (ii-b-5) as further said active chemical structure.

[0059] Preferred embodiments of the method are described in the dependent claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 is a schematic diagram of beads suitable for use in the method of the invention, which itself forms part of the invention.

[0061] Figure 2 is a schematic diagram of another type of bead suitable for use in the method of the present invention.

[0062] Figure 3 is a schematic diagram of the incubation medium containing beads and cell targets. DETAILED DESCRIPTION

[0063] Compared with the prior art methods, the method of the present invention first separates and pools all beads (whether hit or miss) from all incubation media or aliquots thereof from all assay compartments (whether "positive" or not), and then identifies hit beads among the pooled beads. In the method of the present invention, the P of the sorted beads is h is uniform and has nothing to do with the average bead population λ present in the microcompartment. In the immediate process, it is not important whether more beads have to be identified based on probe reactions or based on DNA sequencing: for the former, there are very powerful sorting automation tools (e.g., FACS in the case of fluorescent probes) and for the latter, there are, for example, automated next generation sequencing (NGS). Furthermore, it is possible to check only a portion of the bead pool for hit beads. This portion of the bead pool can be a representative portion thereof, for example comprising a number of beads that is the number of chemical structures present in the library multiplied by the "library equivalent" ε as defined in the introduction, where ε can typically be in the range of 1 to 100, preferably 5 to 50.

[0064] The advantageous effects of first pooling all beads and then sorting out the hit beads will be explained in the following four paragraphs.

[0065] The prior art methods mentioned in the introduction first sort out the hit microcompartments (e.g. hit droplets). Therefore, all microcompartments, whether containing beads or not, need to be checked for activity by the sorter. h hit beads, then the number of necessary microcompartments M that must be examined for activity in prior art methods is 现有技术 Yes, using the above formula (7),

[0066]

[0067] The method of the present invention first separates and pools all beads (hit and miss) from all microcompartments (incubation medium or its aliquots), and then sorts out the hit beads. The number of microcompartments required for separating and pooling beads is assumed to be M. 本发明 The number of separated and pooled beads (hits and misses) is λM 本发明 , where λ is the average bead population of the above microcompartment calculated according to the above formula (2). Then the hit beads are sorted from the pool, and the number of hit beads obtained is rλM 本发明 , where r is the above-mentioned “library hit rate”. For comparison with the above-mentioned prior art methods, this number of hit beads is again designated as B h . Based on this, we get

[0068]

[0069] From the comparison between (9) and (10), it can be seen that in order to obtain the required quantity B h The method of the present invention requires the number of microcompartments M 本发明 The number of microcompartments M required by the prior art method is 现有技术 Less so, in those cases where:

[0070]

[0071] If the threshold K above is assumed to be 1, then e can be simply obtained from the above inequality -λ <1

[0072] It is independent of N, ε, and r and holds for any λ > 0. As mentioned before, λ must also be less than K. For K = 1, the useful range of λ is therefore 0 < λ < 1, and in this case, to obtain a given number B h The method of the present invention is superior to the above-mentioned prior art method in terms of the number of microcompartments required.

[0073] Therefore, a preferred embodiment of the method of the present invention is wherein:

[0074] 1a) in step (1-a), the incubation medium has an upper threshold K of 1 for the number of beads that can be contained therein, i.e., the incubation medium is not allowed to contain more than one bead, or

[0075] 1b) in step (ib), each aliquot from the incubation medium has an upper threshold value K of 1 for the number of beads that may be contained therein, i.e., each aliquot from the incubation medium is not allowed to contain more than one bead,

[0076] also

[0077] 2) Average bead group λ, defined as

[0078]

[0079] in

[0080] (k m ) i is an integer representing the number of beads in the i-th incubation medium or the i-th aliquot of the incubation medium; M is the number of incubation media or aliquots of the incubation medium, respectively; and the sum covers all incubation media or all aliquots of the incubation medium, respectively,

[0081] The average bead population λ is greater than 0 and less than 1.0.

[0082] The above requirement of K = 1 is easily achieved with rigid microcompartments, such as microwells of appropriately small volume, because their rigidity strengthens K. For flexible microcompartments, such as droplets, droplets with beads can be immediately generated with a sufficiently small volume to ensure K = 1. Alternatively or in addition, the volume of the droplets can be further reduced by a "droplet separator" as used in ACS Comb. Sci. 21, pp. 425-435 (2019).

[0083] refer to Figure 1 、 2 and 3, the present invention provides a solution to the above problem by binding a small molecule in the form of a chemical structure 2 to a bead 1, wherein the small molecule is connected to the bead 1 via a cleavable structural linker 3. The structural linker 3 includes a cleavable structural linker site (3a). The bead 1 also includes a DNA barcode 4 connected to the bead 1 via a tag linker 5. The tag linker 5 includes a cleavable tag linker site 5a ( Figure 1 ). In the screening process of the present invention, after incubation with the cellular target and separation and pooling of the beads, all DNA barcodes 4 are cleaved from the beads 1 using the cleavable tag adapter site 5a, allowing them to be sequenced and associated with the corresponding chemical structure of the library. Figure 1 In the embodiment of the present invention, the tag linker 5 and / or the cleavable tag linker site 5a and / or the coding DNA 4 are assumed to be cleavable by the response molecule 12 generated during the incubation process, so the bead 1 does not have (or is not necessary to have) a probe. Alternatively, if the tag linker 5, the cleavable tag linker site 5a and the coding DNA 4 are not cleavable by the response molecule 12, the bead 1 further includes a chemical probe 7 / 8 / 9 connected to the bead 1 via a spacer 10, such as Figure 2 As shown in the implementation plan. Figure 2 As shown, the chemical probe 7 / 8 / 9 herein is preferably composed of a fluorophore 7 and a quencher 8 for the fluorophore 7, wherein the fluorophore 7 and the quencher 8 are linked together by a spacer 9 that can be cleaved by a response molecule. Figure 3 , the cellular target 11 is bound or co-encapsulated with the bead 1 in an aliquot of the incubation medium 13 or its sample compartment, and the chemical structure 2 is released from the bead 1 by cleavage of the cleavable structure attachment site (3a). Reaction of the chemical probe with the response molecule ( Figure 2 ) or the continued presence of the uncleaved form of the tag adapter ( Figure 3 The molecule 12 (not shown in the figures, but implicitly assumed to be associated with the beads 1) records the presence and / or activity of response molecules 12 released (or released at different levels) by cellular targets 11 in the incubation medium 13 or aliquots thereof. Figure 3Only one bead 1 in a compartment is shown, which is a preferred embodiment of the invention, but there may be multiple beads, each containing the same chemical structure. Similarly, there may be multiple instances of the cellular target 11, rather than the only instance shown.

