GB2409678A - A ligand of Glucokinase useful in the treatment of hyperglycaemia - Google Patents

Abstract

3-Cyclopentyl-2-pyridin-4-yl-N-thiazol-2-yl-propionamide and pharmaceutically acceptable salts thereof. This compound is an allosteric ligand of mammalian Glucokinase and therefore may be useful in therapy, in particular for the reduction of hyperglycaemia in type II diabetes. Pharmaceutical compositions comprising this compound may be prepared and it may also be used to assist the formation of co-crystals of Glucokinase.

Description

CRYSTALS OF GLUCOKINASE AND METEIODS OF GROWING TOM

AND LIGANDS OF GLUCOK1NASE The invention relates to crystalline forms of Glucokinase of sufficient size and quality to obtain structural data by Xray crystallography and to methods of growing such crystals. s

Glucokinase (GK) is one of four hexokinases found in mammals [Colowick, S. P., in The Enzymes, Vol. 9 (P. Boyer, ea.) Academic Press, New York, NY, pages 1-48, 1973]. The hexokinases catalyze the first steel in the metabolism of glucose, i.e., the conversion of glucose to glucose-6-phosphate. Glucokinase has a limited cellular lo distribution, being found principally in pancreatic p-cells and liver parenchymal cells. In addition, GK is a Mte-controlling enzyme for glucose metabolism in these two cell types that are known to play critical roles in whole-body glucose homeostasis [Chipkin, S.R., Kelly, K.L., and Rudernan, N.B. in Joslin's Diabetes (C.R. Khan and G.C. Wier, eds.), Lea and Febiger, Philadelphia, PA, pages 97-115, 1994]. The concentration of glucose at Is which GK demonstrates half-maximal activity is approximately 8 rnM. The other three hexokinases are saturated with glucose at much lower concentrations (<1 mM).

Therefore, the flux of glucose through the GK pathway rises as the concentration of glucose in the blood increases from fasting (5 mM) to postprandial (10-15 mM) levels following a carbohydrate-containing meal [Printz, R.G., Magnuson, M.A., and Granner, zo D.K. in Ann. Rev. Nutrition Vol. 13 (R.E. Olson, D.M. Bier, and D.B. McCormick, eds.), Annual Review, Inc., Palo Alto, CA, pages 463496, 1993]. These findings contributed over a decade ago to the hypothesis that GK functions as a glucose sensor in p-cells and hepatocytes (Meglasson, M.D. and Matschinsky, F.M. Amer. J. Physiol. 246, El-E13, 1984). In recent years, studies in transgenic animals have confirmed that GK does indeed z play a critical role in whole-body glucose homeostasis. Animals that do not express GK die within days of birth with severe diabetes while animals overexpressing GK have improved glucose tolerance (Grupe, A., Hultgren, B., Ryan, A. et al., Cell 83, 69-78, 1995; Ferrie, T., Riu, E., Bosch, F. et al., FASEB J., 10, 1213-1218, 1996). An increase in glucose exposure is coupled through GK in,8-cells to increased insulin secretion and in so hepatocytes to increased glycogen deposition and perhaps decreased glucose production.

The finding that type II maturity-onset diabetes of the young (MODY-2) is caused by loss of function mutations in the GK gene suggests that GK also functions as a glucose sensor in humans (Liang, Y., Kesavan, P., Wang, L. et al., Biochem. J. 309,167-173,1995).

Additional evidence supporting an important role for GK in the regulation of glucose metabolism in humans was provided by the identification of patients that express a mutant form of GK with increased enzymatic activity. These patients exhibit a fasting hypoglycemia associated with an inappropriately elevated level of plasma insulin (Glaser, B., Kesavan, P., Heyman, M. et al., New England J. Med. 338, 226-230, 1998). While lo mutations of the GK gene are not found in the majority of patients with type II diabetes, compounds that activate GK and, thereby, increase the sensitivity of the GK sensor system will still be useful in the treatment of the hyperglycemia characteristic of all type II diabetes. Glucokinase activators will increase the flux of glucose metabolism in p-cells and hepatocytes, which will be coupled to increased insulin secretion. Such agents would ts be useful for treating type II diabetes.

In an effort to elucidate the mechanisms underlying kinase activation, the crystal structure of such proteins is often sought to be determined. The crystal structures of several hexokinases have been reported. See, e. g. A. E. Aleshin, C. Zeng, G. P. Bourenkov, to H. D. Bartunik, H. J. Fromm & R. B. Honzatko 'The mechanism of regulation of hexokinase: new insights from the crystal structure of recombinant human brain hexokinase complexed with glucose and glucose-6-phosphate' Structure 6, 39-50 (1998); W. S. Bennett, Jr. & T. A. Steitz 'Structure of a complex between yeast hexokinase A and glucose I. Structure determination and refinement at 3.5 resolution' J. Mol. Biol. 140, 2; 183-209 (1978); and S. Ito, S. Fushinobu, I. Yoshioka, S. Koga, H. Matsuzawa & T. Wakagi 'Structural Basis for the ADP-Specificity of a Novel Glucokinase from a Hyperthermophilic Archaeon' Structure 9, 205-214 (2001). Despite these reports, researchers armed with the knowledge of how to obtain crystals of related hexokinases have attempted to obtain crystals of any mammalian Glucokinase without success. - 2

