Patent Application
20050260730
This application claims priority to application number 0324465.4 filed Oct. 20, 2003 filed in the United Kingdom. The contents of the aforementioned application is hereby incorporated by reference.
The present invention relates to a method of forming a crystal. More specifically, the invention relates to a method of forming a crystal of CDK2/cyclin A and a cyclin binding groove ligand. The invention further relates to novel crystal complexes of CDK2/cyclin A with various peptide oligomers.
Human cancer cells are characterised by the loss of cell cycle checkpoint regulation, leading to cell proliferation under conditions where non-transformed cells cannot enter and pass through the cell cycle. CDKs and their natural inhibitors, the CDK tumour suppressor proteins (CDKIs), are central to cell cycle regulation and their functions are commonly altered in tumour cells (Ortega et al., 2002). Deregulation of CDK2 and CDK4 through inactivation of CDKIs such as p16INK4a, p21WAF1, p27KIP1, and p57KIP2, thus provides a means for cancer cells to override the G1 checkpoint. Reinstatement of CDK inhibition therefore represents an opportunity for pharmacological interference with tumour progression. This is particularly attractive as one consequence of unchecked CDK2 activity is persistent E2F transcriptional activity, which, unless terminated in a timely fashion during S-phase, constitutes a powerful apoptotic signal (Lees and Weinberg, 1999). Because E2F deactivation occurs through phosphorylation by CDK2/CA, tumour cells will be selectively sensitive to inhibition. One method of CDK2/ CA inhibition is through the use of peptides that block binding of both pRb and E2F to the CDK2/CA complex (Fischer and Lane, 2000).
Recognition of both of these substrates, as well as CDKIs, by the CDK2/CA complex depends on a shallow hydrophobic surface binding site in the cyclin A subunit, termed the cyclin groove (Schulman et al., 1998). Recognition of this groove is mediated through certain sequences, designated the CBM (Chen et al., 1996). This motif represents a consensus of the cyclin groove binding sequences found in a variety of CDK2/CA partner proteins, including the CDKIs, pRb proteins, E2F transcription factors, p53, HDM2, the CDK-activating kinase CDK7, etc. This sequence is commonly referred to as the RXL or ZRXL (SEQ. ID No: 7) motif, where Z and X denote basic residues (Fischer et al., 2003). Following a detailed study with peptides representing various sequences from cyclin A binding proteins, the definition of the motif has recently been refined (McInnes et al., 2003): ZRXLYY′ (SEQ. ID No: 8), where Y and Y′ are hydrophobic residues. Further studies have concluded that the cyclin binding motif from p21 is more accurately described as a cluster of hydrophobic residues covering the LYY′ portion of the sequence above (Wohlschlegel et al, 2001). However, other positions in the motif are seen and differences are observed in the relative importance of particular residues, most probably due to differences in experimental design.
When CBM peptides are conjugated to cellular delivery vectors (Fischer et al., 2001), the resulting peptide constructs cause cell cycle arrest and selective apoptosis in tumour cells in vitro (Ball et al., 1996; Bonfanti et al., 1997; Chen et al., 1999) and, as shown more recently, in vivo (Mendoza et al., 2003). Inhibition of CDK2 activity through blocking of substrate recruitment is also likely to provide kinase selectivity, which is more difficult to achieve with traditional ATP-competitive molecules (Fischer, 2001), since the cyclin-dependent substrate recognition, and hence phosphorylation, appears to be unique to CDKs 2 and 4.
A number of CDK2/CA complex X-ray crystal structures with CBM peptides have been reported. The first of these included a 68-residue p27KIP1 fragment containing the 30RNLFG34 (SEQ. ID No: 9) sequence (Russo et al., 1996). A later study used a peptide with the sequence 658RRLFGEDPPKE668 (SEQ. ID No: 10) from p107, a pRb-family protein (Brown et al., 1999). The most recent complexes reported included the peptides 376STSRHKKLMFK386 (SEQ. ID No: 11) (p53), 868PPKPLKKLRFD878 (SEQ. ID No: 12) (pRb), 25KPSACRNLFGP35 (SEQ. ID No: 13) (p27KIP1), 87PVKRRLDLE95 (SEQ. ID No: 14) (E2F), and 653AGSAKRRLFGE663 (SEQ. ID No: 15) (p107) (Lowe et al., 2002). However, to date there have been no reports of crystallographic complexes of CDK2/CA with various peptides having the p21WAF1 motif 155RRLIF159 (SEQ. ID No: 16).
A comparison of a series of octapeptides derived from the p21WAF1 CBM-containing sequences, as well as those found in p107, E2F1, and p27KIP1, showed (Zheleva et al., 2002) that the p21WAF1 55RRLIF159 (SEQ. ID No: 16) motif was the most effective in cyclin A inhibitor peptides. In this motif the Arg155 residue was demonstrated to contribute extensively to binding. The optimised bioactive peptide H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH2, (SEQ. ID No: 17) a low nanomolar CDK2/CA inhibitor (Zheleva et al., 2002), corresponds to the p21WAF1(152-159) sequence, with a Ser153-Ala mutation, which was found to enhance potency significantly. These observations rendered this sequence a good candidate for structural studies, as it provides the best template for the design of peptidomimetics.