[0084] The screening process of the present invention uses beads modified with the chemical structure of the library, the corresponding DNA barcodes and optionally probes of the response molecules. For the purposes of the present invention, any type of particulate, solid or gel-like material can be used as a "bead" as long as the particulate material

[0085] a) is inert to the incubation medium, particularly an aqueous medium, in which the screening process of the invention is carried out ("inert" means that the material does not react with the incubation medium and is substantially insoluble therein), and

[0086] b) have a particle surface comprising chemically reactive moieties that allow covalent bonding of chemical structures, chemical probes and DNA barcodes to the surface of the bead via respective linkers.

[0087] The bulk of the beads may be an inorganic particulate material (e.g. silica or alumina), in which case the chemically reactive moieties will be primarily hydroxyl groups. In another more preferred embodiment, the bulk of the beads is an organic polymer, particularly polystyrene, which may be modified on the surface by the introduction of chemically reactive moieties. There are a number of such surface-modified organic bead materials available on the market, for example Rapp Polymere under the trade name Beads are commercially available. These beads typically contain a surface loading of chemically reactive moieties in the range of 0.1 to 0.5 mmol / g of beads. The beads are preferably approximately spherical, preferably with an average diameter in the range of 50 μm to 500 μm. Such beads, even those whose surfaces are modified with chemically reactive moieties, are conventional.

[0088] As used herein, the term "chemical structure" refers to the small molecule in free form or a derivative of the small molecule bound to a bead, whereby it is clear from the context in which the term is used which of the two meanings applies.

[0089] The screening process of the present invention first cleaves the chemical structure from the bead, allowing the free, DNA-free chemical structure to penetrate the cellular target. Once the chemical structure has penetrated the cellular target, it can interact with, react with, interfere with, enhance, or inhibit any system present in the cellular target. Such systems can include, for example, any type of receptor, as well as systems involved in cell differentiation, transcription, translation, respiration, membrane construction, and mitosis.

[0090] The structural linker that connects the chemical structure to the beads can be any organic divalent group that is bound to the bead surface on one side and to the chemical structure on the other. The only required feature is that the structural linker contains a cleavable structural linker site, which allows the chemical structure to be released from the beads in the incubation medium. Preferably, the cleavable structural linker site is immediately adjacent to the chemical structure so that upon cleavage, the chemical structure contains as few residues from the structural linker as possible.

[0091] Preferred examples of such cleavable structural linker sites are provided in Table 1 below (* indicates a valency that is preferably attached to the remainder of the structural linker, while ** indicates a valency that is preferably attached directly to the chemical structure):

[0092] Table 1

[0093]

[0094]

[0095] These cleavable structural linker sites are themselves conventional, with further examples in the literature.

[0096] The beads used in the screening process of the present invention first have a library chemical structure covalently bound to the bead surface via a cleavable structural linker. The chemical structure can be any chemical compound found in nature, produced by synthetic means or by (modified) biological activity (such as transcription). The molecular weight of the chemical structure can, for example, range from a few hundred daltons to several thousand or tens of thousands of daltons.

[0097] Preferred examples of chemical structures that may form part of a screening library are any pharmaceutically acceptable compounds that satisfy all of a) to d) below:

[0098] a) they contain up to five hydrogen atoms capable of participating in hydrogen bonding and are derived from hydroxyl groups (providing one such hydrogen atom), primary amino groups (providing two such hydrogen atoms) and secondary amino groups (providing one such hydrogen atom);

[0099] b) they contain up to ten oxygen and nitrogen atoms capable of acting as hydrogen bond acceptors (moieties containing electron lone pairs capable of participating in hydrogen bonds);

[0100] c) they have a molecular weight of at most 500 Daltons; and

[0101] d) the negative decimal logarithm of its octanol / water partition coefficient (-log(c 正辛醇 / c 水 ), where c 正辛醇 is the molar concentration of the compound in n-octanol, c 水is its molar concentration in water, the n-octanol solution of the compound and the aqueous solution of the compound being in contact with each other at 25° C. and in thermodynamic equilibrium) is at most +5; preferably in the range of -0.4 to +5.

[0102] Compounds that meet the above conditions are generally defined as "Rule 5" compounds.

[0103] Preferably, the screening process of the present invention can rely on a library of known chemical structures as candidates for attachment to beads. Table 1 of J.Med.Chem.59, pp.6629-6644 (2016) provides an overview of known libraries. In order to be used as chemical structures in the present method using beads, as shown in the table of the publication, the DNA tags directly attached to the chemical structure via the amino group will be replaced by a cleavable structural linker and beads attached thereto, as described herein. In addition, the split-merge method described herein will be used to simultaneously construct fragments and components of the chemical structure and the associated DNA tag fragments.

[0104] The beads contain a "plurality" of such chemical structures attached thereto. The "plurality" must be large enough so that the chemical structure, once released from even a single bead, is in sufficient quantity, or in a sufficiently high concentration in the incubation medium, to cause a detectable release of the response molecule from the cellular target. The "plurality" is limited by the number of reaction sites present on the surface of the beads. If it is not possible to obtain a detectable release of the response molecule from the cellular target even when all the reaction sites of the beads are attached to the chemical structure, then the chemical structure may be a priori ineffective for its intended purpose and can be discarded from the analysis. Furthermore, the "plurality" of chemical structures can be in the range of 0.001 to 0.01 molar equivalents, based on the above-mentioned surface loading of the beads with the chemically reactive portion.

[0105] Secondly, the beads used in the method of the present invention have a coding DNA covalently linked to the surface of the beads via a tag linker. The tag linker can also be any suitable organic bivalent linker, as long as it contains a cleavable tag linker site, which allows the DNA barcode to be released from the beads after the beads are incubated with the cell target, separated and collected therefrom. Examples of cleavable tag linker sites and their related cleavage reagents can be cleavable structural linker sites and their related cleavage reagents as described above, or 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 contains a recognition site for the restriction endonuclease. Many examples of suitable restriction endonucleases and their related recognition sites are known in the literature. For example, Table 2 of WO2010 / 94036A1 discloses various restriction endonucleases and their related recognition and cleavage sites in the second column. The most preferred restriction endonuclease as a cleavage agent is Stu1.

[0106] In the case of a cleavable tag linker site that can be cleaved by a restriction endonuclease, it is further preferred that the tag linker further comprises a PEG divalent spacer of 5-10 ethylene glycol units (preferably 8 ethylene glycol units) in length immediately adjacent to the cleavable tag linker site. This may facilitate cleavage of the restriction endonuclease recognition site.

[0107] Preferably, the cleavable tag adapter site is immediately adjacent to the encoding DNA so that upon cleavage the encoding DNA contains as few residues from the tag adapter as possible. Preferably, the tag adapter itself is constructed by a so-called "click chemistry" reaction.