Applicants have discovered protocols which allow crystallization of mammalian Glucokinase with or without a bound allosteric ligand. The crystal structure has been solved by X-ray crystallography to a resolution of 2.7 A. See Figures 3 and 4. Thus the invention relates to a crystalline form of Glucokinase and a crystalline form of a complex of Glucokinase and an allosteric ligand. The invention further relates to a method of forming crystals of Glucokinase, with or without a bound allosteric ligand.

-

Figure 1 shows Glucokinase co-crystals having P6(5)22 symmetry.

lo Figure 2 shows the amino acid sequence of an expressed Glucokinase used for crystallization.

Figure 3 shows a ribbon diagram of the structure of Glucokinase showing the oc-helices and,8-sheets.

Figure 4 shows the atomic structure coordinates for Glucokinase bound to 3-Cyclopentyl- 2-pyridin-4-yl-N-thiazol-2-yl-propionamide.

The present invention relates to crystalline forms of mammalian Glucokinase, with to or without a ligand bound in the allosteric site, where the crystals are of sufficient quality and size to allow for the determination of the three-dimensional X-ray diffraction structure to a resolution of about 2.0 to about 3.5 A. The invention also relates to methods for preparing and crystallizing the Glucokinase. The crystalline forms of Glucokinase, as well as information derived from their crystal structures can be used to analyze and modify glucokinase activity as well as to identify compounds that interact with the allosteric site.

The crystals of the invention include apo crystals and co-crystals. The apo crystals of the invention generally comprise substantially pure Glucokinase. The co-crystals generally comprise substantially pure Glucokinase with a ligand bound to the allosteric site.

It is to be understood that the crystalline Glucokinases of the invention are not limited to naturally occurring or native Glucokinases. Indeed, the crystals of the invention include mutants of the native Glucokinases. Mutants of native Glucokinases are obtained by replacing at least one amino acid residue in a native Glucokinase domain lo with a different amino acid residue, or by adding or deleting amino acid residues within the native polypeptide or at the N- or C- terminus of the native polypeptide, and have substantially the same three-dimensional structure as the native Glucokinase from which the mutant is derived.

is By having substantially the same three-dimensional structure is meant having a set of atomic structure coordinates from an apo- or co-crystal that have a root mean square deviation of less than or equal to about 2 when superimposed with the atomic structure coordinates of the native Glucokinase from which the mutant is derived when at least about 50% to about 100% ofthe alpha carbon atoms ofthe native Glucokinase are to included in the superposition.

In some instances, it may be particularly advantageous or convenient to substitute, delete and/or add amino acid residues to a native Glucokinase domain in order to provide convenient cloning sites in cDNA encoding the polypeptide, to aid in purification of the polypeptide, etc. Such substitutions, deletions and/or additions which do not substantially alter the three dimensional structure of the native Glucokinase will be apparent to those having skills in the art. - 4

It should be noted that the mutants contemplated herein need not exhibit glucokinase activity. Indeed, amino acid substitutions, additions or deletions that interfere with the kinase activity of the glucokinase but which do not significantly alter the three-dimensional structure of the domain are specifically contemplated by the invention.

Such crystalline polypeptides, or the atomic structure coordinates obtained therefrom, can be used to identify compounds that bind to the native domain. These compounds may affect the activity or the native domain.

The derivative crystals of the invention generally comprise a crystalline glucokinase lo polypeptide in covalent association with one or more heavy metal atoms. The polypeptide may correspond to a native or a mutated Glucokinase. Heavy metal atoms useful for providing derivative crystals include, by way of example and not limitation, gold and mercury. Alternatively, derivative crystals can be formed from proteins which have heavy atoms incorporated into one or more amino acids, such as selenomethionine The co-crystals of the invention generally comprise a crystalline Glucokinase polypeptide in association with one or more compounds at an allosteric site of the polypeptide. The association may be covalent or non-covalent.

The native and mutated glucokinase polypeptides described herein may be isolated from natural sources or produced by methods well known to those skilled in the art of molecular biology. Expression vectors to be used may contain a native or mutated Glucokinase polypeptide coding sequence and appropriate transcriptional and/or translational control signals. These methods include in vitro recombinant DNA 2s techniques, synthetic techniques and in viva recombination/genetic recombination. See, for example, the techniques described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

A variety of host-expression vector systems may be utilized to express the Glucokinase coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmic DNA expression vectors containing the Glucokinase coding sequence; yeast transformed with recombinant yeast expression vectors containing the Glucokinase coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g. baculovirus) containing the Glucokinase coding sequence; plant cell systems infected with recombinant virus expression vectors (e. g., cauliflower mosaic virus, CaMVi tobacco mosiac virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti lo plasmid) containing the glucokinase coding sequence; or animal cell systems. The expression elements of these systems vary in their strength and specificities. Depending on the host/vector system utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters such as pL of bacteriophage L, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be usedi when Coning in insect cell systems, promoters such as the baculovirus polybedrin promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g., heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses (e.g., the 35 S RNA promoter of CaMV; the coat protein promoter of TMV) may be used; when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used; when generating cell lines that contain multiple copies of the glucokinase coding sequence, SV40-, BPV- and EBV- based vectors may be used with an appropriate selectable marker.

