ARRAY, SOAKING SOLUTIONS AND METHOD OF SELECTING SOAKING CONDITIONS FOR SMALL MOLECULES IN BIOLOGICAL MACROMOLECULAR CRYSTALS

Abstract

Subject-matter of the present invention is an array comprising soaking solutions for soaking a biological macromolecular crystal. Further, the subject of the invention is a rule-based method of selecting specific soaking solution compositions having a specific composition comprising composite solute(s), water (w), crystallization solution (crs) and/or organic solvent(s). Additionally, the subjects matter of the invention the soaking solutions obtained by the method of the invention and a screening method for small molecules comprising molecular probes, fragments and drug-size molecules using the soaking solutions and the use of the soaking solutions in a screening method for small molecules on a macromolecular crystal.

Claims

1. Method of selecting a soaking solution for soaking the crystal form of a biological macromolecule wherein the soaking solution comprises an organic solvent (os), a compatible solute (cs), and a crystallization solution (crs) and/or water (w); comprising the steps of 1) preparing a first series of 1n to xn individual soaking solutions by mixing os, cs, crs and w wherein: a) x is at least 2 b) each soaking solutions of the first series comprises the same organic solvent (os), the same compatible solute (cs), and the same crystallization solution (crs), and c) Vos and Vcs are the same in each solution, or, alternatively, Mos/Mcs is the same in each solution, and d) Vcrs and Vw are changed in each solution, and in the solution in, V.sub.crs is the minimum V.sub.crs (V.sub.crs min) and the V.sub.w is the maximum V.sub.w (V.sub.w max), and in the solution xn, V.sub.w is the minimum V.sub.w (V.sub.w min) and the V.sub.crs is the maximum V.sub.crs(V.sub.crs max), and when x>2, in any additional solutions between in and xn, the V.sub.crs is varied between V.sub.crs min and V.sub.crs max and, inversely, Vw is varied between V.sub.w min and V.sub.w max 2)transferring at least one crystal of the biological macromolecular crystal per compartment into x compartments, each compartment comprising one of the solutions prepared in step 1); or transferring each solution prepared in step 1) into x compartments, each compartment comprising at least one crystal of the biological macromolecular crystal from its crystallization solution. 3) controlling the crystal in each compartment 4) selecting the soaking solution(s) which is/are suitable for soaking the crystal; wherein the compatible solute is selected from the group comprising polyols, amino acids, methylamines, and mixtures thereof, and wherein the organic solvent is selected from the group comprising liquid carbohydrates, protic or aprotic, of low reactivity, that can serve to solve small molecules and, mixtures thereof, and wherein the crystallization solution crs is obtained from the crystallization process of said crystal or is equivalent thereto in terms of osmotic net pressure, permittivity, solubility of the according biological macromolecule, and effect on its structural conformation, comprising pH and buffer materials, additives, and precipitants.

2. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol a or a mixture thereof, and said organic solvent (os) is a liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof, that can serve to solve small molecules, more preferably, said compatible solute (cs) is a poly(oxyethylene) and said organic solvent (os) is selected from the group comprising a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute (cs) is selected from the group comprising the poly(oxyethylene)s polyethylene glycol 400, polyethylene glycol 600 or a mixture thereof and said organic solvent (os) is selected from the group comprising dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous solution V.sub.crs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos; Vcs)).

3. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol or a mixture thereof, and said organic solvent is a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide, or a mixture thereof, more preferably, said compatible solute (cs) is a polyhydric alcohol or a mixture thereof and said organic solvent (os) is selected from the group comprising a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute (cs) is selected from the group comprising the polyhydric alcohols propane-1,2,3-triol (glycerol), ethane-1,2-diol (ethylene glycol), 2-Methylpentane-2,4-diol (2-Methylpentane-2,4-diol),(3R,4S,5S,6R)-2-(2,3-dihydroxypropoxy)-6-(hydroxymethyl)oxane-3,4,5-triol (1-Glucosylglycerol) or a mixture thereof, and said organic solvent (os) is selected from the group comprising dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous solution V.sub.crs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos; Vcs)).

4. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a polyol a or a mixture thereof, and said organic solvent (os) is a liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof, that can serve to solve small molecules, more preferably said compatible solute (cs) is a mono-, di-, tri, oligo- or polysaccharide or a mixture thereof and said organic solvent (os) is selected from the group comprising a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide or a mixture thereof, most preferably, the compatible solute cs is selected from the group comprising the saccharides (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxane-3,4,5-triol (trehalose) or β-D-Fructofuranosyl α-D-glucopyranoside (sucrose) or a mixtures thereof, and said organic solvent (os) is selected from the group comprising dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the molar ratio of compatible solute to organic solvent is the same and the molar ratio is 1:10000 to 10000:1, preferably between 100:1 to 1:100 and more preferably between 10:1 to 1:10, and wherein the ratio of volume crystallization solution or equivalent aqueous solution V.sub.crs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 to 1:100 and most preferably between 85:15 to 1:1 (V.sub.crs: (Vos; Vcs)).

5. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is a methylamine or a mixture thereof and said organic solvent (os) is liquid carbohydrate protic or aprotic, of low reactivity, or a mixture thereof that can serve to solve small molecules, more preferably said compatible solute (cs) is selected from the group comprising N,N-dimethylmethanamine oxide (Trimethylamine N-oxide or TMAO), 2-trimethylammonioacetate (trimethylglycine or betaine), 2-(Methylamino)acetic acid (N-methylglycine or sarcosine), or a mixture thereof and said organic solvent (os) is a (alkylated) sulfoxide, cyclic non-aromatic ether, straight or branched chain monohydric aliphatic alcohol, alkylated formamide, or a mixture thereof, most preferably said compatible solute (cs) is selected from the group comprising N,N-dimethylmethanamine oxide (Trimethylamine N-oxide or TMAO), 2-trimethylammonioacetate (trimethylglycine or betaine), 2-(Methylamino)acetic acid (N-methylglycine or sarcosine), or a mixture thereof and said organic solvent (os) is selected from the group comprising dimethyl sulfoxide, 1,4-dioxane, dimethylformamide, methanol, ethanol, or a mixture thereof and wherein the molar ratio of organic solvent (os) to compatible solute (cs) Mos/cs is the same and the molar ratio Mos/cs is 1:10000 to 10000:1, preferably between 100:1 to 1:100 and more preferably between 10:1 to 1:10, and wherein the ratio of volume crystallization solution or equivalent aqueous solution V.sub.crs to the combined volumes of organic solvent Vos and compatible solute Vcs is 1:1000000 to 1000000:1, preferably between 100:1 to 1:100 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos;Vcs)).

6. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is polyethylene glycol 400 and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs polyethylene glycol 400 to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution V.sub.crs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) polyethylene glycol 400 is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos:Vcs)).

7. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is ethane-1,2-diol (ethylene glycol) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs ethane-1,2-diol (ethylene glycol) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution V.sub.crs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) ethane-1,2-diol (ethylene glycol) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos:Vcs)).

8. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is propane-1,2,3-triol (glycerol) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs propane-1,2,3-triol (glycerol) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs:Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution V.sub.crs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) propane-1,2,3-triol (glycerol) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos:Vcs)).

9. Method of selecting the composition of a soaking solution according to claim 1, wherein said soaking solution comprises a crystallization solution (crs) or an equivalent aqueous salt solution thereto and an organic solvent (os) and a compatible solute (cs), wherein said compatible solute (cs) is 2-methylpentane-2,4-diol (MPD) and said organic solvent (os) is dimethyl sulfoxide, wherein the ratio of volume Vcs 2-methylpentane-2,4-diol (MPD) to volume Vos dimethyl sulfoxide is the same and the volume ratio is 1:1000000 to 1000000:1, preferably between 8:1 to 1:8 and more preferably between 3:1 to 1:1 (Vcs: Vos); and wherein the ratio of volume crystallization solution or equivalent aqueous salt solution V.sub.crs to the combined volumes of the organic solvent (os) dimethyl sulfoxide and the compatible solute (cs) 2-methylpentane-2,4-diol (MPD) is 1:1000000 to 1000000:1, preferably between 99:1 to 1:99 and most preferably between 85:15 to 1:1 (V.sub.crs:(Vos:Vcs)).

10. Method of selecting the composition of a soaking solution of claim 1 further comprising the step of 1) preparing m series of 1n to yn individual soaking solutions by mixing os, cs, crs and w wherein m is at least 1 and wherein in each of the m series a) y is at least 2 b) os and crs are the same as in the first series, and c) Vos and Vcs are the same as in the first series, or, alternatively, Mos/Mcs is the same in the first series, d) the cs is changed and is different from the cs of the first series, and wherein in each individual solutions of each of the m series e) Vos and Vcs are conserved, or, alternatively, Mos/Mcs is conserved, and f) V.sub.crs and Vw are changed in each solution, and in the solution 1n, V.sub.crs is the minimum V.sub.crs (V.sub.crs min) and the Vw is the maximum V.sub.w (V.sub.w max), and in the solution yn, V.sub.w is the minimum V.sub.w (V.sub.w min) and the V.sub.crs is the maximum V.sub.crs(V.sub.crs max), and when y>2, in any additional solutions between in and yn, the V.sub.crs is varied between V.sub.crs min and V.sub.crs max and, inversely, V.sub.w is varied between V.sub.w min and V.sub.w max 2) transferring at least one crystal of the biological macromolecular crystal per compartment into y compartments, each compartment comprising one of the solutions prepared in step 1); or transferring each solution prepared in step 1) into y compartments, each compartment comprising at least one crystal of the biological macromolecular crystal from its crystallization solution.