[0108] The above preferred variants of the tag linker, cleavable tag linker site and DNA barcode, optionally with said PEG spacer forming part of the tag linker and immediately adjacent to the cleavable tag linker site, are preferably constructed according to the following synthetic scheme:

[0109]

[0110] In this scheme, Fmoc-PEG derivatives are commercially available, for example, from JenKem (USA), Abbexa (UK) or Iris Biotech (Germany). Wherein, n is 5 to 10, preferably 8. m is 0 to 1, and k is 0 to 2; preferably, m and k are both 0, or m is 0 and k is greater than 0. C6-amino-terminated deoxythymidine (dT) derivatives are also commercially available, for example, from GeneLink (USA). Thereafter, the Fmoc-protected amine portion can be deprotected, and the free amine can be attached to a DNA tag linker already attached to the beads, or the free amine can be directly attached to beads with carboxyl surface functional groups, in which case the PEG linker itself will form a DNA tag linker. One or two oligomers such as X and / or Y can serve as headpiece DNA, and in the separation and merging methods described herein, other coding DNA fragments can be continuously attached to the headpiece DNA. If both X and Y are DNA oligomers, this may 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 the deoxythymidine moiety into a vicinal diol using alkaline hydrogen peroxide, which will then be a cleavable tag linker site that can be cleaved by NaIO4 (see Table 1 above). Alternatively, as described above, oligomers such as X and / or Y can contain restriction endonuclease recognition sites as cleavable tag linker sites.

[0111] For the methods and beads of the invention, the cleavable structural linker site and the cleavable tag linker site are "orthogonal" in reactivity, i.e., the cleavable structural linker site is not cleavable under reaction conditions that cleave the cleavable tag linker site, and vice versa;

[0112] The tag adapter and / or the cleavable tag adapter site and / or the encoded DNA can be cleaved by the response molecule generated during the incubation process, in which case a chemical probe is unnecessary. The cleavable tag adapter site itself must then first be cleaved by the cleavage agent and optionally by the response molecule. Herein, the cleavage agent and the response molecule can be different or the same; preferably, they are different.

[0113] The coding DNA itself is preferably hairpin DNA. This allows for the construction of a fully double-stranded DNA from only one DNA strand, as it can pair itself to form a double strand. Therefore, only one coding DNA sequence needs to be constructed. In addition to the actual coding sequence characteristics of the chemical structure to be encoded, the coding DNA may also include leader and / or tail sequences that may be necessary or beneficial when connecting the coding DNA to beads and / or cutting the tag adapter at a cleavable tag adapter site. These further leader and / or tail sequences are typically the same for all encoded DNA instances.

[0114] The beads contain a "plurality" of such encoding DNA instances. This number must be large enough so that the encoding DNA instances (even from a single bead) are numerous enough to allow amplification (e.g., by PCR) and subsequent sequencing. Furthermore, based on the above-described surface loading of the beads with chemically reactive moieties, the "plurality" of encoding DNA instances can be in the range of 0.001 to 0.01 molar equivalents.

[0115] The pearl used in the present invention optionally comprises a chemical probe, which is sensitive to the response molecule and is connected to the pearl. The chemical probe can be a substrate for the response molecule released, which can be an enzyme, protein or molecule so that the pearl fluoresces (or stops fluorescing) after being incubated with the released substance and a suitable additive. Alternatively (or in combination with it), the released enzyme can be degraded to change the DNA barcode combined with the pearl, thereby regulating its detection by amplification or hybridization. The probe can include a chemical indicator, including a reporter molecule. For example, it can be a colorimetric assay (i.e., causing a colored reaction product between the response molecule and the probe, which absorbs light within the visible light range), fluorescent (e.g., based on the response molecule and the probe being converted into an enzyme that emits fluorescence when excited by light of a specific wavelength) and / or luminescent (e.g., based on bioluminescence, chemiluminescence and / or photoluminescence). Preferably, the probe passes through a fluorescent reaction when contacted with the response molecule, and is therefore a combination of a fluorophore or a fluorophore and a related quencher, wherein the response molecule cuts the quencher from the fluorophore. However, the chemical probe is only present if the bead does not contain a tag linker, which itself can be cleaved by the response molecule.

[0116] The spacers that connect the probes to the beads can also be used for structural linkers and / or tag linkers as described above.

[0117] The cellular target is preferably a prokaryotic or eukaryotic cell. More preferred examples of prokaryotic cells are Gram-positive bacteria, Gram-positive cocci, Gram-negative cocci, and Gram-negative bacteria. More preferred examples of eukaryotic cells are fungal cells and animal cells, particularly human cells. The cellular target contains proteins, receptors, or other substances that readily interact with chemical structures that permeate the cellular medium from outside the cellular target. Following this interaction, the cellular target may initiate production of the response molecule, increase production of the response molecule, or reduce or even cease production of the response molecule.

[0118] The beads used in the screening process of the present invention are synthesized according to known chemical methods, or by directly bonding the encoding DNA to the linker through a tag linker and a chemical structure to form the beads.

[0119] Alternatively, the encoding DNA and chemical structure can be synthesized stepwise directly on the beads, for example by split-and-pool synthesis. In this alternative, the "backbone" (the chemical core structure common to all small molecules in the library to be tested) is initially attached to the beads via a cleavable linker as described above, and no tag linker of the encoding DNA is also attached to the beads. The backbone is typically a ring-containing structure containing a set of diversity sites, for example, typically 1 to 3 diversity sites. Each such diversity site is a functional group on which a variety of different substituents can be attached through the same synthetic pathway. If there are more than one diversity site, the reactivity of the diversity sites is "orthogonal", that is, one diversity site can react with the various substituents under its specific reaction conditions without affecting other diversity sites, which are only reactive under reaction conditions different from those of the one diversity site. The batch of beads containing the backbone thus introduced is divided into as many subbatches as the first substituents in the chemical library, and each subbatch is modified with one such first substituent, and simultaneously or subsequently the associated first encoding DNA increment is added to the tag linker. The subbatches are then re-pooled into a single batch. This is split again into subbatches equal to the number of another substituent in the library, and the additional substituent and the associated encoding DNA are incrementally ligated to the DNA tags already bound to the beads using a conventional ligase such as T4 ligase. This re-pooling is repeated multiple times until more substituents are attached to the backbone, that is, until all diversity sites in the backbone are modified with the corresponding substituents from the chemical library.

[0120] Examples of diverse sites of the backbone are shown in Table 2 below (* indicates possible connection points between the diverse sites and the backbone):

[0121] Table 2:

[0122]

[0123]

[0124] The above-mentioned diversity sites and reactions are known to be compatible with DNA moieties present in the same molecule or reaction medium and can therefore be modified in the presence of encoding DNA (see, AL, Cai, J., Chen, Y., Goodnow, R., Gruber, F., Kowalczyk, A., Petersen, A., Naderi-Oboodi, G., Orzechowsky, L., Strebel, Q., in Bioconjugate Chemistry 26,, pp. 1623ff. (2015)).