The present invention relates to an approach for developing potent, smaller, and less peptidic inhibitors of CDK2/CA. In particular, the invention seeks to provide an improved method of preparing crystals complexes of CDK2/CA with p21WAF-derived peptide analogues. The invention further seeks to use X-ray crystallography in conjunction with in vitro activity results to provide a comprehensive description of the requirements of each position in the CBM and to define a series of design principles for the structure-guided design of small molecule cyclin groove inhibitors.
A first aspect of the invention provides a method of preparing a crystal comprising CDK2/cyclin A and a ligand, L, said method comprising the steps of:
Further aspects of the invention relate to a crystal obtainable by the above method, and the use thereof in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
Another aspect of the invention relates to the use of a crystal obtainable by the above method in an assay for identifying further ligands capable of binding to the cyclin binding groove of CDK2/cyclin A.
Yet another aspect of the invention relates to a crystal comprising CDK2/cyclin A and a cyclin binding groove peptide selected from the following:
Another aspect relates to a method of screening for a ligand capable of binding to the cyclin binding groove of CDK2/cyclin A, wherein said method comprises the use of a crystal according to the invention, or obtainable by the method according to the invention. The invention also relates to a ligand identified by such a method.
A further aspect relates to the use of a ligand identifed by said method in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
Another aspect relates to the use of a crystal according to the invention in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
As mentioned above, in one aspect, the invention relates to method of preparing a crystal comprising CDK2/cyclin A and a ligand, L.
Advantageously, the co-crystallisation of protein with a ligand, followed by “ligand exchange” provides a way of studying binding sites involved in protein-protein interactions that would otherwise be difficult to elucidate using conventional techniques such as “soaking” (for example, where native protein crystals are not readily obtainable, or where protein-recognition sites are occupied by neighbouring molecules in the crystal lattice), or “co-crystallisation”.
In particular, the present invention provides a general method of preparing crystals of CDK2/CA and a ligand, L, for complexes that are particularly difficult to prepare (or are indeed unobtainable) by standard techniques known in the art.
As used herein, the term “crystal” means a structure (such as a three dimensional (3D) solid aggregate) in which the plane faces intersect at definite angles and in which there is a regular structure (such as internal structure) of the constituent chemical species. Thus, the term “crystal” can include any one of: a solid physical crystal form such as an experimentally prepared crystal, a 3D model based on the crystal structure, a representation thereof such as a schematic representation thereof or a diagrammatic representation thereof, or a data set thereof for a computer.
In one aspect, the crystal is usable in X-ray crystallography techniques. Here, the crystals used can withstand exposure to X-ray beams used to produce a diffraction pattern data necessary to solve the X-ray crystallographic structure.
In one preferred embodiment, step (ii) comprises eluting the crystal obtained in step (i) with a solvent.
Preferably, step (iii) comprises soaking the crystal obtained in step (ii) with a solution of ligand, L.
In one preferred embodiment, steps (ii) and (iii) are carried out sequentially.
In an alternative preferred embodiment, steps (ii) and (iii) are carried out simultaneously.
Preferably, step (ii) comprises eluting said first crystal with a solvent to remove substantially all of said first ligand, L′.
The presently described method of ligand exchange is effective in the case where L has a stronger affinity for the cyclin binding groove than L′. Surprisingly, the presently described method is also effective in the case where L has a weaker affinity than L′. In other words, using the present method it is possible to replace a higher-affinity ligand with a lower-affinity ligand.
In a preferred embodiment, said first ligand, L′, and said second ligand, L, are different and are each capable of binding to the cyclin binding groove of CDK2/cyclin A.
The ligands of the present invention may be natural or synthetic. The term “ligand” also refers to a chemically modified ligand.
As used herein, the term “ligand” includes, but is not limited to, a compound which may be obtainable from or produced by any suitable source, whether natural or not. The ligand may be designed or obtained from a library of compounds which may comprise peptides, as well as other compounds, such as small organic molecules and particularly new lead compounds. By way of example, the ligand may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic ligand a semi-synthetic ligand, a structural or functional mimetic, a peptide, a peptidomimetic, a derivatised ligand, a peptide cleaved from a whole protein, or a peptides synthesised synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof), a recombinant ligand, a natural or a non-natural ligand a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof.
The ligand may also comprise “modified peptides”, which are peptides derived from unnatural amino acids. The term “unnatural amino acid” refers to a derivative of an amino acid and may for example include alpha and alpha-disubstituted amino acids, N-alkyl amino acids, lactic acid, halide derivatives of natural amino acids such as trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, p-I-phenylalanine, L-allyl-glycine, β-alanine, L-α-amino butyric acid, L-γ-amino butyric acid, L-α-amino isobutyric acid, L-ε-amino caproic acid, 7-amino heptanoic acid, L-methionine sulfone, L-norleucine, L-norvaline, p-nitro-L-phenylalanine, L-hydroxyproline, L-thioproline, methyl derivatives of phenylalanine (Phe) such as 4-methyl-Phe, pentamethyl-Phe, L-Phe (4-amino), L-Tyr (methyl), L-Phe (4-isopropyl), L-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxyl acid), L-diaminopropionic acid and L-Phe (4-benzyl).