The apo, derivative and co-crystals of the invention can be obtained by techniques well-known in the art of protein crystallography, including batch, liquid bridge, dialysis, vapor diffusion and hanging drop methods (see e.g. McPherson, 1982, Preparation and Analysis of Protein Crystals, John Wiley, NY; McPherson, 1990, Eur. J. Biochem. 189:1-23; so Webber, 1991, Adv. Protein Chem. 41:1-36; Crystallization of Nucleic Acids and Proteins, Edited by Arnaud Ducruix and Richard Giege, Oxford University Press; Protein Crystallization Techniques, Strategies, and Tips, Edited by Terese Bergfors, International University Line, 1999). Generally, the apoor co-crystals of the invention are grown by placing a substantially pure Glucokinase polypeptide in an aqueous buffer containing a precipitant at a concentration just below that necessary to precipitate the protein. Water is then removed from the solution by controlled evaporation to produce crystallizing conditions, which are maintained until crystal growth ceases.

In a preferred embodiment of the invention, apo or co-crystals are grown by vapor diffusion. In this method, the polypeptide/precipitant solution is allowed to equilibrate in a closed container with a larger aqueous reservoir having a precipitant concentration optimal for producing crystals. Generally, less than about 10 pL of subtantially pure lo polypeptide solution is mixed with an equal volume of reservoir solution, giving a precipitant concentration about half that required for crystallization. This solution is suspended as a droplet underneath a coverslip, which is sealed onto the top of a reservoir.

The sealed container is allowed to stand, from one day to one year, usually for about 2-6 weeks, until crystals grow.

is For crystals of the invention, it has been found that hanging drops containing about 2-5 p1 of Glucokinase (9-22 mg/ml in 20 mM tris pH 7.1 measured at room temperature, mM NaCl, 50 mM glucose, 10 mM DTT and optionally 0.2 mM EDTA) and an equal amount of reservoir solution (16-25% w/v polyethylene glycol with an average molecular weight from about 8000 to about 10000 Daltons, 0.1-0.2 M Iris or bistris or Hepes or to ammonium phosphate buffer, pH 6.9-7.5,8-10 mM DTT, O - 30% saturated glucose) suspended over 0.5 to 1.0 mL reservoir buffer for about 3-4 weeks at 4-6 C provided crystals suitable for high resolution X-ray structure determination. Particularly preferred conditions were: about 2-5 p1 of Glucokinase ( 10 mg/ml in 20 mM tris pH 7.1 measured at room temperature, 50 mM NaCl, 50 mM glucose, 10 mM DTT and optionally 0.2 mM EDTA) and an equal amount of reservoir solution (22.5% w/v polyethylene glycol with an average molecular weight of about 10000 Daltons, 0.1 M tris pH 7.08,10 mM DTT, 20% glucose) were suspended over 0.5 to 1.0 mL reservoir buffer for about 3-4 weeks at 4 6 C. - 7

The optimum procedure for growing crystals large enough to collect data from involved first streaking 3-4 Ill of protein solution on the coverslip, followed by streaking 3-4 Ill of well solution across the elongated droplet of protein, forming a droplet shaped like the letter 'X'. Before discovering this crossed droplet technique, most droplets yielded showers of small crystals which were not large enough for data collection purposes. The crossed droplets allow gradients of protein and precipitating agent to form as the two solutions slowly mix, and the resulting kinetics of crystal nucleation and growth are optimal for the growth of a small number of large crystals in each crossed droplet. S mply mixing the protein and precipitant solutions together in a single round droplet often lo produced an overabundance of nuclei which grew to a final size too small for data collection purposes. Crystals usually appeared within 5 days of setup. The crystals grow in the form of hexagonal bipyramids, reaching dimensions of 0.2 x 0.2 x 0.4 mm typically, although larger crystals are often observed. Figure 1 shows grown crystals.