[0125] In the methods of the present invention, incubation of the beads and the cell targets in an incubation medium can be accomplished by any of the following methods: Method a): providing an incubation medium and combining one or more beads comprising multiple instances of a uniquely defined chemical structure and its associated encoding DNA with the cell targets therein and incubating them in the unique incubation medium (this alternative requires one incubation medium for each chemical structure and each type of cell target). "Incubation medium" is understood here to mean a volume of medium that is small enough to be contained in a microcompartment, such as a microwell or a droplet dispersed in oil or micro-dotted or inkjetted, but large enough to accommodate at least one bead.

[0126] Mode b): Provide an incubation medium in which all beads and cell targets containing multiple instances of all chemical structures of the library are combined, and separate aliquots from this incubation medium (this alternative requires one incubation medium for all chemical structures and each type of cell target). Here, "aliquot of incubation medium" is understood to be a small volume portion separated from the macroscopic incubation medium and does not necessarily represent a defined volume fraction of the macroscopic incubation medium, which is small enough to be contained in a microcompartment, such as a microwell or a droplet dispersed in oil or microspotted or inkjetted, but which is large enough to contain at least one bead.

[0127] If the beads are of different types, having two or more beads in one aliquot may give ambiguous results in prior art analyses after incubation. However, this is not detrimental to the method of the present invention because the method of the present invention first separates all the beads (hits and misses) and only then separates the hits and misses. There will always be many other aliquots, each containing a single bead of the different types described. Typically there will be dozens of aliquots containing beads of a given type, either alone (the main case) or in combination with one or more other types of beads. The aliquots can be present in sample compartments, which can take any form, including wells in a microplate, microfabricated nanopores, aqueous droplets in oil, or compartments consisting of lipid bilayers. Such sample compartments are conventional. Aliquots can be prepared by co-spotting cell targets and beads in an incubation medium by mechanical microspotting, in which case the sample compartments can be simply defined as aliquots of droplets separated from each other by spatial distances. Alternatively, aliquots can be generated from a bulk incubation medium containing the cellular target, beads, and optional additives as aqueous droplets of a water-in-oil emulsion, wherein each monodisperse droplet preferably contains only one bead.

[0128] In each incubation medium or aliquot thereof, only one bead is sufficient to perform the assay of the present invention. Therefore, an average bead population λ of about 1.0, preferably about 0.2 to about 0.9, is a preferred embodiment, wherein λ is defined and calculated as described in the introduction.

[0129] The cellular target may preferably be added to the incubation medium before it is divided into said aliquots. On the other hand, if the cleavage conditions used to cleave the chemical structure from the beads are harmful to the cellular target, it may be preferable to add the cellular target to the already divided aliquots after the chemical structure has been cleaved from the beads contained therein, and optionally even after any residues of the harmful cleavage chemical have been inactivated.

[0130] However, cleavage of the chemical structure from the beads must be performed after said aliquoting to maintain confinement of the cleaved chemical structure and the bead-bound encoding DNA tag.

[0131] The incubation medium itself is typically an aqueous medium, typically containing other adjuvants and / or nutrients (including enzymes and potential modulators that cleave chemical structures) required for performing the assay and / or maintaining the activity of the cellular target. Incubations are typically performed under conditions that allow the viability of the cellular target to be maintained, for example at or near room temperature and physiological pH and salt concentration, for a period of time until a possible release or change in release of the response molecule is observed in the incubation medium or one or more aliquots thereof, whether by reaction of probes attached to beads, the appearance of cleaved DNA in the incubation medium, or any other technique suitable for detecting the presence or amount of the response molecule in the incubation medium (e.g., GC-MS). In the methods of the present invention, it is preferred or even necessary that any reaction of the response molecule with the probe, or, as the case may be, any cleavage of the tag linker and / or cleavable tag linker site and / or encoding DNA by the response molecule is kinetically irreversible. That is, in the case of a reversible reaction, the reacted probe will be converted back to an unreacted probe; or in the case of a cleaved tag adapter and / or cleaved tag adapter site and / or cleaved encoding DNA, as described above, once the response molecule is separated from the beads, these will reattach by simply reestablishing thermodynamic equilibrium.

[0132] After incubation, two alternative methods can be used to pool beads, isolate hits, and characterize the encoded DNA and its association with the library chemistry:

[0133] Alternative method a (if the tag adapter and / or cleavable tag adapter site and / or DNA sequence is cleavable by the response molecule): Once any released encoding DNA or fragment thereof is detected in the incubation medium or any aliquot thereof, or in a plurality of aliquots, the beads are separated from all of the incubation medium or from all aliquots thereof and then pooled. This pooling eliminates the P discussed in the introduction h Dependence on the average bead population λ present in the incubation medium or its aliquots. This separation is also used to separate beads from any residues of the response molecule, and this separation cannot adopt the method described in the prior art. Separating beads from aliquots quenches the analysis at the desired time point, then allowing subsequent analysis (i.e. PCR amplification and sequencing) to the beads under the pooled state. Adopting the method described in the prior art, this subsequent analysis is essentially impossible. In the absence of this separation, the response molecule will continue to interact with the beads collected and further cut the DNA linker attached to all the collected beads, which will make it impossible to identify the beads attached with active chemical structures. In this alternative a), the DNA of all separated beads is released from the beads by cutting the cleavable tag linker site. Subsequently, all released DNA materials are analyzed and sequenced. In most cases, there will be full-length examples of the coding DNA released (if during incubation, the response molecule neither cuts the tag linker nor cuts the cleavable tag linker site or coding DNA). Therefore, the full-length examples of the coding DNA released have been released from the beads with the chemical structure, and the chemical structure is inactive during incubation. In addition, there will be released fragments of coding DNA (if the response molecule cleaves the coding DNA itself during the incubation process) and coding DNA sequences that are completely absent from the released DNA material (if the response molecule cleaves the tag adapter or cleavable tag adapter site during the incubation process). Therefore, these released coding DNA fragments and any full-length instances of coding DNA that are completely absent from the released DNA material are from beads with chemical structures that are active during the incubation process. Therefore, the correlation with the active chemical structure in the library is composed of fragments of coding DNA that are present in the released DNA material and full-length instances of coding DNA that are completely absent from the released DNA material.

[0134] During the incubation process of the screening bead batch, the degree of removal of the encoding DNA can be based on a comparison with a control bead batch. The control batch is simply subjected to DNA sequencing without any incubation, or optionally, the control batch is incubated before DNA sequencing under the same conditions as the screening batch (except that the cellular target is not present). Screening and control are both performed using the same total number of beads. More preferably, the number of beads in the screening batch and the control batch herein is 20-100 times the number of chemical structures in the analytical chemical library, so that the ε defined in the introduction is within the range of 20-100. If at least 5 beads carrying a given chemical structure are found in the control batch herein (that is, the complete corresponding DNA tag can be found in these at least 5 beads), and less than 2 beads carrying the chemical structure are found in the screening batch (that is, the complete corresponding DNA tag can only be found in these less than 2 beads), then the chemical structure can be considered a possible hit. If substantially all beads carrying a given chemical structure are found in the control batch (i.e., the corresponding DNA tag is found intact in a number of beads substantially corresponding to the number of beads used for each chemical structure), and substantially no beads carrying that chemical structure are found in the screening batch (i.e., substantially no corresponding DNA tag is found intact in the beads of the screening batch), then that chemical structure can be considered a clear hit.