The term “derivative” or “derivatised” as used herein includes the chemical modification of a ligand. Typical chemical modifications may include, for example, the replacement of a hydrogen by a halo group, an alkyl group, an acyl group or an amino group.
Once a ligand capable of interacting with a key amino acid residue in the cyclin binding groove has been identified, further steps may be carried out either to select and/or to modify compounds and/or to modify existing compounds, to modulate the interaction with the key amino acid residues.
By way of example, once a ligand has been optimally selected or designed, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analyzed for efficiency of fit to the cyclin binding groove by the same computer methods described above.
Preferably, positions for substitution are selected based on the predicted binding orientation of a test compound to the cyclin binding groove of CDK2/cyclin A. The ligand of the present invention or mimetics thereof may be produced using chemical methods to synthesize the ligand in whole or in part. For example, peptides can be synthesized by solid phase techniques, cleaved from the resin, and purified by preparative high performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York N.Y.). The composition of the synthetic peptides may be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure; Creighton, supra).
Direct synthesis of the ligand or mimetics thereof can be performed using various solid-phase techniques (Roberge J Y et al (1995) Science 269: 202-204) and automated synthesis may be achieved, for example, using the ABI 43 1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Additionally, the amino acid sequences obtainable from the ligand, or any part thereof, may be altered during direct synthesis and/or combined using chemical methods with a sequence from other subunits, or any part thereof, to produce a variant ligand.
In an alternative embodiment of the invention, the coding sequence of the ligand or mimetics thereof may be synthesized, in whole or in part, using chemical methods well known in the art (see Caruthers M H et al (1980) Nuc Acids Res Symp Ser 215-23, Horn T et al (1980) Nuc Acids Res Symp Ser 225-232).
Hence, the ligands may be chemically synthesised or they may be prepared using recombinant techniques.
In one preferred aspect, the ligand is prepared by the use of chemical synthesis techniques.
In another preferred aspect, the ligands of the present invention may be produced from host cells using recombinant techniques.
A wide variety of host cells can be employed for expression of the nucleotide sequences encoding the ligands of the present invention. These cells may be both prokaryotic and eukaryotic host cells. Suitable host cells include bacteria such as E. coli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the expression products to produce an appropriate mature polypeptide. Processing includes but is not limited to glycosylation, ubiquitination, disulfide bond formation and general post-translational modification.
In one embodiment of the present invention, the ligand may be a chemically modified ligand.
The chemical modification of a ligand and/or a key amino acid residue of the present invention may either enhance or reduce hydrogen bonding interaction, charge interaction, hydrophobic interaction, Van Der Waals interaction or dipole interaction between the ligand and the key amino acid residue(s) of the cyclin binding groove.
More preferably, said first ligand, L′, and said second ligand, L, are each cyclin binding groove inhibitors.
Preferably, the cyclin binding groove is defined by the structural coordinates of the following amino acid residues of human CDK2/cyclin A: Met210, Ile213, Leu214, Trp217, Leu253, Glu220, Val221, Ile281 and Gln254, or a homologue thereof.
As used herein, the term “structural co-ordinates” refer to a set of values that define the position of one or more amino acid residues with reference to a system of axes.
As used herein, the term “homologue” refers to the ligand binding domain of the cyclin binding groove, or a portion thereof, which may have deletions, insertions or substitutions of amino acid residues as long as the binding specificity of the molecule is retained. In this regard, deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the binding specificity of the ligand binding domain is retained.
In one particularly preferred embodiment, said first ligand, L′, and said second ligand, L, are each peptides.
More preferably, the peptides are p21WAF-derived peptide analogues.
Previous studies by the applicant delineated the components of the p21WAF1 CBM (Zheleva et al., 2002) and arrived at a complete description of the motif through both extensive residue replacements, as well as by molecular modelling (McInnes et al., 2003). It was proposed that residues C-terminal to the ZRXL (SEQ. ID NO: 7) motif are also critical for cyclin A binding and hence the motif would be better described by the sequence ZRXLYY′ (SEQ. ID NO: 8), where either Y and/or Y′ are hydrophobic residues. The studies described herein confirm this crystallographically and in addition, reveal that the ZRXLYY′ (SEQ. ID NO: 8) motif can be further truncated to RXLYY′ (SEQ. ID NO: 18), since the pentapeptide H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID NO: 2) retains considerable potency. In the present study, several CBM peptides, including those identified previously (McInnes et al., 2003), containing unnatural amino acids and which resulted in potency enhancements, were crystallised with CDK2/CA, and their interactions with the cyclin groove described. The structural interpretation of the potency differences between various CBM peptides illustrates the determinants of cyclin groove binding, and shows that small substitutions in the peptide structures can result in profound differences in activity and in the bound conformation.
The peptide residue numbering system used throughout this work is based on the minimally active pentapeptide sequence, where amino acid residues are denoted Arg1-Arg2-Leu3-Ile4-Phe5 (SEQ. ID NO: 16) and residues extending from the N- and C-termini are numbered −1 and 6, respectively. The cyclin groove components involved in binding specific residues are denoted similarly, e.g. site 1 for elements interacting with the residue at position (P1) of the peptide ligand.
