MICROBEADS FOR TAGLESS ENCODED CHEMICAL LIBRARY SCREENING

Abstract

Disclosed is an encoded chemical library microbead, which microbead has immobilized thereon and/or therein: (i) an encoding tag; and (ii) a target assay system reporter moiety, wherein the reporter moiety exists in a first state in the absence of activity against the target and in a second state in the presence of said activity, and wherein said microbead further comprises a clonal population of one or more chemical structure(s) releasably linked thereto and encoded by said tag.

Claims

1-83. (canceled)

84. An encoded chemical library microbead, which microbead has immobilized thereon and/or therein: (i) an encoding tag; and (ii) a target assay system reporter moiety, wherein the reporter moiety exists in a first state in the absence of activity against the target and in a second state in the presence of said activity, and wherein said microbead further comprises a clonal population of one or more chemical structure(s) releasably linked thereto and encoded by said tag.

85. The microbead of claim 84, wherein said encoding tag also encodes the target assay system reporter moiety.

86. The microbead of claim 84, wherein said encoding tag also encodes the target.

87. The microbead of claim 84, wherein the chemical structures are present at a loading of between 1 and 1013 molecules per microbead.

88. The microbead of claim 84, wherein the microbead comprises non-DNA tags, non-RNA tags, modified nucleic acid tags, peptide tags, light-based barcodes (e.g. quantum dots), RFID tags, reporter chemicals linked by click chemistry and mass spectrometry-decodable tags.

89. The microbead of claim 84, wherein the chemical structures are releasably linked to the microbead by a cleavable linker.

90. The microbead of claim 89, wherein the linker is scarless, such that the chemical structure(s) can be cleaved from the microbead in a form in which they are completely or substantially free of linker residues.

91. The microbead of claim 84, wherein the chemical structures are indirectly or directly linked to the microbead.

92. The microbead of claim 84, wherein the target assay system reporter moiety is substrate and chemical structures which function as chromophore coatings can be identified by decoding the tags of microbeads having chromatic reporter moieties.

93. The microbead of claim 84, wherein the target assay system reporter moiety is substrate and chemical structures which function as a substrate coating can be identified by decoding the tags of microbeads having coated reporter moieties.

94. A chemical library microcompartment which contains a microbead as defined in claim 84 and a solvent, for example an aqueous solvent.

95. An encoded chemical library (ECL) comprising a plurality of microcompartments as defined in claim 93, wherein each of the microcompartments contains a different chemical structure.

96. The ECL of claim 95, which comprises a number n of different clonal populations of chemical structures, each clonal population being confined to n discrete library microcompartments.

97. A method for screening an ECL as defined in claim 95 for chemical structures having activity against a target, the method comprising the steps of: (a) providing said ECL; (b) releasing the chemical structures from the microbeads to produce a plurality of free, tagless chemical structures (TCSs) dissolved in the solvent and contained within microcompartments together with the microbeads from which they were released, such that a spatial association between each TCS and its encoding tag is maintained; (c) assaying the TCSs by incubating the ECL microcompartments of step (b) under conditions such that the state of the reporter moieties immobilized in or on the microbeads contained therein is determined by the level of activity against the target; (d) releasing the assayed microbeads by opening the microcompartments; and (e) screening the released and assayed microbeads by determining the state of the reporter moieties, whereby chemical structures having activity against the target can be identified by decoding the tags of microbeads having reporter moieties in the second state.

98. The method of claim 97 wherein step (d) further comprises stopping the incubation, for example by heat denaturation, freezing, addition of inhibitors or breaking of the microcompartments.

99. The method of claim 98 wherein the microcompartments are broken by centrifugation, sonication and/or filtration or by the addition of solvents and/or surfactants.

100. The method of claim 97 further comprising the step of isolating the microbeads released in step (d) during or prior to screening step (e).

101. The method of claim 97 wherein the screening step comprises fractionation and/or selection of the released and assayed microbeads.

102. The method of claim 97 wherein the screening step (e) comprises determining the level of activity against the target by measuring the ratio of reporter moieties in the first state to the second state.

103. The method of claim 97 wherein the microbead comprises a clonal population of a plurality of chemical structures and said encoding tag also encodes the loading of the chemical structures, and wherein the screening step (e) comprises determining the level of activity against the target by correlating the loading of the chemical structures with the ratio of reporter moieties in the first state to the second state.

Description

FIGURE LEGENDS

[0303] FIG. 1: Schematic representation of release of free chemical structure using a self-immolative linker.

[0304] FIG. 2: Schematic representation of split-and-pool DECL using a self-immolative dipeptide linker.