Crystals maybe frozen prior to data collection. The crystals were cryoprotected with either (a) 20-30% saturated glucose present in the crystallization setup, (b) ethanol added to 15-20%, (c) ethylene glycol added to 10-20% and PEG10,000 brought up to 25%, or (d) glycerol added to 15%. The crystals were either briefly immersed in the cryo protectant or soaked in the cryo-protectant for periods as long as a day. Freezing was accomplished by immersing the crystal in a bath of liquid nitrogen or by placing the crystal in a stream of nitrogen gas at 100 K. The mosaic spread of the frozen crystals could sometimes be reduced by annealing, wherein the stream of cold nitrogen gas is briefly blocked, allowing the frozen crystal to thaw momentarily before re-freezing in the nitrogen gas stream. Another technique which was sometimes helpful in data collection was to center one of the ends of the hexagonal bipyramid in the x-ray beam, rather than the mid portion of the crystal. The mosaic spread could sometimes be reduced by this technique. - 8

Diffraction data typically extending to 2.7 was collected from the frozen crystals at the synchrotron beamline X8C of the National Synchrotron Light Source in Brookhaven, New York. Under optimum conditions, data extending to 2.2 was recorded. See Figures 3 and 4 for solution. The space group of the crystals was determined to be P6(5)22 during the course of the solution of the crystal structure. The crystals have unit cell dimensions a = b = 79.62 +/- 0.60 A, c = 321.73 +/- 3.70 A, oy = = 90 , = 120 .

The crystals are in a hexagonal system with P6(5)22 symmetry.

Of course, those having skill in the art will recognize that the abovedescribed lo crystallization conditions can be varied. Such variations may be used alone or in combination, and include polypeptide solutions containing polypeptide concentrations between 1 mg/mL and 60 mg/mL, any commercially available buffer systems which can maintain pH from about 6. 5 to about 7.6, Tris-HC1 concentrations between 10 mM and mM, dithiothreitol concentrations between O mM and 20 mM, preferably between 8 and 10 mM, substitution of dithiothreitol with beta mercapto ethanol or other art recognized equivalents, glucose concentrations between 0% w/v and 30% w/v, or substitution of glucose with other sugars known to bind to Glucokinase; and reservoir solutions containing polyethylene glycol (PEG) concentrations between about 10% and about 30%, polyethylene glycol average molecular weights between about 1000 and about 20,000 daltons, any commercially available buffer systems which can maintain pH from about 6.5 to about 7.6, dithiothreitol concentrations between 0 mM and 20 mM, substitution of dithiothreitol with beta mercapto ethanol or other art-recognized -SH group containing equivalents, or substitution of glucose with other sugars known to bind to Glucokinase, and temperature ranges between 4 and 20 C.

Derivative crystals of the invention can be obtained by soaking apo or cocrystals in mother liquor containing salts of heavy metal atoms, according to procedures known to those of skill in the art of X-ray crystallography.

Q _

Co-crystals of the invention can be obtained by soaking an apo crystal in mother liquor containing a ligand that binds to the allosteric site, or can be obtained by co crystallizing the Glucokinase polypeptide in the presence of one or more ligands that bind to the allosteric site. Preferably, co-crystals are formed with a glucokinase activator disclosed in US Pat. No. 6,320,050; US Pat. Appl. 09/532,506 filed March 21, 2000; US Pat. Appl. 09/675,781 filed September 28,2000; US Pat. Appl. 09/727, 624, filed December 1,2000; US Pat. Appl. 09/841,983, filed April 25,2001; US Pat. Appl. 09/843,466, filed April 26,2001; US Pat. Appl. 09/846,820, filed May 1, 2001; US Pat. Appl. 09/846,821, filed May 1,2001; US Pat. Appl. 09/905, 152, filed July 13,2001; US Pat. Appl. 09/924,247, lo filed August 8, 2001; US Provisional Pat. Appl. 60/251,637, filed December 6, 2000; or US Provisional Pat. Appl. 60/318,715, filed September 13,2001, each of which is incorporated herein by reference.

Methods for obtaining the three-dimensional structure of the crystalline glucokinases described herein, as well as the atomic structure coordinates, are well-known in the art (see, e.g., D. E. McRee, Practical Protein Crystallography, published by Academic Press, San Diego (1993), and references cited therein).

The crystals of the invention, and particularly the atomic structure coordinates obtained therefrom, have a wide variety of uses. For example, the crystals and structure coordinates described herein are particularly useful for identifying compounds that activate Glucokinases as an approach towards developing new therapeutic agents. One such compound is 3-CyHopentyl-2-pyridin-4-yl-N-thiazol-2-yl-propionamide and pharmaceutically acceptable salts thereof Pharmaceutical compositions of said compounds can be developed, and said compounds can be used for the manufacture of a medicament comprising said compound for the treatment of hyperglycemia in type II diabetes.

The structure coordinates described herein can be used as phasing models in determining the crystal structures of additional native or mutated glucokinases, as well as the structures of co-crystals of such glucokinases with allosteric inhibitors or activators bound. The structure coordinates, as well as models of the three-dimensional structures obtained therefrom, can also be used to aid the elucidation of solution-based structures of native or mutated glucokinases, such as those obtained via NMR. Thus, the crystals and atomic structure coordinates of the invention provide a convenient means for elucidating the structures and functions of glucokinases.

For purposes of clarity and discussion, the crystals of the invention will be described by reference to specific Glucokinase exemplary apo crystals and co-crystals. Those skilled in the art will appreciate that the principles described herein are generally applicable to lo crystals of any mammalian Glucokinase, including, but not limited to the Glucokinase of Figure 2.