In one especially preferred embodiment, said first ligand, L′, and said second ligand, L, are selected from:
wherein L and L′ are different.
In one particularly preferred embodiment, said first ligand, L′, is selected from:
In another particularly preferred embodiment, said second ligand, L, is selected from:
In one particularly preferred embodiment, said first ligand, L′, and said second ligand, L, are each pentamers.
Preferably, residues 1, 3 and 5 of ligand L are capable of interacting with the cyclin binding groove of CDK2/cyclin A.
More preferably, residue 1 of ligand L is capable of interacting with Glu220 of the cyclin binding groove of CDK2/cyclin A.
Even more preferably, residue 1 is Arg.
Preferably, residue 3 of ligand L is capable of interacting with Gln254, Trp217 and Leu 214 of the cyclin binding groove of CDK2/cyclin A.
More preferably, residue 3 is Leu.
In one preferred embodiment of the invention, the crystal is of space group P21212 1.
In one particularly preferred embodiment, ligand L is H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH2 (SEQ ID NO. 1) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=114.6 Å, c=157.0 Å.
In another particularly preferred embodiment, ligand L is H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ ID NO. 2) and said crystal comprises a unit cell having unit dimensions a=74.2 Å, b=113.9 Å, c=155.3 Å.
In yet another particularly preferred embodiment, ligand L is Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH2 (SEQ ID NO. 3) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=114.0 Å, c=156.2 Å.
In another particularly preferred embodiment, ligand L is H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH2 (SEQ ID NO. 4) and said crystal comprises a unit cell having unit dimensions a=73.5 Å, b=113.0 Å, c=153.1 Å.
In another particularly preferred embodiment, ligand L is H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH2 (SEQ ID NO. 5) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=113.5 Å, c=154.5 Å.
Another aspect of the invention relates to a crystal obtainable by the above described method.
A further aspect of the invention relates to the use of a crystal obtainable by the above-described method in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
As used herein the phrase “preparation of a medicament” includes the use of a crystal directly as the medicament in addition to its use in a screening programme for further therapeutic agents or in any stage of the manufacture of such a medicament.
Another aspect relates to the use of a crystal obtainable by the above-described method in an assay for identifying further ligands capable of binding to the cyclin binding groove of CDK2/cyclin A.
Yet another aspect of the invention relates to a crystal comprising CDK2/cyclin A and a cyclin binding groove peptide selected from the following:
Preferably, the crystal is of space group P212121.
Preferably, the cyclin binding groove is defined by the structural coordinates of the following amino acid residues of CDK2/cyclin A: Met210, Ile213, Leu214, Trp217, Leu253, Glu220, Val221, Ile281 and Gln254, or a homologue thereof.
Preferably, residues 1, 3 and 5 of the cyclin binding groove peptide are capable of interacting with the cyclin binding groove of CDK2/cyclin A.
In one preferred embodiment, residue 1 of the cyclin binding groove peptide is capable of interacting with Glu220 of the cyclin binding groove of CDK2/cyclin A.
In another preferred embodiment, residue 3 of the cyclin binding groove peptide is capable of interacting with Gln254, Trp217 and Leu214 of the cyclin binding groove of CDK2/cyclin A.
In one particularly preferred embodiment, ligand L is H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH2 (SEQ ID NO. 1) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=114.6 Å, c=157.0 Å.
In another particularly preferred embodiment, ligand L is H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ ID NO. 2) and said crystal comprises a unit cell having unit dimensions a=74.2 Å, b=113.9 Å, c=155.3 Å.
In another particularly preferred embodiment, ligand L is Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH2 (SEQ ID NO. 3) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=114.0 Å, c=156.2 Å.
In another particularly preferred embodiment, ligand L is H-Arg-Arg-Leu-Asn-(p-F-Phe)-NH2 (SEQ ID NO. 4) and said crystal comprises a unit cell having unit dimensions a=73.5 Å, b=113. Å, c=153.1 Å.
In another particularly preferred embodiment, ligand L is H-Cit-Cit-Leu-Ile-p-F-Phe)-NH2 (SEQ ID NO. 5) and said crystal comprises a unit cell having unit dimensions a=74.5 Å, b=113.5 Å, c=154.5 Å.
Yet another aspect relates to the use of a crystal according to the invention in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
Crystal Soaking and Ligand Exchange
Despite considerable efforts, crystals of uncomplexed CDK2/cyclin A could not be obtained in the required crystal form. Initially, published crystallisation conditions (Jeffrey et al., 1995) were employed. However, attempts to introduce peptides by soaking of these crystals were not successful and resulted in crystal lattice disorder and X-ray diffraction at a resolution of 5 Å at best.