11. Method of selecting the composition of a soaking solution of claim 1 wherein an equivalent aqueous salt solution different from the crystallization solution is used, and wherein the salt selected from the group comprising disodium; 2,3-dihydroxybutanedioate (sodium tartrate), (2R,3R)-2,3-dihydroxybutanedioate (dipotassium tartrate), azanium; phosphates (ammonium phosphates), diazanium; sulfate (ammonium sulfate), triazanium; 2-hydroxypropane-1,2,3-tricarboxylate (ammonium citrate), sodium; acetate (sodium ethanoate), azanium; acetate (ammonium acetate), dilithium; sulfate, lithium; chloride, lithium; acetate, lithium; formate, lithium; nitrate, sodium; nitrate, sodium; chloride, potassium; chloride, sodium; formate, monophosphate from sodium and potassium, di- and polyphosphates from sodium and potassium, sodium citrates (sodium citrates as sodium dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate)), magnesium; diformate, magnesium; dichloride, magnesium; sulfate, calcium; dichloride; dihydrate, disodium; butanedioate (sodium succinate), cadmium(II) sulfate (cadmium sulfate), disodium; propanedioate (sodium malonate), magnesium; diacetate (magnesium acetate), zinc; diacetate (zinc acetate), calcium; diacetate (calcium acetate) and wherein the salt for the equivalent solution is preferably selected from the group comprising lithium; acetate, lithium; chloride, lithium; formate, lithium; nitrate, dilithium; sulfate, sodium; chloride, potassium; chloride, magnesium; dichloride, diazanium; sulfate (ammonium sulfate), sodium; acetate (sodium ethanoate), sodium citrates (sodium citrates as sodium dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (monosodium citrate), disodium hydrogen 2-hydroxypropane-1,2,3-tricarboxylate (disodium citrate), trisodium 2-hydroxypropane-1,2,3-tricarboxylate (trisodium citrate), magnesium; diformate, sodium; nitrate.

12. Method of selecting the composition of a soaking solution of claim 1 wherein the os contains small molecules including small fragments and small probes to be analyzed, which diffuse into the biological macromolecular crystal.

13. Method of small molecule screening for a biological macromolecular crystal wherein the screening is performed in a soaking solution selected according to the method of claim 12.

14. Soaking solution obtainable by the method of selecting the composition of a soaking solution as claimed in claim 1.

15. (canceled)

16. Array arranged to perform the method as claimed in claim 1, wherein said array comprises a first dimension of at least two individual soaking solutions (1n to xn) and a second dimension of at least two individual soaking solutions (1m to ym) wherein each of said soaking solutions is located in a separated compartment of said array, and wherein each of said soaking solution comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs) and water, and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same within a series of soaking solutions in said first dimension, or, alternatively, the molar ratio of organic solvent (os) to compatible solute (cs) Mos/Mcs is the same within a series of soaking solutions in said first dimension, and wherein the individual soaking solutions of said first dimension comprise the same organic solvent and the same compatible solute within a series of said soaking solutions in the first dimension, respectively, and wherein the ratio of the volume of water Vw and the volume of the crystallization solution Vcrs is varied over the individual soaking solutions of the series in the first dimension and wherein one of the individual soaking solutions of the series in the first dimension has a minimal or zero Vcrs and a maximum Vw and another individual soaking solution of said first dimension has a minimal or zero Vw and a maximum Vcrs and wherein the other individual soaking solutions of the series in said first dimension take values of Vcrs and Vw in between the two before-mentioned values.

17. A method for obtaining the solutions as claimed in claim 12, by an array that comprises a first dimension of at least two individual soaking solutions (1n to xn) and a second dimension of at least two individual soaking solutions (1m to ym) wherein each of said soaking solutions is located in a separated compartment of said array, and wherein each of said soaking solution comprises an organic solvent (os), a compatible solute (cs), a crystallization solution (crs) and water, and wherein the ratio of volume compatible solute Vcs to volume organic solvent Vos is the same within a series of soaking solutions in said first dimension, or, alternatively, the molar ratio of organic solvent (os) to compatible solute (cs) Mos/Mcs is the same within a series of soaking solutions in said first dimension, and wherein the individual soaking solutions of said first dimension comprise the same organic solvent and the same compatible solute within a series of said soaking solutions in the first dimension, respectively, and wherein the ratio of the volume of water Vw and the volume of the crystallization solution Vcrs is varied over the individual soaking solutions of the series in the first dimension and wherein one of the individual soaking solutions of the series in the first dimension has a minimal or zero Vcrs and a maximum Vw and another individual soaking solution of said first dimension has a minimal or zero Vw and a maximum Vcrs and wherein the other individual soaking solutions of the series in said first dimension take values of Vcrs and Vw in between the two before-mentioned values.

Description

FIGURE DESCRIPTION

[0454] FIG. 1: 1A is a picture of a screening plate used for soaking selection; FIG. 1B shows the details of one of the wells of a 24 well plate and on FIG. 1C a schematic representation of the well during a soaking experiment is depicted.