As used herein, "allosteric site" refers in general to any ligand binding site on a mammalian Glucokinase other than the active site of the enzyme.

As used herein, "apo crystal" refers to crystals of mammalian Glucokinase formed without a bound allosteric ligand.

As used herein, "allosteric ligand" refers to any molecule which specifically binds an allosteric site on a mammalian Glucokinase.

EXAMPLES

Example 1: Expression and Purification of Glucokinase Expression of GK Glucokinase (GK) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The amino-acid sequence of the fusion protein is given in Figure 2.

The expression construct is based on the pGEX-3X vector from Pharmacia, as described lo in Y. Liang, P. Kesavan, L. Wang, K. Niswender, Y. Tanizawa, M. A. Permutt, M. A. Magnuson, F. M. Matschinsky, Biochem. 1. 309, 167 (1995). The construct codes for one of the two liver isozymes of human GK. The GST tag is at the N-terminus of the construct, and is separated from the coding sequence for GK by a Factor Xa cleavage site.

After purification of the GST fusion protein, the GST fusion tag was removed with Factor Xa protease, which also removes five residues from the N-terminus of GK.

Purification of GK E. cold cells expressing GST-GK were suspended in lysis buffer (50 mM Iris, 200 mM NaCl, 5 mM EDTA, 5 mM DTT, 1% NP-40, pH 7.7) in the presence of protease inhibitors, incubated with lysozyme at 200 p/ml for 30 minutes at room temperature, and sonicated 4x30 sec. at 4 C. After centrifugation to remove insoluble material, the supernatant was loaded onto glutathione-Sepharose, washed with lysis buffer and then with lysis buffer minus NP-40. GST-GK was eluted with lysis buffer (minus NP-40) containing 50 mM D-glucose and 20 mM glutathione. The eluted protein was concentrated and dialyzed into 20 mM tris, 100 mM NaCl, 0.2 mM EDTA, 50 mM D glucose, lmM DTT, pH 7.7. Factor Xa was added at a protein ratio of 1:100 GST-GK followed by the addition of CaCI2 to 1 mM, and the sample was incubated at 4 C for 48 hours. The sample was added to glutathione Sepharose and the unbound fraction collected and concentrated. The sample was then incubated with benzamidine Sepharose to remove Factor Xa, and the unbound fraction was collected and loaded on a Q Sepharose column equilibrated with 25 mM bis-tris propane, 50 mM NaCl, 5 mM DTT, 50 mM D-glucose and 5% glycerol (pH 7.0). The protein was eluted with a NaC1 gradient from 50-400 mM. Fractions containing purified GK were pooled and concentrated and filtered.

Example 2: Formation of apo Crystal - o 4 pi of glucokinase and 4 pi of precipitant were mixed and equilibrated against the precipitant solution at 4 C. The glucokinase solution consisted of 22 mg/ml glucokinase prepared in Example 1 in 20 mM hepes pH 7.5, 50 mM NaCI, 10 mM Did, and 50 mM glucose. The precipitant consisted of 22.5% PEG10000, 0.1 M Iris pH 7.08, mM DTT, 20% glucose; the precipitant solution contained seed crystals in order to microseed the droplets. Crystals appeared in the droplets after leaving the crystallization plates at 4 C. Example 3: Formation of Co-crystal with 3- Cyclopentyl-2-pyridin-4-yl-N-thiazol-2 yl-propionamide 3(a) 4 p1 of glucokinase and 4 pi of precipitant were mixed and equilibrated against the precipitant solution at 4 C. The glucokinase solution consisted of 13 mg/ml glucokinase prepared in Example 1 in 20 mM tris pH 7.0, 50 mM NaCI, 10 mM DTT, 50 mM glucose, and the glucokinase activator 3-Cyclopentyl-2-pyridin-4-yl-N-thiazol-2-yl-propionamide at a concentration 5 times that of the protein. The precipitant consisted of 22.5% PEG10000, 0.1 M tris pH 7.08, 10 mM DTT, 20% glucose. Crystals appeared in the droplets after leaving the crystallization plates at 4 C. 3(b) Alternatively, crystals were grown as in Example 3(a) with the following changes: instead of 4 pi glucokinase and 4,ul precipitant, 2 foul of each were used; the glucokinase solution contained 11 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant 18% PEG8000 was used; the precipitant solution contained seed crystals in order to microseed the droplets. 3(c):

In another alternative, crystals were grown as in Example 3(a) with the following lo changes: instead of 4 pi glucokinase and 4 Ill precipitant, 2 p1 of each were used; the glucokinase solution contained l l mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution induded 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant 20% PEG8000 was used; the precipitant solution contained seed crystals in order to microseed the droplets. 3(d)

In yet another alternative, crystals were grown as in Example 3(a) with the following changes: instead of 4 Ill glucokinase and 4 Ill precipitant, 2 Ill of each were used; the glucokinase solution contained 12 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant 16% PEGlOOOO was used; glucose was not present as a component of the precipitant; the precipitant solution contained seed crystals in order to microseed the droplets. 3(e):