All peptide complex structures presented here were solved using a novel crystal form, which has space group P212121 and contains two copies of the binary protein complex in the crystallographic asymmetric unit. Of the two peptide ligands in this complex, one (
Peptides 2 and 4 were co-crystallised with CDK2/cyclin A, while the remaining peptide complexes were prepared using the so-called “ligand exchange” procedure. A crystal containing H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID NO: 2) was soaked extensively against precipitant solution to elute the original peptide. Examination of electron density difference maps around the cyclin binding groove regions showed that the original co-crystallised peptide (
In general, the time required for ligand soaking experiments can vary between seconds and days. In systems where no protein rearrangement is necessary (e.g. dipeptide ligands soaked into crystals of cyclophilin), binding was found to take place within seconds (Wu et al., 2001). This contrasts with soaking times of days required for the binding of some CDK2 active site inhibitors, which require significant changes in the protein conformation before binding can occur (Wu et al., 2003).
Diffusion of peptides in and out of the CDK2/CA crystals was slow and in some cases, even after a period of three days of soaking in new peptide solution, the original peptide could still be observed to occupy the binding groove. However, occasional agitation of the crystallisation mixture during the three-day period led to complete ligand exchange. Solution studies for peptides binding to the CBG using a competitive binding assay (McInnes et al, 2003) demonstrated that equilibrium is reached within a few seconds. Buffer and salt conditions in these solution studies and in the crystal exchange experiments described here were very similar, apart from the high PEG concentrations in the crystal soaking experiments. It therefore seems that diffusion rates play a major role during ligand exchange in crystals. Fortuitously, the crystal system described has two ligand binding sites with different crystallographic environments, thereby leading to unambiguous results for the accessible site. The results with the cyclin-CDK2 system described herein suggest that the ligand exchange approach may be of general use in different protein systems where native protein crystals are not readily obtainable, or where protein-recognition sites are occupied by neighbouring molecules in the crystal lattice.
Interactions between Ligand Peptides and Cyclin A
The binding site for the CBM peptides is comprised of a shallow groove resulting from the exposed surface of the cyclin A α1, α3, and α4 helices. The residues lining the groove form a number of subsites including the LYY′ hydrophobic pocket (Met210, Ile213, Leu214, Trp217, Leu253), the arginine site (RX; Glu220), and a secondary lipophilic pocket we call the alanine pocket (Val221, Ile281). In addition, binding is also facilitated by a number of H-bond interactions with the peptide backbone, including a pair of interactions with the side-chain carboxamide of Gln254. Despite the well-documented observation that the CDK2 kinase subunit of the CDK2/CA complex undergoes significant conformational changes upon cyclin binding and T-loop phosphorylation, the cyclin subunit structure is essentially the same in the free and complexed forms (Brown et al., 1995). In our peptide complex structures, most of the cyclin A binding groove (CBG) atoms are rigid and their average B factors do not change upon ligand binding. This was confirmed by comparison of the temperature factors of the CBG atoms with the rest of the cyclin A chain, as well as by comparison of the unliganded and peptide-liganded CDK2/CA structures. All main chain atoms of the residues in the groove have low B factors in both the peptide-bound and unliganded structures, although there are some significant differences in the side chains of the hydrophilic residues of the binding groove.
All residues in all of the peptide ligands complexes presented here are well defined by electron density. The electron density difference maps of the peptide complexes are shown in
An overlay of all the solved peptide structures is shown in
Peptide Potency and Design
Analysis of peptide 2 complexed with cyclin A shows that the bound conformation is stabilised through a number of contacts with the CBG, involving the side chains of Arg1, Leu3, and Phe5, as well as the peptide backbone. (
P1 Site
A key interaction in the CBM, and a requirement for good inhibitory potency of relevant peptides, is the ion pair between Glu220 and Arg1. The importance of Arg in this position was also borne out in previous structures (Lowe et al., 2002) and mutation of Arg1 always results in a decrease in biochemical inhibitory activity (McInnes et al., 2003). Even the isosteric substitution of Arg1 with Cit1, i.e. replacement of the guanidine —NHC═NH(NH2) with an urea —NHC═O(NH2), leads to a significant decrease in potency. Replacement of the ω imino function in Arg with the carbonyl group in Cit results in a system with similar H-bonding ability but unlike the Arg guanidine, the Cit urea is not charged under physiological conditions and therefore does not participate in ion pair interactions. Analysis of the peptide 5 complex crystal structure shows that the distance between Cit1 NH and the Glu220 side chain carbonyl O is 5.1 Å, whereas the corresponding distance in the peptide 4 structure is 4.5 Å. This difference effectively results in loss of the contacts between Glu220 and the Cit-containing peptide. A similar picture emerges from comparison of the average cyclin A Glu220 side chain atom B factors in both the peptide 5 and peptide 2 complex structures. In the latter structure this factor lies only 20.5% above the average for all main chain atoms, whereas in the unliganded and peptide 5-liganded complexes the value rises to 45 and 67%, respectively. This is a clear indication of large displacement of Glu220 side chain atoms when peptide 5 binds in the CBG.