[0455] FIG. 2 shows a preparation scheme according to the method of the invention for selection of soaking solutions comprising CS: compatible solute; OS—organic solvent; CB (crs)—crystallization buffer (solution); W—water. The lines (solute A-D) represent the first dimension and the columns (1-6) the second dimension. Alternatively, the lines (solute A-D) can represent the second dimension and the columns (1-6) the first dimension

[0456] FIG. 3 shows an example of a scheme according to the method of the invention for selection of soaking solutions comprising for fragment screening on PKA. PEG400—polyethylene glycol 400; MPD—2-methylpentane-2,4-diol; EG—ethane-1,2-diol (ethylene glycol); Gly-propane-1,2,3-triol (glycerol); OS—organic solvent=DMSO; CB (crs)—crystallization buffer (solution); W—water.

[0457] FIG. 4 shows photographs of crystals after 24 h in the soaking solutions of FIG. 3

[0458] FIG. 5: shows an example of the visual difference between suitable crystal (A) and crystals suffering from a severe quality loss (B) due to detrimental soaking conditions/solution

[0459] FIG. 6 shows a template for the visual inspection of crystals under i.e. a microscope.

[0460] FIG. 7: shows the criteria used for visual inspection and to control and select the solutions having minimal impact on the crystal

[0461] FIG. 8: shows the results of the visual inspection of PKA crystals after 24 h in the tested soaking solutions of FIG. 3. Each series was prepared with different composite solutes: PEG400; MPD; EG—ethane-1,2-diol (ethylene glycol) or (glycerol); S&C stands for surface and cracks; EDG stands for edges; COL stands for colour; SMS stands for smooth surface; SME stands for smooth edges; Y− stands for yes but little; Y+ stands for yes many; LCR stands for longitudinal cracks; CCC stands for criss-cross cracks; ROS stands for rough surface; FRE stands for frayed edges; N stands for no; DIS stands for dissolving; X stands for no data.

[0462] FIG. 9 shows an image of a diffraction pattern after an X-Ray experiment.

[0463] FIG. 10 shows a template for the X-Ray inspection of crystals.

[0464] FIG. 11: shows the criteria used for X-Ray inspection and to control and select the solutions having minimal impact on the crystal.

[0465] FIG. 12 shows the results of x-ray examination of crystals after 24 h in the soaking solutions of FIG. 3. RES stands for resolution; MOS stands for mosaicity; TWI stands for twinning; ICE stands for ice rings.

[0466] FIG. 13 highlights the most suitable soaking conditions on the scheme thus providing a range/scope of suitable soaking parameters. The star indicates one of the soaking conditions which was chosen for the subsequent fragment screening.

[0467] FIG. 14 shows a template scheme for fragment screening.

[0468] FIG. 15 shows the possible scheme using one of the compositions highlighted in FIG. 13 (PEG 400, 3rd column) for the fragment screening on PKA.

[0469] FIG. 16 shows the possible scheme using one (with the star) of the compositions highlighted in FIG. 13 (PEG 400, 1st column) for the fragment screening on PKA. The ending “Frag”+number indicates individual fragments.

[0470] FIG. 17 shows an example of a potential workflow representing the different step of the processes of the invention performed in a soaking selection experiment with subsequent fragment screening.

[0471] FIG. 18 shows the distribution of hits over the example plate illustrated in FIG. 16. The plate comprises the first 24 fragments out of 87 fragments. Five fragments form this plate turned out to be PKA-binders (see boxes A1, A4, B6, D3 and D5). RES and MOS indicate the data set's quality in terms of resolution (RES) and mosaicity (MOS).

[0472] FIG. 19 shows a 3D-model of PKA representing the protein and an overlay of all identified binders distributed over different locations on the protein. The magnification in the circle shows the crowd of ATP-pocket binders in more detail.

[0473] FIG. 20 shows the excerpt of a reconstructed electron density.

[0474] FIG. 21 shows the same as FIG. 20 but completed with the protein's amino acid sequence and structurally fixed water molecules (spheres).

[0475] FIG. 22: shows the same as FIG. 20 but with highlighted (ellipse) unassigned electron density that is representative for a small molecule fragment-hit.

[0476] FIG. 23 shows the same as FIG. 20 but completed with the representation of the respective small molecule fragment in the formerly unassigned electron density highlighted in FIG. 19.

[0477] FIG. 24 shows the excerpt of the structure elucidating the binding mode in terms of hydrogen bonds (dotted lines; numbers represent the length of the hydrogen bonds) of the small molecule fragment to the amino acid side chains of the protein and to structurally fixed water (dot).