[0305] FIG. 3: Schematic representation of release of free chemical structures using a Val-Cit-PAB self-immolative peptide linker.

[0306] FIG. 4: A self-immolative process showing decrease of substrate A and increase of product B.

[0307] FIG. 5: Proportional compound loading on beads, showing clear distinct populations for each of loading sample.

[0308] FIG. 6: Schematic representation of enzyme activity measurement in micro-droplets using fluorescent reporter moiety.

[0309] FIG. 7: Relative intensity of FITC signal in micro-droplets using fluorescent reporter moiety.

EXAMPLE 1: ‘SCAR-LESS’ RELEASE OF A SMALL MOLECULE COMPOUND FROM A BEAD-LINKAGE-COMPATIBLE LINKER

[0310] Stock solutions of test substrate A was made up at 10 mg/ml in DMSO and NADPH (Sigma Aldrich) at 10 mg/ml (11.9 mM) in 40 mM MOPS, pH 7.5, 150 mM NaCl.

[0311] 20 μl substrate A solution was combined with 480p1 of 40 mM MOPS pH 7.5 150 mM NaCl buffer and 500 μL NADPH solution. Reaction was initiated by adding 3 μL (29.6 mg/ml) nitroreductase (Prozomix) and incubated for 20 minutes at room temperature. 50 μl reaction solution was combined with 200 μl 3:1 ACN: H.sub.2O+0.1% formic acid run on an LCMS (Agilent Technologies, Infinity 1290).

[0312] A clear decrease of substrate A and increase of product B could be observed (see FIG. 4) demonstrating a self-immolative process where the desired ‘drug molecule’ was released at 3 minutes. This result demonstrates controllable enzymic cleavage of a useful and specific linker resulting in ‘scar-less’ release of a small molecule. This linker is therefore useful for releasable attachment of small molecules to beads for use in activity assays.

EXAMPLE 2: ACCURATE PROPORTIONAL COMPOUND LOADING ON BEADS

[0313] Preparation

[0314] 50 ml of a 100 μM stock was made of 4-(aminomethyl) fluorescein hydrochloride (AMF.HCl) solution using 1.99 mg of AMF.HCl in 50 ml of 100 mM MOPS, 150 mM NaCl. This solution was diluted using 100 mM MOPS pH 7.5, 150 mM NaCl to 10 μM and 3 μM as required.

[0315] The following solutions were placed into 5×50 ml falcon tubes (1 mL each) for loading test.

[0316] In all cases DMTMM (4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, Sigma Aldrich) was used as a coupling reagent (assuming 10% of all acids on bead)=4.77×10−7 mol—Volume of 4.8 mM stock with 100 μl added to each falcon:

[0317] Falcon 1-0.11% loading—5.247×10.sup.−9 mol of AMF required—Volume of 100 μM (AMF.HCl) (Fisher) stock used=52.5 μL;

[0318] Falcon 2-0.033% loading—1.574×10.sup.−9 mol of AMF required—Volume of 100 μM AMF.HCl stock used=15.7 μL;

[0319] Falcon 3-0.011% loading—5.247×10.sup.−1° mol of AMF required—Volume of 10 μM AMF.HCl stock used=52.5 μL;

[0320] Falcon 4-0.0033% loading—1.574×10.sup.−1° mol of AMF required—Volume of 3 μM AMF.HCl stock used=15.7 μL;

[0321] Falcon 5-0.0011% loading—5.247×10.sup.−11 mol of AMF required—Volume of 10 μM AMF.HCl stock used=5.25 μL.

[0322] Procedure

[0323] 3.9 ml 100 mM MOPS pH 7.5, 150 mM NaCl was added to 5×50 mL falcons. 100 μL of freshly made 4.8 mM DMTMM stock solution was added and mixed thoroughly. 1 ml of 50 μm bead suspension was added (35 beads/μl) to each falcon and mixed for 60 minutes. In five separate 50 mL falcons AMF.HCl was added as shown above.

[0324] Beads were reacted with DMTMM for 60 mins, then poured into associated AMF 50 mL falcon and reacted overnight at room temperature.

[0325] Beads were pelleted at 1000 g for 10 minutes and as much solvent as possible was removed. 15 ml fresh buffer was added, mixed and wash steps repeated a further 2 times. Bead samples were analysed using FACS (MoFloXDP, Beckmann Coulter) exciting at Excite AFM on the beads using 488 nM laser and measuring emission using a 540 nm+/−20 nm filter. Samples were measured individual and a pool on logarithmic scale.