In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 11 mg/ml glucokinase in tris

-

buffer at pH 7.1 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant 25% PEG10000 was used. 3(fl:

In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 11 mg/ml glucokinase in tris buffer at pH 7.1 instead of 7.0i the glucokinase solution included 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant 21.25% PEG10000 was usedi in place of tris buffe ed at pH 7.08 in the precipitant tris buffered at pH 7.52 was used. 3(g): lo In still another alternative, crystals were grown as in Example 3(a)

with the following changes: the glucokinase solution contained 12 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of tris buffered at pH 7.08 in the precipitant, hopes buffered at pH 6.89 was usedi in place of 20% glucose in the precipitant, 200 mM glucose was used. 3(h):

In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 12 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 0.1 M tris buffered at pH 7.08 in the precipitant, 0.2 M ammonium phosphate buffered to at pH 7.03 was used; in place of 20% glucose in the precipitant, 200 mM glucose was used. 3(i)

In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 10 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant, 20% PEG10000 was usedi in place of tris buffered at pH 7.08 in the precipitant, tris buffered at pH 7.05 was used; in place of 10 mM DTT in the precipitant, 8 mM DTT was used; glucose was not present as a component of the precipitant. \ 3(j):

In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 12 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution induded 0.2 mM EDTA; in place of 22.5% PEG10000 as precipitant, 22% PEG8000 was used; glucose was not present as a component of the precipitant; the precipitant solution contained seed crystals in order to microseed the droplets. 3(k)

lo In still another alternative, crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 11 mg/ml glucokinase in tris buffer at pH 7.1 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of 20% glucose in the precipitant, 30% glucose was used.

Example 4: Formation of Co-crystal with N-(5-Bromo-pyridin-2-yl)-2-(3chloro-4 methanesulfonyl-phenyl)-3-cyclopentyl-propionamide Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 9 mg/ml glucokinase in Iris buffer at pH 7.1 instead of to 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator N-(5-Bromo-pyridin-2-yl)-2-(3-chloro-4methanesulfonyl-phenyl)-3 -cyclopentyl propionamide; in place of 20% glucose in the precipitant, 200 mM glucose was used.

Example 5: Formation of Co-crystal with 2-(3-Chloro-4-methanesulfonylphenyl)-3 cyclopentyl-N- (5-trifluoromethyl-pyridin-2-yl)-propionamide Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 10 mg/ml glucokinase in tris buffer at pH 7.1 instead of so 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator 2- (3 -Chloro-4-methanesulfonyl-phenyl)-3-cyclopentyl-N-(5 -trifluoromethylpyridin-2-yl)- propionamide; in place of 22.5% PEG10000 as precipitant, 21.25% PEG10000 was used.

Example 6: Formation of Co-crystal with (2S)-2-[3-Cyclopentyl-2-(3,4dichloro phenyl)-propionylamino]-thiazole-4-carboxylic acid methyl ester Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 10 mg/ml glucokinase in tris buffer at pH 7.1 instead of lo 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator (2S)-2-[3-Cyclopentyl-2-(3,4-dichloro-phenyl)-propionylamino]-thiazole-4carboxylic acid methyl ester; in place of 22.5% PEG10000 as precipitant, 21.25% PEG10000 was used; in place of tris buffered at pH 7.08 in the precipitant, bistris buffered at pH 7.0 was used.

Example 7: Formation of Co-crystal with (2S)-{2-[3-Cyclopentyl-2-(3,4dichloro- phenyl)-propionylamino]-thiazol-5-yl}-oxo-acetic acid ethyl ester so Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 10 mg/ml glucokinase in tris buffer at pH 7.1 instead of 7.0; the glucokinase solution included 0.2 rnM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator (2S)2-[3-Cyclopentyl-2-(3,4-dichloro-phenyl)-propionylamino]-thiazol-5- yl} -oxo s acetic acid ethyl ester; in place of 22.5% PEG10000 as precipitant, 21.25% PEG10000 was used.

Example 8: Formation of Co-crystal with (2S)-{3-[3-Cyclopentyl-2-(3,4dichloro- phenyl)-propionyl]-ureido}-acetic acid methylester Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 9 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator (2S)- { 3-[3-Cyclopentyl-2-(3,4-dichloro-phenyl)-propionyl]-ureido} - acetic acid methylester; in place of 20% glucose in the precipitant, 200 mM glucose was used.

Example 9: Formation of Co-crystal with (2S)-1-[3-Cyclopentyl-2-(3,4dichloro- phenyl)-propionyl] -3-(3-hydroxy-propyl)-urea is Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 14 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator (2S)-1-[3-Cyclopentyl-2-(3,4-dichloro-phenyl)-propionyl]-3-(3-hydroxypropyl)-urea; in place of 20% glucose in the precipitant, 200 mM glucose was used.