EXAMPLES

Example 1: Screening of 87 Fragments Using Crystal of the Protein Kinase a (PKA)

[0478] Crystallization of PKA

[0479] A PKA solution was concentrated to 8-10 mg/mL by filtration centrifugation. At the same time a buffer exchange was conducted against 100 mM Mes-Bis-Tris buffer (pH 6.9) containing 1 mM DTT, 0.1 mM EDTA (ethylenediaminetetraacetic acid), and 75 mM LiCl. Afterwards the solution was sterile-filtrated. Of this solution, a volume of 72 μL was mixed with 8 μL of 1 M Mes-Bis-Tris buffer (pH 6.9), and 2 μL of 10 mM Mega 8 solution and centrifuged 15 min. Crystallization was performed by the vapor-diffusion method at 4° C. using 3 μl-sitting drops of the master-mix against 400 μL of methanol/water solutions with methanol concentrations of 14-23% (v/v).

[0480] Selection of the Suitable Soaking Solution

[0481] The crystals were harvested via a cryo-loop and transferred to the wells of an experimental plate that was prepared and distributed in two dimensions according to the method of the invention (FIG. 3).

[0482] Preparation of a first series of 6 individual soaking solutions by mixing os, w and crs and cs. As organic solvent DMSO was used in a concentration of 10% (Vos). Compatible solutes are polyethylene glycol 400 (PEG 400) and 2-methylpentane-2,4-diol (MPD) at a concentration of 25%, ethane-1,2-diol (ethylene glycol; EG) or propane-1,2,3-triol (glycerol; Gly) at concentrations of 10%. As third component dilutions in water (W) of the crystallization buffer (crs named CB in FIG. 3) were used according to the method of the invention.

[0483] The series of line A which was prepared with PEG400 as cs can be designated as the first series according to the invention. Each soaking solutions of the first series comprises the same organic solvent (os) (DMSO) and the same compatible solute (cs) and the Vos 10% (1 μl) and Vcs 25% (2.5 μl) remains the same in all solutions of the series. However, the volume/proportion of Vcrs (CB) and Vw are changed in each solution of the first series, and one of the solution is composed of Vwmin is 0% (0 μl) and Vcrsmax is 65% (6.5 μl) (see column 1), and another solution is composed of Vcrsmin is 0% (0 μl) and the Vwmax is 65% (6.5 μl) (see column 6) and in the other solutions (see columns 2-5) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax in any additional solutions (see column 2-5 of line PEG400) and in any additional solutions, the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 13% (1.3 μl) between each solution of the first series.

[0484] The solutions were distributed in the wells of a line (line A on FIG. 3) of an array thus constituting a first dimension. The solutions were preferably distributed following an order from the lowest Vw to the highest Vw but the order of the distribution in the array can be random.

[0485] Then or simultaneously, the following step were performed as follows Preparation of an additional series of 6 individual soaking solutions by mixing os, w, crs (CB) and cs. In this example of FIG. 3, the number of individual soaking solutions 6 is conserved and is the same as the number of individual soaking solutions of the first series. In the additional series (line B), os is the same as in the first series (DMSO), but the cs is changed (line B: BMPD). Within the additional MPD series, each soaking solutions of the first series comprises the same organic solvent (os) (DMSO) and the same compatible solute (cs) (MPD) and the Vos and Vcs remains the same in all solutions of the series. However, the volume/proportion of Vcrs (CB) and Vw are changed in each solution of the series, and one of the solution is composed of Vwmin is 0% (0 μl) and Vcrsmax is 65% (6.5 μl) (see column 1), and another solution is composed of Vcrsmin is 0% (0 μl) and the Vwmax is 65% (6.5 μl) (see column 6) and in the other solutions (see columns 2-5 of line B) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax (see column 2-5 of line B), the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 13% (1.3 μl) between each solution. In this example, the number of individual soaking solutions i.e. 6 in the MPD series (line B) is the same as the number of individual soaking solutions x of the first series (line A); and the increment of Vcrs between Vcrsmin and Vcrsmax and of Vw between Vwmin and Vwmax is the same in the first (PEG400) series and in the MPD series.

[0486] The solutions were distributed in the wells of additional line (line B on FIG. 3) of an array thus constituting a second dimension. The solutions were preferably distributed following an order from the lowest Vw to the highest Vw but the order of the distribution in the array can be random.

[0487] On the same array of FIG. 3 (but another array could be used), another “first” series (line C with EG, C series) and its additional series (line D with Gly, D series) were prepared and distributed. The series of line C which was prepared with EG as cs can be designated as the first series according to the invention. Each soaking solutions of the C series comprises the same organic solvent (os) (DMSO) and the same compatible solute (EG) and the Vos 10% (10 I) and Vcs 10% (10 μl) remains the same in all solutions of the C series. However, the volume/proportion of Vcrs (CB) and Vw are changed in each solution of the first series, and one of the solution is composed of Vwmin is 0% (0 μl) and Vcrsmax is 80% (8 μl) (see column 1), and another solution is composed of Vcrsmin is 0% (0 μl) and the Vwmax is 80% (8 μl) (see column 6) and in the other solutions (see columns 2-5) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax in any additional solutions (see column 2-5 of line C) and in any additional solutions, the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 16% (1.6 μl) between each solution of the first series.