[0326] As shown in FIG. 5, clear distinct populations were seen for each of the relative loading samples, showing increases in fluorescence exactly proportional to the relative AMF.HCl bead loading with CV's <6.5%. This means distinct amounts of a chemical compounds can be accurately loaded onto beads allowing quantitative dose responses to be performed in activity assays.

EXAMPLE 3: ENZYME ACTIVITY MEASUREMENT IN MICRO-DROPLETS

[0327] 500 μl of —COOH magnetic beads (Bang Beads Inc) were washed 5×1 ml with 10 mM MOPS pH 5.5, 150 mM NaCl and then finally resuspended in 500 μl of the same buffer. Oligos 1 and 2 were each resuspended in Nuclease free water to a concentration of 100 μM. [0328] Oligo 1—/5AmMC6/ATGC/iFluroT/ACGTGCATCCAAGCA/3IABkFQ/ [0329] Oligo 2—TGC TTG GAT GCA CGT AGC AT

[0330] 5AmC6=C6 Amine Linker, iFluroT is a FITC-Fused dT, 3IAbkDQ is a Black Hole Quencher on the 3′ End. Underlined is the BstCl Restriction Enzyme Site.

[0331] Oligos 1 and 2 (for bead attachment) were first annealed by mixing equal ratios of each oligo in 100 μl total volume, consisting of 40 μl each oligo, 10 μl of 10×T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl.sub.2, 10 mM Dithiothreitol, 1 mM ATP, pH 7.5) and 10 μl of Nuclease free water. The mixture was heated to 95° C. in a hot block for 10 minutes and then allowed to cool to room temperature (25° C.) slowly by switching off the block but leaving the sample in the aluminium block.

[0332] For double-stranded oligo coupling to the beads: 30 mg of EDC was dissolved in 400 μl of 10 mM MES pH 5.5, 150 mM NaCl. Beads were pulled down with the magnet and then resuspended in the EDC-containing buffer. Once resuspended, the oligo mixture was added and incubation performed with end-over-end mixing at 37° C. for 2 hours.

[0333] The beads were washed 5× with 1 ml of 10 mM Tris 1 mM, EDTA pH 7.5 buffer then 2×1 ml with water.

[0334] Digest samples with and without inhibitors were set up: Each reaction was in 25 μl, 2.5 μl of Outsmart buffer 3.1 (New England Biolabs, UK), 17.5 μl of beads in water which was then made up to 25 μl with inhibitor and/or water added in order, enzyme added last in every case. [0335] Sample 1+5 μl Nuclease Free Water no enzyme [0336] Sample 2+4 μl Nuclease free water and 1 μl of BstCl [0337] Sample 3+3.5 μl Nuclease free water, 0.5 μl 0.5M EDTA (inhibitor 1) and 1 μl of BstCl [0338] Sample 4+3.5 μl Nuclease free water, 50 mM Spermidine (inhibitor 2, Sigma Aldrich) and 1p1 of BstCl

[0339] Samples were immediately emulsified; emulsification was performed using a vortex in bulk with 200p1 of mineral oil (73% Tegosoft DEC, Evonik, 7% Abil EM 90, Evonik and 10% Light mineral oil, Sigma Aldrich). This step can also be performed using a microdroplet-generating device.

[0340] The emulsified mixtures containing the reaction/bead-containing microdroplets were incubated with mixing for 30 minutes at 37° C. and then the emulsion broken. 500 μl of 100% ethanol was added and samples were then centrifuged for 1 minute at 14000 g to pellet the beads. These were then washed 3× with 1 ml of 10 mM Tris, 1 mM EDTA pH 7.5 buffer and resuspended in 500 μl of the same buffer.

[0341] The beads were then diluted to be roughly 1 million/ml by diluting 20-fold in buffer and run on a Cytoflex flow cytometer (Beckman Coulter). The machine was calibrated to detect small particles (i.e. bacteria). The excitation was at 488 nM with detection at 525 nm+/−40 nm; 10,000 events were detected and the average fluorescence plotted.

[0342] The results are shown in FIG. 7. Sample 2 was active showing 3 times the control signal suggesting cleavage of the oligo retained on the beads and in the microdroplets, while samples with inhibitors and negative control (no enzyme) were similar, showing that inhibition of an enzyme that cleaves a bead-retained target substrate can be performed and clearly detected using the microdroplet incubation system.

[0343] It will be appreciated that fluorescent/non-fluorescent beads can be easily be separated by FACS. Thus, initial loading of the beads with compound would permit encoded identification of the specific inhibitor compounds and their respective bead loading.

EQUIVALENTS

[0344] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.