[0135] Alternative method b (if the beads have a chemical probe sensitive to a response molecule covalently attached thereto): As described above, once the incubation in the incubation medium or aliquot thereof has been sufficiently carried out, all beads are separated from all the incubation medium or aliquot thereof, and hence from any remnants of the response molecule, and then pooled. This pooling eliminates P hDependence on the average bead population λ present in the incubation medium or its aliquots. This separation is also used to separate the beads from any remnants of the response molecules. Without this separation, the response molecules would continue to interact with the probes of further collected beads and produce further reactions, which would make it impossible to identify beads with active chemical structures attached thereto. In addition, this separation is not possible using the methods described in the prior art. Separating the beads from the aliquots quenches the analysis at the desired time point, which then allows the sorting of the collected beads to be optimized and completed at a later time of choice. In this alternative b), the beads with the reacted probes are then separated from the beads with the unreacted probes. If the reacted probes are fluorescent and the unreacted probes are not (or vice versa), then this sorting can be done using a commercially available fluorescence-activated cell sorter (100 million beads can be sorted in a few hours, 100 times faster than described in the prior art). When the methods described in the prior art are employed, it is not possible to use a commercially available fluorescence-activated cell sorter, and its higher throughput is required to query a chemical library with larger values. In this alternative b), all probe-reacted DNA from the isolated beads is released from the beads by cleaving the cleavable tag linker site. Subsequently, all released DNA material is analyzed and sequenced. Only the full-length instances of the released coding DNA will be present. Thus, the correlation with the active chemical structures in the library is composed of all full-length coding DNA instances present in the released DNA material.

[0136] In alternative b), the number of beads used for each chemical structure of the library is preferably in the range of 5-10, and thus ε is defined in the introduction as being in the range of 5-10.

[0137] In either of the above separation alternatives a) or b), the incubation reaction can be terminated, if desired, before or as a first step in separating the beads, for example by destroying or inactivating the adjuvant and / or the response molecule. Such destruction or inactivation can be accomplished, for example, by denaturing agents (e.g., ethanol), heating, evaporation, precipitation, changing the pH, oxidative destruction, or salting out.

[0138] In either of the above separation alternatives a) or b), the incubation medium from all used (incubation variant a) or from all used aliquots thereof (incubation variant b) is pooled.

[0139] In the above-described 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 provide 200 million sequence reads per sequencing channel. In this way, a tenfold increase in the number of samples investigated has little impact on processing time, so 10 million small molecules can be investigated just as easily as 1 million. Therefore, the methods of the present invention are scalable in terms of both logistics and processing time.

[0140] The screening method of the present invention can be used, for example, to screen:

[0141] a) The lethality of the chemical structure of the compound library to the pathogenic cell target. In this case, the response molecule can be a protein that indicates the death or apoptosis of the cell target, or it can be a degradation product of the cell target itself. The onset of release of the response molecule during incubation with the cell target is an indication of lethality.

[0142] b) Beneficiality of the compound library chemical structure in the cell target. In this case, the response molecule can be any compound known to be released at a certain level by healthy cell targets and known to be released at a higher (or lower) level in damaged cell targets. A decrease (or increase) in the release of the response molecule during incubation with the cell target indicates benefit.

[0143] c) Suitability of the chemical structure as a prodrug that can penetrate the cellular target, in cases where the drug itself cannot penetrate the cellular target.

[0144] A first preferred application of the methods of the present invention is a secreted embryonic alkaline phosphatase (SEAP) reporter assay. This assay utilizes genetically engineered reporter cells that indirectly report internal activation of SEAP through a chemical structure, which in turn secretes it from the target cell into the incubation medium or an aliquot thereof. Such genetically engineered reporter cells can be custom designed according to known techniques. Published examples are, for example, the Huh7.5-EG (_4B5A) SEAP cell line described by Pan KL., Lee JC., Sung HW., Chang, TY., Hsu, JTA; Antimicrob. Agents. Chemother. 53 (11) pp. 4825-4834 (2009) (secreting SEAP upon infection with hepatitis C virus) and the 293E / CRE-SEAP cell line described by Durocher D., Perret S., Thibaudeau E., Gaumond MH., Kamen A., Stocco R., Abramovitz M., Anal. Biochem. 284, pp. 316-326 (2000) (secreting SEAP upon activation of G protein-coupled receptors, an important target for many known drugs). Such engineered cells are even commercially available in some cases. Thus, an example of a cell line is HEK-Blue TM IFN-α / β (expresses and secretes SEAP upon stimulation with human IFN-α or IFN-β), the designed cell line is THP1-Blue sold by InvivoGen TM ISG cell lines (which express and secrete SEAP upon activation of the stimulator of interferon genes (STING), which is important in the treatment of cancer and infectious diseases) can also be used in the SEAP assay of the present invention.

[0145] The chemical structures used as candidates and attached to the beads in the SEAP assay of the present invention can, on the one hand, be compounds of the "Rule 5" type described above. As a second preferred category are any compounds synthesized using known chemicals that are compatible with DNA. A third preferred category of chemical structure candidates are macrocyclic peptides. For example, a library of cyclic peptides has been disclosed in ACS Chem.Biol.13, pp.53-59 (2017). For use as chemical structures in alternative methods and beads, the DNA tag directly attached to the cyclic peptide at the amide group, as shown in this publication, will be replaced by a cleavable structural linker and beads attached thereto, as described herein. Another exemplary subset of chemical structure candidates can be, for example, based on one of the following libraries shown in Table 3 below

[0146] Table 3

[0147]

[0148]

[0149]

[0150] The diversity sites X that appear in them 1 and X 2 Examples may be, independently of one another, such as -O-, -NH-, -(CH2-), natural or non-natural amino acids (preferably linked to the respective backbone carbonyl groups via their amino groups and to the respective backbone amino groups via their carboxyl groups); and dipeptides or tripeptides consisting of natural amino acids (preferably linked to the respective backbone carbonyl groups via their N-termini and to the backbone amino groups via their C-termini).

[0151] Attachment of these chemical structures to the cleavable structural linker can be performed in a manner similar to that outlined in Table 1 above.