[0488] The solutions were distributed in the wells of a line (line C on FIG. 3) of an array thus constituting a first dimension. The solutions were preferably distributed following an order from the lowest Vw to the highest Vw but the order of the distribution in the array can be random.

[0489] Simultaneously or not, the following step were performed as follows

[0490] Preparation of an additional series of 6 individual soaking solutions by mixing os, w, crs (CB: crystallization buffer) and cs. In this example of FIG. 3, the number of individual soaking solutions 6 is conserved and is the same as the number of individual soaking solutions of the first series. In the additional series (line D, series D), os is the same as in the first series (DMSO), but the cs is changed (Gly). Within the D series, each soaking solutions of the first series comprises the same organic solvent (os) (DMSO) and the same compatible solute (cs) (Gly) and the Vos and Vcs remains the same in all solutions of the D series. However, the volume/proportion of Vcrs (CB) and Vw are changed in each solution of the D series, and one of the solution is composed of Vwmin is 0% (0 μl) and Vcrsmax is 80% (8 μl) (see column 1), and another solution is composed of Vcrsmin is 0% (0 μl) and the Vwmax is 80% (8 μl) (see column 6) and in the other solutions (see columns 2-5 of line D) the Vw is varied between Vwmin and Vwmax and, inversely, Vcrs is varied between Vcrsmin and Vcrsmax. In this example, Vcrs is incrementally varied between Vcrsmin and Vcrsmax and, inversely, Vw is incrementally varied between Vwmin and Vwmax (see column 2-5 of line D), the increment of Vw between Vwmin and Vwmax and of Vcrs between Vcrsmin and Vcrsmax remains the same, 16% (1.6 μl) between each solution. In this example, the number of individual soaking solutions i.e. 6 in the MPD series (line D) is the same as the number of individual soaking solutions x of the first series (line C); and the increment of Vcrs between Vcrsmin and Vcrsmax and of Vw between Vwmin and Vwmax is the same in the first (EG) series and in the Gly series.

[0491] Then at least one PKA crystal prepared in a) above was placed in each compartments of the array of FIG. 3. The crystal was placed under microscope for control by visual analysis.

[0492] After the transfer of the crystal(s) in each well every well was photographed for visual control and present crystals characterized immediately after the transfer (0 h), after 1 h and 24 h. Pictures of crystals after 24 h are shown in FIG. 4 and the results of the visual inspection of the surfaces, edges and colours in FIG. 8 (data at 0 h and after 1 h are not shown). FIG. 4 shows clearly that after 24 h crystals are still present in row A in wells 1-6, in row B in wells 1-4 and in row C in well 3. Some remnants of crystals are visible in row B in wells 5 and 6 and in row C in wells 2 and 4 where these crystal remnants exhibit signs of advanced dissolution. In row C in wells 1, 5 and 6 as well as in row D no crystals are left.

[0493] From visual inspection crystals, the solutions suitable for soaking of the crystal were selected. Crystals in wells 1 to 4 in the first row A (PEG400) appeared suitable for diffraction tests as well as crystals in wells 1 and 2 in row B (MPD)(FIGS. 4 and 8). From visual inspection it appeared not expectable that crystals in well 5 and 6 in row A, in wells 3 and 4 in row B and crystals from the C row will diffract and therefore these solutions were not selected.

[0494] For further controls and selections, the crystals were harvested using cryo-loops, vitrified in liquid nitrogen and subjected to an inhouse x-ray measurement. For each crystal two perpendicular diffraction pattern were measured (position 0° and 90°). The results as exhibited in FIG. 12 show good diffraction quality for the crystals obtained from wells 1 to 4 in row A and wells 2 in row B. As expected, crystals from well 5 and 6 in row A, from wells 3 and 4 in row B and crystals from row C did not show diffraction. Against expectations, crystals from well 1 in row B did not show diffraction, too. The choice of the compatible solutes, organic solvent and the range of concentrations proved suitable to determine the scope of parameters affecting the protein's crystal integrity, stability and quality, respectively. In FIG. 13, the resulting suitable conditions are mapped on the scheme and have been highlighted. The mapping reveals a broad spectrum (4 solutions) of suitable conditions when using PEG400 and one single condition when using MPD.

[0495] Fragment Screening on PKA.

[0496] For the fragment screen on PKA the composition of the soaking solution of the well 1 in row A was selected (indicated with a star in FIG. 13). The soaking solution with the same proportion of cs (25% of PEG400), crs (65% of CB) and os (10% of DMSO) was prepared. Several 24-well sitting drop experimental plates were filled with the solution (FIG. 16). Instead of DMSO alone, 1 M fragment in DMSO was used (end concentration in each well 100 mM). Afterwards, freshly prepared crystals were transferred to the wells using cryo-loops. Overall, the experiment subjected PKA crystals to 87 different fragments.