Another factor contributing to the decrease in activity is the loss of intramolecular contacts with Leu3. The peptide 5 2Fo-1Fc map shows an absence of density for the Leu3 side chain beyond the Cβ atom, indicating free rotation about the Cα-Cβ bond. By contrast, all atoms of the Leu3 side chain are clearly visible in the electron density difference maps for the peptide 2 and peptide 4 structures (
The effect of acetylation of the N-terminal nitrogen was also investigated. In the peptide 2 complex structure, the peptide amino terminus forms a H-bond (3 Å) with the carbonyl of Ile281 and a similar H-bond is also observed in peptide 4 and peptide 3 structures. The weakening of this H-bond (3.4 Å) upon acetylation of the peptide N-terminus is consistent with the reduced biological activity of acetylated peptides (Table 2). An additional interesting observation relating to this involves the Arg1 side chain, which cannot make the same contacts in the acetylated and non-acetylated peptides. In the acetylated peptide a series of contacts are observed that are absent in peptides with free N-termini and involve the acetyl O, a water molecule, the Glu220 side chain, as well as the NH group of the Arg1 side chain.
P2 Site
Analysis of the peptide activity data for different amino acid residues in this position suggests that it is comparatively tolerant of modification. This finding is supported by the complex structures, which do not show any strong interactions between the P2 side chain and the protein. In contrast to P1, replacement of the charged side chain at P2 has comparatively little effect on biological activity, as is apparent from the Arg2-Cit mutation. Inclusion of certain uncharged residues at P2 is also tolerated to some extent, e.g. Gln (Table 2) or Ser (McInnes et al., 2003). The observed 3-fold reduction in potency, however, is consistent with the decrease in the Coulombic interaction energies calculated upon substitution of the Arg2 with Asn.
P3 Site
The necessity of a hydrophobic side chain at this position, as indicated by the activity data, is obvious from all structures reported here. The Leu3 side chain participates in a network of van der Waals contacts with Gln254, Trp217, and Leu214 of cyclin A, in addition to several intramolecular contacts with P1 and P5 (
P4 Site
The major role of this residue (as encountered in the p21WAF1 and p53 CBMs), is to act as a linker between the P3 and P5 hydrophobic groups. Incorporation of a linker is favourable for binding and in the peptides tested improves potency relative to the p27KIP1 CBM, where the P4 residue is absent (Leu3-Phe5-Gly6). The extra 3 atoms of the Ile4 residue in the backbone structure allow P5 to adopt a more favourable position in the groove and make more complementary contacts with the hydrophobic pocket (
P5 Site
This site was probed with a variety of residues leading to the conclusion that certain aromatic side chains are optimal (McInnes et al., 2003). The most potent Phe derivatives were found to be para-substituted (Table 2). The crystal structures of three peptides in which P5 is m-Cl-Phe or p-F-Phe in complex with the cyclin A groove demonstrate that P5 always occupies the same site (
Computational Analysis
In an attempt to understand better the observed differences in affinity for p21WAF1 versus p27KIP1-derived CBM peptides, intermolecular non-bonded energetics were calculated using the X-ray complex structures of the peptide 2 (p21WAF1) and H-Arg-Asn-Leu-Phe-Gly-OH 4 (p27KIP1; 1H27.pdb) (SEQ. ID No: 9) peptide (Lowe et al., 2002) (Table 3). The results help to explain the individual contributions for the p21WAF1 and p27KIP1 CBM peptides in terms of binding energy and indicate that a major contribution to the observed difference in affinity is due to the presence of a second charged Arg at P2 in p21WAF1. Although this residue does not appear to make specific protein contacts, it does contribute significantly via Coulombic energy due to its proximity with Asp282. In addition, there also appears to be considerable contribution to binding from the surrounding solvent-mediated H-bonds. Whereas the p27KIP1 peptide structure (Russo et al., 1996) contains a single water molecule interacting with the peptide, we refined our structures to include as many crystallographically defined water molecules as could be found, especially in the binding groove, for the purpose of using these in drug design. Specifically, the energetic analysis suggests that a water-mediated H-bond from the Leu3 carbonyl to the Gln254 side chain provides 14 kcal/mol to the binding energy of the p21WAF1 sequence. We also carried out energy minimisation calculations on the bound conformations for peptide 2 and the H-Arg-Arg-Leu-Phe-NH2 (SEQ. ID No: 20) peptide in isolation from cyclin A. The energy difference between the minimised peptide conformation and that observed when bound to the CBG is an indication of the energetic penalty a peptide has to pay during binding. Since peptide 2 has to form non-optimal intramolecular contacts and torsion angles in order to form complementarity with the cyclin groove, there is an unfavourable enthalpic contribution to the overall free energy. This was shown from the observation that the energy difference between the bound and minimised bound peptides was much greater for the p27KIP1 (54 kcal/mol) versus the p21WAF1 sequence (17 kcal/mol). As the binding energetics are comparable for peptide 2 and H-Arg-Arg Leu-Phe-NH2, (SEQ. ID No: 13) this factor most likely explains the more than 10-fold potency decrease for the latter peptide. Those two peptides adopt a very similar conformation for the first three residues up to Leu3 (
Assay
A further aspect of the invention relates to an assay for screening one or more ligands capable of binding to the cyclin binding groove of CDK2/cyclin A, said assay comprising contacting a crystal as defined above, or obtainable by the above-described method, with a candidate ligand, L″, and detecting any change in the interaction between ligand L and CDK2/cyclin A.
In a preferred embodiment, the assay is a competitive binding assay.