[0152] In the SEAP probes of the present invention, the probes attached to the beads can, on the one hand, be organic, particularly aromatic or polyaromatic, hydroxyl-containing fluorophores or chromophores, wherein the hydroxyl groups have been converted to phosphates that are readily released by SEAP cleavage. Upon cleavage by SEAP released into the incubation medium or aliquot, the phosphate groups are hydrolyzed, causing the fluorophore to fluoresce or the chromophore to stain or change color. Known examples of such phosphate-quenched fluorophores include 1-oxo-3',6'-diphosphinooxy-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 also contain a carboxylic acid group that can be attached to the beads by forming an amide group using a suitable amino-terminated linker already attached to the beads. As with corresponding similar prior art assays, the existing fluorescence is readily detected in the pooled beads. The intrinsic color or intrinsic color change of the bead-bound chromophore can be measured on the pooled beads, for example by reflectance spectroscopy, rather than conventional spectroscopy used in corresponding similar prior art assays.

[0153] The second preferred application of the screening method of the present invention is to screen antibiotics. The cellular target here is a pathological bacterium that releases a nuclease as a response molecule when the cell dies. In this first application, the beads can preferably contain multiple instances of a single chemical structure and multiple instances of the coding DNA sequence covalently connected to the beads by a tag linker, wherein the tag linker contains a portion that can be cut by the nuclease. In addition, the beads do not have any other chemical parts that are sensitive, cut and / or reactive to the response molecule. In other words, the beads may consist of a bead core, a connected chemical structure and a connected coding DNA, but nothing else. Such beads themselves are the purpose of the present invention. Here, multiple possible antibiotic candidates can be connected to the same number of beads (or the same number of bead batches) in one step, and the corresponding coding DNA tag can be connected to each such bead (or the beads in each bead batch). Alternatively, beads can be gradually constructed using a suitable skeleton that is likely to produce an antimicrobial active chemical structure after being modified by other substituents. Here, such a skeleton can be, for example, a cyclic oligopeptide containing, for example, lysine, asparagine and / or serine units, which provides, for example, hydroxyl and / or amino groups as diversity sites for connecting other substituents.

[0154] A third preferred application of the present screening method is to screen small molecules for potential anticancer drugs. An effective anticancer drug can induce apoptosis in targeted cancer cells. This apoptosis is accompanied by the release of caspases. In this second particularly preferred application, the beads of the present invention can preferably include as probes, similar to the microparticle-based systems described by Yozwiak CE, Hirschhorn, T., and Stockwell, BR in ACS Chem. Biol. 2018, 13, 761-771, a portion of the following formula:

[0155]

[0156] Wherein "spacer" is a divalent residue; R 1 is the fluorophore, R 2 It is a quencher that acts on the fluorophore through fluorescence resonance energy transfer (FRET) or contact quenching (R 1 ). The peptide sequence is SEQ ID NO. 3. When the rightmost D amino acid in the DEVD sequence releases caspase-3, the probe is cleaved, which removes the quencher (R 2 ), and make R 1Able to emit fluorescence. Fluorescent beads can be sorted using a commercial fluorescence activated cell sorter (FACS). The positively sorted beads are subjected to DNA amplification and sequencing to find which small molecule initially attached to these beads and caused cell apoptosis and caspase release. Preferably, in the above formula, R is selected according to a row in the table below 1 and R2:

[0157]

[0158]

[0159] The invention will now be illustrated by the following non-limiting examples.

[0160] Example 1: Analysis of the antibacterial activity of fluoroquinolones

[0161] The test uses fluoroquinolones, such as ciprofloxacin or levofloxacin, which may induce oxidative stress in bacteria. Oxidative stress may lead to DNA damage, which in turn releases endonucleases, such as BapE DNA endonuclease, from oxidatively stressed bacteria.

[0162] Step 1: Prepare barcoded DNA and attach it to beads (one protocol for each individual barcoded DNA corresponding to one fluoroquinolone to be analyzed)

[0163] Individual headdress DNAs were provided, each containing a unique nucleotide sequence to "tag" a respective fluoroquinolone and functionalized with 8 PEG chains and primary amines as described in the Summary section of this application. To a solution of such functionalized headdress DNAs (1 mM in H2O) was added at 0°C. Base (5 equivalents) and (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (3 equivalents) were provided as 0.2 M solutions in NMP. After completion of the reaction, as judged by ESI-TOF mass spectrometry, the reaction was purified according to the reported precipitation procedure. The resulting functionalized DNA headpiece (in DNA-BCN below) was resuspended in water at a concentration of 1 mM and used without further purification.

[0164] Tentagel beads functionalized with azidoglutaric acid (loaded at 0.29 mmol / g) were divided into batches corresponding to the number of fluoroquinolones to be analyzed. Each batch of beads was first washed briefly with PBS, then with water, and finally with CH3CN. Each DNA-BCN (0.004 equivalents) obtained in the previous step was dissolved in a 1:1 PBS:CH3CN mixture (alternatively, a 10% aqueous pyridine solution could be used) and then added to one of the batches with azide. Strain-promoted alkyne azide cycloaddition (SPAAC) was then performed in an incubator at 45°C for approximately 24 hours. The batches of DNA-BCN functionalized beads were washed with CH3CN, PBS, and finally with molecular biology grade water and kept in an ice bath at 0°C until further use.

[0165] Step 2: Attaching fluoroquinolones to beads

[0166] All DNA-BCN functionalized bead batches obtained in step 1 were treated with triphenylphosphine in DCM and H2O at room temperature to reduce the remaining azide moieties to primary amine moieties.

[0167] Each bead batch thus obtained is reacted with a corresponding fluoroquinolone antibiotic according to the following scheme. The beads are subjected to standard peptide coupling and functionalized with an acid-labile Rink linker, followed by a PEG8 linker and two lysine residues. Each batch of beads (1.0 equivalents, 0.0096 mmol) is treated with 4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy)butyric acid (a secondary alcohol photolabile linker; 4.0 equivalents, 38.4 μmol), HATU (3.8 equivalents, 36.5 μmol) and Hünig's base (6.0 equivalents, 57.6 μmol) in a pre-activated solution of 100 μl of NMP at room temperature for 2-12 hours. The beads are then carefully washed and analyzed by acid-mediated cleavage with LCMS. Fluoroquinolone antibiotic solution (10.0 equivalents, 48.0 μmol), DIAD (15.0 equivalents, 72.0 μmol) triphenylphosphine (15.0 equivalents, 72.0 μmol) were dissolved in 150 μL of anhydrous THF and a small amount of NMP to help dissolve the prepared fluoroquinolone, and the formation of the Mitsunobu complex was carried out at 0°C. The solution prepared in this way was poured onto the beads (1.0 equivalents, 0.0048 mmol). The reaction was initially carried out at 0°C and then at room temperature for 12 hours to replace the hydroxyl group of the photolabile linker already bound to the beads with the carboxylate of the fluoroquinolone. The beads were then carefully washed and the analytical samples were checked by LCMS by acid-mediated cleavage. Finally, all batches of beads were pooled to obtain a single pool containing beads, each of which was modified with a unique fluoroquinolone / DNA-BCN combination.