[0497] On FIG. 15, another example of plate for screening is shown using the selected composition of an alternative suitable soaking solution made of 25% cs (PEG400), 39% crs (CB), 26% water and 10% os (DMSO) selected according to the method of the invention—see line A column 3 of FIG. 13.

[0498] After 24 h of soaking time the soaked crystals were harvested using cryo-loops and vitrified in liquid nitrogen. Data collection took place at a synchrotron. Data evaluation yielded 55 binders at resolutions of 1.3-1.8 Å. FIG. 18 shows the distribution of hits over the example plate of FIG. 16. The refinement of according data yielded 3D-models of the interaction of 55 fragments with PKA at different interaction sites from which 27 fragments bind to the ATP-binding pocket. FIG. 19 shows an overlay of the protein with respective binders. The study is just one example of a very successful application of the method of the invention to determine soaking parameters for a small molecule screening (in this case with fragments). Among the hits were several unexpected chemotypes for kinase binders. That proves that the method of the invention technology is a very successful means to perform rapid and reliable small molecule-screenings and delivers high-quality data.

Example 2—A Rule-Based Method of Selection of the Suitable Soaking Solution and Subsequent Small Molecule Screening

[0499] In order to perform a small molecule fragment screening, crystals of the protein of interest (POI) must be obtained (step a of workflow in FIG. 17). The crystallization is conducted according to the vapor diffusion approach in commercially available 24-well sitting drop crystallization plates (or any other plate format)(FIG. 1). For this purpose, the reservoirs of each 24-well plate used are filled with a buffer according to a beforehand established crystallization protocol. Furthermore, a crystallization drop is prepared in the indentation on top of the column of each of the wells by adding some volume of a protein solution to some volume of the buffer which is also used in the reservoir. Usually, but not always, the volumes are in a ratio of 1:1 (FIG. 1). The resulting protein concentration is usually, but not always, in the range of 2 to 30 mg/ml but can also reach concentrations of 250 mg/ml or more. Afterwards the wells are sealed using an adhesive film, so called sealing tape. All these steps are conducted in an according to the crystallization protocol correctly tempered laboratory, usually at 4° C., 10° C., 18° C., 20° C. The plates are stored on a shelf in this laboratory for the time needed for crystallization, usually a few days to a few weeks.

[0500] After the crystallization process is finished, a selection of the soaking solution according to the present invention is conducted in order to map conditions for a small molecule fragment screening (same temperature). For this purpose, a 24-well crystallization plate (or any other plate format) is utilized, and the indentation of each well are provided with possible soaking solutions prepared according to the present invention as is exemplified in FIG. 1-3 (step b of workflow in FIG. 17). Subsequently, the film used for sealing the 24-well plates is removed from the plates used for crystallization of the POI. The meanwhile grown crystals are removed from their growth solutions using a special laboratory tool usually equipped with a nylon loop for crystal picking or any other appropriate tool for the so-called “crystal fishing” and transferred to the possible soaking solutions in the indentations of the new 24-well plate that was prepared according to an instantiation of the protocol (step c of workflow in FIG. 17). These plates are also sealed using sealing tape.

[0501] The purpose of the next steps is to control the crystals quality, the maximal soaking time and a proper relation between soaking time and crystal quality for small molecule fragment screenings. Within a certain time, usually the next 24 hours, pictures of each indentation are taken under a microscope at least 1 time, usually three times, sometimes more, under a microscope in order to document the crystals conditions for example immediately after the transfer, after 1 hour and after 24 hours (FIG. 4-5). In parallel, usually notes are taken in order to supplement the information in the pictures. (A template for such notes is exemplified in FIG. 6). Notes refer to different types of cracks crystals can show (longitudinal, lateral, crisscross), to the appearance of the crystals surface (e.g. smooth, rough) or edges (e.g. sharp, roundish) and their behavior in polarized light (strong color, some color, no color) (FIG. 8, exemplified for 24 h). In the most cases not all of the crystals survive in all the conditions. Some crystals dissolve over time or are damaged while other crystals are in good condition. Therefore, an X-Ray evaluation of the crystal's quality is recommended (step d of workflow in FIG. 17). For this purpose, the sealing tape is removed from the plate in order to transfer the remaining crystals from the plate into liquid nitrogen or another vitrification medium. In case that the X-Ray study is conducted at room temperature, previous vitrification is not required. The crystals' vitrification takes place in the nylon loops mentioned before or any other equivalent transfer-equipment like litho-loops or capillaries. The vitrified crystals in the nylon loops or other transfer-equipment are stored in so called “cryo-vials” under liquid nitrogen or another vitrification medium until they are subjected to an X-Ray examination. Crystals can also be stored in any other appropriate storage container.