Preferably, any change in the interaction between ligand L and CDK2/cyclin A can be monitored by detecting the displacement of ligand L from the CDK2/cyclin A/L complex and/or by monitoring formation of a complex comprising CDK2/cyclin A and candidate ligand L″.
More preferably, the assay is used to screen for a ligand useful as an inhibitor of CDK2/cyclin A.
Preferably, the assay is used to screen for a ligand useful in the prevention and/or treatment of proliferative disorder such as cancers, leukaemias and other disorders associated with uncontrolled cellular proliferation such as psoriasis and restenosis. Preferably, the proliferative disorder is a cancer or leukaemia. The term proliferative disorder is used herein in a broad sense to include any disorder that requires control of the cell cycle, for example cardiovascular disorders such as restenosis, cardiomyopathy and myocardial infarction, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema, alopecia, and chronic obstructive pulmonary disorder. In these disorders, the ligands of the present invention may induce apoptosis or maintain stasis within the desired cells as required.
The ligands of the invention may inhibit any of the steps or stages in the cell cycle, for example, formation of the nuclear envelope, exit from the quiescent phase of the cell cycle (G0), G1 progression, chromosome decondensation, nuclear envelope breakdown, START, initiation of DNA replication, progression of DNA replication, termination of DNA replication, centrosome duplication, G2 progression, activation of mitotic or meiotic functions, chromosome condensation, centrosome separation, microtubule nucleation, spindle formation and function, interactions with microtubule motor proteins, chromatid separation and segregation, inactivation of mitotic functions, formation of contractile ring, and cytokinesis functions. In particular, the compounds of the invention may influence certain gene functions such as chromatin binding, formation of replication complexes, replication licensing, phosphorylation or other secondary modification activity, proteolytic degradation, microtubule binding, actin binding, septin binding, microtubule organising centre nucleation activity and binding to components of cell cycle signalling pathways.
More preferably, the assay is used to screen for a ligand useful in the prevention and/or treatment of cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
Ligands which are identified using the crystal of the present invention can be screened in assays such as those well known in the art. Screening can be, for example, in vitro, in cell culture, and/or in vivo. Biological screening assays preferably centre on activity-based response models, binding assays (which measure how well a compound binds to the receptor), and bacterial, yeast and animal cell lines (which measure the biological effect of a compound in a cell). The assays can be automated for high capacity-high throughput screening (HTS) in which large numbers of compounds can be tested to identify compounds with the desired activity. The biological assay, may also be an assay for the ligand binding activity of a compound that selectively binds to the LBD compared to other nuclear receptors.
Another aspect of the invention relates to a process comprising the steps of:
Another aspect relates to a process comprising the steps of:
Another aspect of the invention relates to a process comprising the steps of:
Yet another aspect of the invention relates to a ligand identified by the above-described assay. Preferably, the identified ligand may act as a ligand model (for example, a template) for the development of other compounds.
The crystal structure of the present invention can be used to generate a structural model such as a three dimensional (3D) structural model (or a representation thereof). Alternatively, the crystal structure may be used to generate a computer model for the structure.
Thus, for example, the structural co-ordinates provided in the crystal structure and/or model structure may comprise the amino acid residues of the cyclin binding groove, or a portion thereof, or a homologue thereof useful in the modelling and design of test compounds or further ligands capable of binding to the cyclin binding groove.
As used herein the term “three dimensional model” includes both crystal structures as determined by X-ray diffraction analysis, solution structures determined by nuclear magnetic resonance spectroscopy as well as computer generated models. Such computer generated models may be created on the basis of a physically determined structure of a peptide of the present invention bound to cyclin A or on the basis of the known crystal structure of cyclin A, modified (by the constraints provided by the software) to accommodate a peptide of formula I. Suitable software suitable of the generation of such computer generated three dimensional models include AFFINITY, CATALYST and LUDI (Molecular Simulations, Inc.).
Such three dimensional models may be used in a program of rational drug design to generate further candidate compounds that will bind to cyclin A. As used herein the term “rational drug design” is used to signify the process wherein structural information about a ligand-receptor interaction is used to design and propose modified ligand candidate compounds possessing improved fit with the receptor site in terms of geometry and chemical complementarity and hence improved biological and pharmaceutical properties, such properties including, e.g., increased receptor affinity (potency) and simplified chemical structure. Such candidate compounds may be further compounds or synthetic organic molecules.
As used herein, the term “modelling” includes the quantitative and qualitative analysis of molecular structure and/or function based on atomic structural information and interaction models. The term “modelling” includes conventional numeric-based molecular dynamic and energy minimization models, interactive computer graphic models, modified molecular mechanics models, distance geometry and other structure-based constraint models.
In another aspect of the present invention, the structural coordinates comprising the cyclin binding groove, or portion thereof, and the ligand bound thereto, may be applied to a model screening system.
As used herein, the term “model screening system” may be a solid 3D screening system or a computational screening system. Using this model, test compounds may be modelled which fit spatially into the cyclin binding groove.
In one preferred aspect, the test compounds are positioned in the cyclin binding groove through computational docking.
In another preferred aspect, the test compounds are positioned in the cyclin binding groove through manual docking.