[0168] Step 3: Encapsulation and analysis of beads and bacterial cells (protocol for each strain to be tested)

[0169] Using a flow-focusing microfluidic chip, water droplets are generated in oil to produce an emulsion of monodisperse droplets. The aqueous phase contains cells of the bacterial strain in question, fully functionalized and barcoded beads prepared as described above, and additives, so that each droplet contains 0, 1, or a few beads and 0, 1, a few, or up to tens of thousands of bacterial cells. The droplets are collected in a small reaction tube. The droplets with beads are exposed to ultraviolet light for a few minutes, releasing the fluoroquinolone from all the beads by photolytic cleavage of the above-mentioned photolabile linker. The droplets are incubated for several hours. The droplets are then optionally heat-inactivated (depending on the desired analytical reaction), or the emulsion is disrupted by an organic solvent, which also stops the detection reaction. The beads are collected on a glass frit, and the filtrate is amplified by PCR and sequenced to test for the presence of any headdress DNA that may be cleaved by bacterial endonucleases, indicating that the fluoroquinolone associated with the headdress DNA is active against the bacteria.

[0170] Example 2: Sequential Construction of Bead-Linked Encoding DNA Considering the Cleavage and Pooled Synthesis of Beads Containing a Ligated Library of Chemical Structures and Linked Encoding DNA

[0171] To the headdress DNA oligomer and primary amine solution functionalized with eight PEG chains and a primary amine (1 mM in H2O) described in the Overview section of this application was added Hünigs' base (5 equivalents) and a 0.2 M solution of (1R,8S,9S)-bicyclo[6.1.0]non-4-yn-9-ylmethyl N-succinimidyl carbonate (3 equivalents) in NMP at 0°C. After completion of the reaction, as judged by ESI-TOF mass spectrometry, the reaction was purified according to the reported precipitation procedure. The resulting functionalized DNA headdress (DNA-BCN) was resuspended in water at a concentration of 1 mM and used without further purification.

[0172] Tentagel beads functionalized with azidoglutaric acid (loaded at 0.29 mmol / g) were briefly washed with PBS, then with water, and finally with CH3CN. The DNA-BCN from the previous step (0.004 equivalents) was dissolved in a 1:1 PBS:CH3CN mixture (alternatively, 10% aqueous pyridine solution can be used) and added to the Tentagel beads. Strain-promoted alkyne azide cycloaddition (SPAAC) was then performed in a 45°C incubator for approximately 24 hours, yielding beads with the initial headdress DNA oligomer attached thereto.

[0173] The beads from the previous step were washed with CH3CN, PBS, and finally molecular biology-grade water. While preparing the ligation mixture, the beads were kept in an ice bath at 0°C. The further labeled DNA to be ligated to the existing DNA tag was diluted relative to the DNA loading from the previous step in DNAse-free water, 10X T4 ligation buffer was added, and the mixture was kept on ice; for 10-15 mgs of beads, the final ligation volume was approximately 150 μL. T4 DNA ligase was then added, and the mixture was poured onto the beads. The reaction was then allowed to proceed at room temperature for 5 to 16 hours.

[0174] The DNA labeling step described in the previous paragraph is performed as many times as there are variable substituents attached to the fixed backbone in the split-pool synthesis.

[0175] Example 3: Classification of beads

[0176] This approach can be applied, for example, to beads used in the assay of the present invention, where the beads contain a fluorophore and an associated quencher as a probe, and the quencher is removed by a response molecule. After the incubation is complete, the beads are separated from the incubation medium and their response molecules, and the separated beads (with fluorescent and non-fluorescent probes) are combined.

[0177] The pooled beads were sorted using a commercial fluorescence-activated cell sorter (FACS) by gating on the desired properties of the beads (fluorescence or non-fluorescence, and potentially other quality control gates). Positively sorted beads were subjected to DNA amplification and sequencing.

[0178] Example 4: Bead-based Secreted Embryonic Alkaline Phosphatase (SEAP) Reporter Assay

[0179] Step 1: Preparation of Quenched Fluorescently Labeled Beads

[0180] A quenched fluorescein derivative (such as 1-oxo-3',6'-diphosphinooxy-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 agent such as DMT-MM (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride) in an aqueous buffer. The DNA-encoded library beads with bead-bound DNA barcodes contain single-stranded DNA ends whose sequence is complementary to that of the fluorescein single-stranded DNA conjugate. Following incubation with the pooled library beads, the fluorescein single-stranded DNA conjugate anneals to the complement on the beads, forming a stable double-stranded DNA duplex.

[0181] 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 DNA-encoded library beads with the bead-bound DNA barcode will terminate with a short overhang of 2-4 bases, and the overhang will complement the overhang of the above-mentioned fluorescein-conjugated double-stranded DNA. The double-stranded DNA oligomer and the barcode are then covalently linked using a ligase, as previously described (see Clark et al.). Using any of the above methods, the library beads can be labeled with any probe as needed.

[0182] Step 2: Beads and Eukaryotic (THP1-Dual TM Encapsulation and analysis of KI-hSTING-S154 (InvivoGen) cells

[0183] Using a flow focusing microfluidic chip, water droplets are generated in oil to produce an emulsion of monodisperse droplets. The aqueous phase contains the desired eukaryotic cells, the fully functionalized and barcoded beads prepared as above (additionally labeled with phosphorylated fluorescein), and additives such that each droplet contains 0, 1, or a few beads and 0, 1, a few, or up to hundreds of eukaryotic cells. Note that for THP1-Dual TM KI-hSTING-S154 cells, additives may include 2'3'-cGAMP. In addition, a positive control (known irreversible small molecule inhibitor H-151, InvivoGen) can also be used as an additive to provide a baseline for subsequent statistical analysis and bead sorting. The droplets are collected in a small reaction tube. The droplets with beads are exposed to ultraviolet light for several minutes, releasing library members from all beads through photolytic cleavage of the above-mentioned photolabile linker. The droplets are incubated for several hours. The droplets are then heat-inactivated, or the emulsion is disrupted by an organic solvent, which also stops the detection reaction. The beads are collected on a glass frit and pooled, and hit beads with low fluorescence are sorted from the pool by FACS. The low-fluorescent beads are then amplified by PCR and sequenced; low fluorescence indicates that the released small molecule associated with the barcoded DNA successfully inhibits STING activation, thereby preventing the release of SEAP. Sequence Listing <110> Franz Haffman-La Roche GmbH <120> Small molecule screening cellular assays using modified beads <130> P44572EP00 (P35234-EP) <140> EP19169563.4 <141> 2019-04-16 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic linker peptide substructure <400> 1 Glu Asn Leu Tyr Phe Gln Gly 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Synthetic adapter DNA cleavage sequence <400> 2 gtaacgatcc agctgtcact 20 <210> 3 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic linker cleavage site <400> 3 Lys Gly Asp Glu Val Asp Gly Ser Gly Lys 1 5 10