[0502] For the X-Ray examination the crystals in the nylon loops or other transfer-equipment are removed from the cryo-vials or other storage containers and mounted on an X-Ray machine in a so-called cryo-stream that maintains the cryogenic temperature around the protein crystal. In case of X-Ray studies at room temperature, no cryo-stream is required. Afterwards, X-Ray data is collected. Using an inhouse X-Ray source two to several pictures are collected. At a synchrotron source a whole data set is collected (preferably on a template of FIG. 10-11), due to its rapid data collection. The raw data (FIG. 12) is evaluated regarding resolution, and mosaicity, the appearance of artifact like data originating from unwanted ice crystals in the protein crystal and sometimes a phenomenon called “twinning” (FIG. 5B). In case of data collected at a synchrotron, the appearance of the so called “electron density” is judged by an experienced crystallographer as suitable or not suitable. Based on this data a decision is made on which soaking condition or conditions is/are to be chosen for a small molecule fragment screening (FIG. 13, marked with a star; step e/g of workflow in FIG. 17). In contrast to traditional optimizations of soaking solutions which result in one single soaking condition, the rule-based method of the invention usually results in a spectrum of soaking conditions. From this spectrum the experimenter can choose alternative soaking solutions to face detrimental effects on the crystal that may be caused by for example special properties of small molecule fragments or pH changes etc.

[0503] Optionally, at this stage of the workflow stress tests can be conducted (step f of workflow in FIG. 17). For this purpose, one or more of the previously obtained soaking solutions is/are selected and prepared in a new 24-well plate. Afterwards, crystals are transferred to these solutions. Instead of pure DMSO, small molecules, for example molecular probes or small molecule fragments, for stress tests dissolved in DMSO are used. They exert different kinds of stress such as pH-stress or osmotic stress on the crystals. Based on the subsequently performed data evaluation (same as in step d of workflow in FIG. 6) one or more final soaking solutions can be chosen.

[0504] Usually, a decision is made for one soaking condition which is subsequently used for the small molecule fragment screening. For a small molecule fragment screening comprising 300 small molecule fragments 13 of the 24-well plates are prepared by pipetting the respective soaking solution composition of the chosen soaking solution without DMSO. Instead of pure DMSO, 300 small molecule species at concentrations of usually 1M dissolved in DMSO are used and added to the prepared 300 wells. This results in final concentrations of the small molecule fragments in each soaking solution of usually 100 mM but may be higher or lower (FIG. 15-16; step h of workflow in FIG. 17). Following that, usually two newly prepared crystals (sometimes just one or more than two) are transferred to each soaking solution and soaked for usually 24 hours (step i of workflow in FIG. 17). Then, the crystals are fished, transferred to liquid nitrogen for vitrification (step j of workflow in FIG. 17), stored in cryo-vials in Uni-pucks or some other applicable storage device and sent to a synchrotron facility, where data collection takes place (step k of workflow in FIG. 17). In case of X-Ray studies at room temperature no vitrification is required.

[0505] In data collection individual data sets for all crystals are obtained comprising of several hundreds to thousands of pictures showing reflections of the X-Ray beams (exemplified in FIG. 9). These reflections stem from interactions between X-Ray and electrons and, therefore, can be exploited to reconstruct the so-called electron densities. Such electron densities define the space in the crystals where electrons are located and are representative for the protein's geometry. Using computational methods important information are extracted from these data sets. To reconstruct the geometry further information is needed, the so-called phases, that are not extractable from this kind of experiment without heavy atom derivatization. This information can be derived in an approach called molecular replacement from previously obtained structural solutions of the same or very similar proteins. The result is a 3-dimensional model of the protein's geometry according to the positions of all the electrons in the protein (FIG. 19). This electron density is merged with the known sequence of amino acids of the respective protein and positions of structurally fixed water molecules are identified. This solution for the structure is a model that must be optimized in several structure refinement cycles in order to obtain a convincing fit between the measured data and the model. The result is an atomically resolved representation of the geometry of the target (FIG. 19; step I of workflow in FIG. 17).

[0506] Each of these structures is examined in order to identify electron density (FIG. 20) that can be assigned to the special small molecule fragment that was used for soaking of this very crystal (FIG. 21-22). If this is the case, this small molecule fragment is judged as a so-called “hit”. Examination of all data sets reveals all small molecule fragment-binders (FIG. 23 maps the hits on the experimental layout on one plate of the screening encompassing five hits). Data sets without additional electron density that is assignable to respective small molecule fragments are discarded. Further refinement of each of these “hit”-data sets reveal the exact geometry of the interaction between the protein and the respective small molecule fragments (FIG. 24). Therefore, the described process provides validated knowledge about small molecule fragments that bind to the protein target and delivers structurally elucidated binding modes that can be used for medicinal-chemical optimization processes. Instead of small molecule fragments also more drug-size molecules can be used for example to validate follow-up compounds resulting from medicinal-chemical optimization. Furthermore, special molecular probes can be used to map properties of the binding sites or to obtain information about putative pharmacophore patterns etc.