Example 10: Formation of Co-crystal with (2S)-{3-[3-Cyclopentyl-2-(3,4dichloro- phenyl)-propionyl]-ureido}-acetic acid ethyl ester Crystals were grown as in Example 3(a) with the following changes: the glucokinase solution contained 14 mg/ml glucokinase in tris buffer at pH 7.08 instead of 7.0; the glucokinase solution included 0.2 mM EDTA; in place of the glucokinase activator of Example 3(a), the glucokinase solution contained the glucokinase activator (2S)-{ 3-[3-Cyclopentyl-2-(3,4-dichloro-phenyl)-propionyl]-ureido}-acetic acid ethyl so ester; in place of tris buffered at pH 7.08 in the precipitant, tris buffered at pH 7.05 was used.

Example 11: Synthesis of 3-Cydopentyl-2-pyridin-4-yl-N-thiazol-2-ylpropionamide 3-Cydopentyl-2-pyridin-4-yl-N-thiazol-2-yl-propionamide can be prepared using well 0S Amide vend 0 - AlkylationI Formation, I H :0--N.: Mg(ocH3)2 N\) commercially IN availableA\\ IS NH2 known organic synthesis techniques according to the following reaction scheme: 3-Cyclopentyl-2-pyridin-4-yl-N-thiazol-2-yl-propionamide is useful as an allosteric activator of Glucokinase and to assist the formation of cocrystals of Glucokinase.

Preferred features of the invention are as follows: 1. A co-crystal of mammalian Glucokinase and a ligand bound to an allosteric site of the Glucokinase, wherein the co-crystal has unit cell dimensions of: a and b are from 79.02 A to 80.22 A; c is from 318.03 A to 325.03 A; a and are 90 ; and y is 120 ; and the co-crystal has P6(5)22 symmetry.

2. A crystal of mammalian Glucokinase, wherein the crystal has unit cell dimensions of: a and b are from 79.02 A to 80.22 A; c is from 318.03 A to 325.03 A; a and are 90 ; and by is 120 ; and the crystal has P6(5)22 symmetry.

3. A process for co-crystalizing mammalian Glucokinase and an allosteric ligand of Glucokinase, the process comprising; providing a buffered, aqueous solution of 9 to 22 mg/ml of the mammalian Glucokinase; adding a molar excess of allosteric ligand to the aqueous solution of mammalian Glucokinase; and growing crystals by vapor diffusion using a buffered reservoir solution between about 10% and about 30% PEG, about 0% w/v and about 30% w/v glucose, and between O and 20 mM OTT, wherein the PEG has an average molecular weight between about 1,000 and about 20,000.

4. The process of feature 3, wherein the step of growing crystals of vapor diffusion comprises: streaking the buffered, aqueous solution of mammalian Glucokinase with added allosteric ligand on a surface to form an elongated droplet of protein solution, and streaking about an equal amount of the buffered reservoir solution across the elongated droplet of protein solution, forming a combined droplet shaped like the fetter 'X'.

5. A crystal produced by the process of features 3 or 4.

6. A compound identified by analysing the structure coordinates of the cocrystal of feature 1, said compound being a ligand that binds to the allosteric site of Glucokinase.

7. The compound a | H No Hand pharmaceutically acceptable salts thereof.

8. A pharmaceutical composition comprising the compound of feature 6.

9. The pharmaceutical composition of feature 8, wherein said compound is the compound of feature 7.

10. Use of the compound of feature 6 for the manufacture of a medicament comprising a compound according to feature 6 for the treatment of hyperglycemia in type 11 diabetes.

11. The use of a feature 10 wherein said compound is the compound of feature 7.

12. A compound according to features 6 or 7, for use as a therapeutic active substance, in particular for the reduction of hyperglycemia in type 11 diabetes.

13. The novel crystals, processes, compounds, compositions and uses as hereinbefore described. \

14. A process according to feature 3 or 4 further comprising the step of freezing the crystals.

15. A method of identifying a ligand that binds to the allosteric site of Glucokinase comprising analysing the structure co-ordinates of a co crystal according to feature 1.

16. Use of a co-crystal according to feature 1 or a crystal according to feature 2 in the identification of a compound which activates Glucokinase.

17. Use of a co-crystal according to feature 1 or a crystal according to feature 2 for elucidating the structure and function of a Glucokinase.

18. A compound according to feature 6 or 7, or a composition according to feature 8 or 9, for use in a method of treatment of human or animal body.

19. Any novel feature or combination of features described herein.

1h the present specification "comprises" means "include. or consists of' and "comprising" means "including or consisting of'.

The features disclosed in the foregoing description, or the following claims, or the accompanying drawings, expressed in their specific forms or in tend of a means for performing the disclosed Unction, or a method or process for attaining the disclosed result, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms [hereof.

SEQUENCE LISTING

<110> F. Hoffmann - La Roche <120> CRYSTALS OF GLUCOKINASE AND METHODS OF GROWING THEM <130> Case 20892 <140> US 60/341988 <141> 2001-12-19 <150> US 601341988 _ <151> 2001-12-19 <160> 1 l0 <170> PatentIn version 3.1 <210> 1 <211> 692 <212> PRT <213> Homo sapiens <220> <221> GK <222> (229)..(692) <223> <300> <308> Genbank U13852 <309> 1994-12-13 <313> (1)..(228) <400> 1.