Claims

1. A method for screening the activity of a DNA-encoded chemical structure library (2) in a cellular target (11); wherein it is known that the cellular target (11) releases or alters the release of a response molecule (12) upon contact with an active chemical structure; wherein the chemical structures (2) of the library and the corresponding encoding DNA (4) are covalently linked to beads (1), and each bead (1) comprises a) Multiple instances of a single chemical structure (2) of the library, each instance being covalently linked to the bead (1) via a structural linker (3), the structural linker (3) being cleavable at a cleavable structural linker site (3a); and b) multiple instances of a DNA sequence (4) encoding the chemical structure (2), each DNA sequence (4) being covalently linked to the bead (1) via a tag linker (5), the tag linker (5) comprising a cleavable tag linker site (5a) and being cleavable by a cleavage agent (6); wherein the cleavable structure linker site (3a) is non-cleavable under the reaction conditions for cleaving the cleavable tag linker site (5a), and vice versa; and if the tag linker (5) and / or the cleavable tag linker site (5a) and / or the DNA sequence (4) are cleavable by the response molecule (12), then the bead (1) does not have a chemical probe (7 / 8 / 9) sensitive to the response molecule; or, if the encoding DNA sequence (4) and the cleavable tag linker site (5a) and the tag linker (5) are non-cleavable by the response molecule (12), then the bead (1) has a chemical probe (7 / 8 / 9) sensitive to the response molecule (12) covalently linked thereto; The method comprises the following steps: (i-b-1) Providing a single incubation medium (13) in which the cellular target (11) and all the beads (1) as defined above are combined, all the chemical structures (2) of the library are linked thereto, and aliquots in the form of droplets large enough to contain at least one bead and containing zero, one or more beads (1) are separated from the incubation medium, provided that the average bead population λ of the separated aliquots is from 0.2 to 0.9, the λ being defined as where (k m ) i is an integer representing the number of beads in the i-th aliquot; M is the number of all the dispersed aliquots; (i-b-2) Releasing the chemical structure (2) from the contained beads (1) in each aliquot by cleaving the structure linker (3) at the cleavable structure linker site (3a), and incubating the cellular target (11) and the released chemical structure (2) in the aliquot of the incubation medium (13); (ii) Either, if the encoding DNA sequence (4) and / or the cleavable tag linker site (5a) and / or the tag linker (5) are cleavable by the response molecule (12), then: (ii-a-1) Monitoring the release of any encoding DNA sequence (4) or a fragment thereof from any bead (1) in the aliquot of the incubation medium (13); and if so, separating all the beads (1) from all the aliquots of the incubation medium (13) and pooling all the separated beads (1); (ii-a-2) Cleaving the cleavable tag linker site (5a) in the pooled beads (1) with a cleavage agent (6) to release any encoding DNA sequence (4) or a fragment thereof; (ii-a-3) Amplify and sequence the released encoded DNA sequence (4) or its fragment to identify any complete DNA sequence of the DNA-encoded library therein; and (ii-a-4) Associate the remaining portion of the complete DNA sequence of the DNA-encoded library that was not identified in step (ii-a-3) because it was released in step (ii-a-1) and the fragments of the encoded DNA sequence (4) identified in step (ii-a-3) with the corresponding chemical structures (2) of the DNA-encoded library; Either, if the beads (1) contain chemical probes (7 / 8 / 9) sensitive to the response molecule, then: (ii-b-1) Monitor any reaction or change in the reaction of any probe (7 / 8 / 9) with the response molecule (12) in aliquots of the incubation medium (13), and if so, separate and pool all the beads (1) from all aliquots of the incubation medium (13); (ii-b-2) Extract the beads (1) that exhibit the probe reaction or the change in the probe reaction from the said pool; (ii-b-3) Cut the cleavable tag linker site (5a) in the beads (1) extracted from the said pool with a cutting agent (6) to release any DNA sequence (4) covalently linked to the separated beads (1); (ii-b-4) Amplify and sequence the released DNA sequence (4); and (ii-b-5) Associate any DNA sequence sequenced in step (ii-b-3) with the corresponding chemical structure (2) of the DNA-encoded library; and (iii) Select any chemical structure (2) so associated in step (ii-a-4) or (ii-b-5) as the further said active chemical structure.

2. The method according to claim 1, wherein the tag linker (5) and / or the cleavable tag linker site (5a) and / or the DNA sequence (4) can be cleaved by the response molecule (12); and steps (ii-a-1), (ii-a-2), (ii-a-3) and (ii-a-4) are carried out.

3. The method according to claim 2, wherein the cell target (11) is a prokaryotic cell, which is known to undergo cell death upon contact with an active chemical structure, thereby releasing a nuclease as the response molecule (12); and the tag linker (5) and / or the cleavable tag linker site (5a) and / or the DNA sequence (4) can be cleaved by the nuclease.

4. The method according to claim 1, wherein the beads (1) comprise chemical probes (7 / 8 / 9) that are sensitive to the response molecule (12) and are covalently linked to the beads (1); and steps (ii-b-1), (ii-b-2), (ii-b-3), (ii-b-4) and (ii-b-5) are carried out.

5. The method according to claim 4, wherein the cell target (11) is a eukaryotic cell, which is modified such that when it is contacted with a suitable chemical structure (2), the response molecule (12) is secreted embryonic alkaline phosphatase (SEAP); and the chemical probe (7 / 8 / 9) is sensitive to secreted embryonic alkaline phosphatase.

6. The method according to claim 4 or 5, wherein the chemical probe (7 / 8 / 9) is a combination of a fluorophore (7) and a quencher (8), the quencher acting on the fluorophore (7) by fluorescence resonance energy transfer (FRET) or by contact quenching; wherein the fluorophore (7) and the quencher (8) are connected to each other on a spacer (9) that can be cleaved by the response molecule (12); and wherein in step (ii-b-1), cleavage of the spacer (9) of the incubation medium (13) or an aliquot thereof by the response molecule (12) is monitored by the incident fluorescence of the fluorophore (7).

7. The method according to claim 1, wherein the cleavable tag linker site (5a) is a nucleotide sequence that can be cleaved by a restriction endonuclease acting as a cleavage agent (6).

8. The method according to claim 7, wherein said label linker (5) comprises a divalent spacer (5b) of structure -O-(CH2-CH2-O)- adjacent to said cleavable label linker site (5a), where n is an integer from 5 to 10. n - where n is an integer from 5 to 10.

9. The method according to claim 1, wherein each bead (1) comprises multiple instances of the chemical structure (2) connected to the bead (1) via an ultraviolet-light-cleavable structural linker (3).

10. The method according to claim 3, wherein the prokaryotic cell is a bacterium.

11. The method according to claim 8, wherein n is 8.

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