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys T_p Arg Asn Lys Lys Phe Glu Leu 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 55 60 Leu Thr Gln Ser Net Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 105 110 lO Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 120 125 Met Leu Lys Net Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 135 140 Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 170 175 Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 185. 190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Ile Glu Gly 210 215 220 Arg Gly Ile His Met Pro Arg Pro Arg Ser Gln Leu Pro Gln Pro Asn 225 230 235 240 Ser Gln Val Glu Gln Ile Leu Ala Glu Phe Gln Leu Gln Glu Glu Asp 245 250 255 Leu Lys Lys Val Met Arg Arg Met Gln Lys Glu Met Asp Arg Gly Leu \ 260 265 270 Arg Leu Glu Thr His Glu Glu Ala Ser Val Lys Met Leu Pro Thr Tyr 275 280 285 Val Arg Ser Thr Pro Glu Gly Ser Glu Val Gly Asp Phe Leu Ser Leu 290 295 300 Asp Leu Gly Gly Thr Asn Phe Arg Val Met Leu Val Lys Val Gly Glu 305 310 315 320 Gly Glu Glu Gly Gln Trp Ser Val Lys Thr Lys His Glo Met Tyr Ser 325 330 ' 335 lO Ile Pro Glu Asp Ala Net Thr Gly Thr Ala Glu Met Leu Phe Asp Tyr 340 345 350 Ile Ser Glu Cys Ile Se Asp Phe Leu Asp Lys His Gln Met Lys His 355 360 365 Lys Lys Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Val Arg His Glu 370 375 380 Asp Ile Asp Lys Gly Ile Leu Leu Asn Trp Thr Lys Gly Phe Lys Ala 385 390 395 400 Ser Gly Ala Glu Gly Asn Asn Val Val Gly Leu Leu Arg Asp Ala Ile 405 410 415 Lys Arg Arg Gly Asp Phe Glu Met Asp Val Val Ala Net Val Asn Asp 420 425 430 Thr Val Ala Thr Met Ile Ser Cys Tyr Tyr Glu Asp His Gln Cys Glu 435 440 44S Val Gly Met Ile Val Gly Thr Gly Cys Asn Ala Cys Tyr Met Glu GLu 450 455 460 Met Gln Asn Val Glu Leu Val Glu Gly Asp Glu Gly Arg Net Cys Val 465 470 475 480 Asn Thr Glu Trp Gly Ala Phe Gly Asp Ser Gly Glu Leu Asp Glu Phe 485 490 495 Leu Leu Glu Tyr Asp Arg Leu Val Asp Glu Ser Ser Ala Asn Pro Gly 500 505 510 Gln Gln Leu Tyr Glu Lys Leu Ile Gly Gly Lys Tyr Met Gly Glu Leu 515 520 525 Val Arg Leu Val Leu Leu Arg Leu Val Asp Glu Asn Leu Leu Phe His 530 535 540 Gly Glu Ala Ser Glu Gln Leu Arg Thr Arg Gly Ala Phe Glu Thr Arg 545 550 555 560 lO Phe Val Ser Gln Val Glu Ser Asp Thr Gly Asp Arg.Lys Gln Ile Tyr 565 570 575 Asn Ile Leu Ser Thr Leu Gly Leu Arg Pro Ser Thr Thr Asp Cys Asp 580 585 590 Ile Val Arg Arg Ala Cys Glu Ser Val Ser Thr Arg Ala Ala His Met 595 600 605 Cys Ser Ala Gly Leu Ala Gly Val Ile Asn Arg Met Arg Glu Ser Arg 610 615 620 Ser Glu Asp Val Met Arg Ile Thr Val Gly Val Asp Gly Ser Val Tyr 625 630 635 640 Lys Leu His Pro Ser Phe Lys Glu Arg Phe His Ala Ser Val Arg Arg 645 650 655 Leu Thr Pro Ser Cys Glu Ile Thr Phe Ile Glu Ser Glu Glu Gly Ser 660 665 670 Gly Arg Gly Ala Ala Leu Val Ser Ala Val Ala Cys Lys Lys Ala Cys 675 680 - 685 Met Leu Gly Gln

Claims (7)

1. Claims 1. The compound O:

   AH

   and pharmaceutically acceptable salts thereof.
2. 2. A pharmaceutical composition comprising the compound of Claim 1.
3. 3. The compound of Claim 1, for use in a method of treatment of human or animal body.
4. 4. A compound according to Claim 1 for use as a therapeutic active substance, in particular for the reduction of hyperglycemia in type 11 diabetes.
5. 5. Use of the compound of Claim 1 for the manufacture of a medicament for the treatment of hyperglycemia in type 11 diabetes.
6. 6. The novel compounds, compositions and uses as hereinbefore described.
7. 7. Any novel feature or combination of features described herein.