As used herein, the term “fits spatially” means that the three-dimensional structure of a ligand is accommodated geometrically in the cyclin binding groove.
Preferably, modelling is performed using a computer and may be further optimized using known methods. This is called modelling optimisation.
Overlays and super positioning with a three dimensional model of cyclin binding groove, and/or a portion thereof, can also be used for modelling optimisation. Additionally, alignment and/or modelling can be used as a guide for the placement of mutations on the cyclin binding groove to characterise the nature of the site in the context of a cell.
The three dimensional structure of a new crystal may be modelled using molecular replacement. The term “molecular replacement” refers to a method that involves generating a preliminary model of a molecule or complex whose structure coordinates are unknown, by orienting and positioning a molecule whose structure coordinates are known within the unit cell of the unknown crystal, so as best to account for the observed diffraction pattern of the unknown crystal. Phases can then be calculated from this model and combined with the observed amplitudes to give an approximate Fourier synthesis of the structure whose coordinates are unknown. This, in turn, can be subject to any of the several forms of refinement to provide a final, accurate structure of the unknown crystal. Lattman, E., “Use of the Rotation and Translation Functions”, in Methods in Enzymology, 115, pp. 55-77 (1985); M. G. Rossmann, ed., “The Molecular Replacement Method”, Int. Sci. Rev. Ser., No. 13, Gordon & Breach, New York, (1972).
Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., Cohen, N. C. et al, “Molecular Modelling Software and Methods for Medicinal Chemistry”, J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia, M. A. and M. A. Murcko, “The Use of Structural Information in Drug Design”, Current Opinions in Structural Biology, 2, pp. 202-210 (1992).
Using the structure coordinates of the crystal complexes provided by this invention, molecular replacement may be used to determine the structure coordinates of a crystalline mutant or homologue of the cyclin binding groove of CDK2/CA or of a related protein.
As used herein, the term “mutant” refers to any organism that has undergone mutation or that carries a mutant gene that is expressed in the phenotype of that organism. A mutation may arise due to a substitution of one nucleotide for another or from a deletion of a nucleotide or an insertion of a nucleotide relative to a referenced wild type sequence. These single nucleotide variations are sometimes referred to as single nucleotide polymorphisms (SNPs). Some SNPs may occur in protein-coding sequences, in which case, one of the polymorphic forms may give rise to the expression of a defective or other variant protein and, potentially, a genetic disease. Other SNPs may occur in noncoding regions. Some of these polymorphisms may also result in defective protein expression (e.g., as a result of defective splicing). Other SNPs may have no phenotypic effects.
Another aspect relates to the use of a ligand identified by the above-described assay in the preparation of a medicament for treating a proliferative disorder.
Another aspect relates to the use of a ligand identifed by the above-described assay in the preparation of a medicament for treating cancer, leukemia, a viral disorder, stroke, glomerulonephritis, alopecia, diabetes, a CNS disorder, rheumatoid arthritis, psoriasis or chronic obstructive pulmonary disorder.
Pharmaceutical Compositions
Another aspect of the invention relates to a pharmaceutical composition comprising a ligand identified by the above assay admixed with one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the ligands (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients, 2nd Edition, (1994), Edited by A Wade and P J Weller.
Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
Salts/Esters
The ligands identified by the assay of the invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
Pharmaceutically acceptable salts of the ligands identified by the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (C1-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen). Enantiomers/Tautomers
In all aspects of the present invention previously discussed, the invention includes, where appropriate all enantiomers and tautomers of ligands identified by the assay of the invention. The man skilled in the art will recognise compounds that possess an optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art.
Stereo and Geometric Isomers
Some of the ligands of the invention may exist as stereoisomers and/or geometric isomers—e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of all the individual stereoisomers and geometric isomers of those agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the ligand or pharmaceutically acceptable salts thereof. An isotopic variation of a ligand of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts threeof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, flourine and chlorine such as, 2H, 3H, 13C , 14C , 15N, 17O, 18O, 31P, 32P, 35S, 18F, and 36Cl respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
Solvates
The present invention also includes the use of solvate forms of the ligands of the present invention. The terms used in the claims encompass these forms.
Polymers
The invention furthermore relates to the ligands of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
Prodrugs
The invention further includes the ligands of the present invention in prodrug form. Such prodrugs are generally compounds wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
Administration
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 250 mg and more preferably from 10-100 mg, of active ingredient per dose.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10-1000 mg, preferably between 10-250 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
Dosage
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
By way of summary, the technique of ligand exchange described herein is an effective way of obtaining crystallographic information on series of compounds without having to depend on successful co-crystallisation. As has been highlighted, many proteins do not yield crystals in the absence of suitable ligands. In the case studied, the use of a suitable starting ligand allowed the formation of crystals, which were subsequently employed to provide structural information for numerous ligands. The results reported here suggest that the ligand exchange approach may be of general use in different protein systems where native protein crystals are not readily obtainable, for example when contacts of surface-bound ligands with neighbouring molecules are required for crystallisation.
The peptides contain residues that are not directly involved in binding but act as spacers and help to orient those groups involved in groove recognition. The charged amino terminus and side chain functions at P1 (Arg) are of prime importance for potency in the context of pentapeptide inhibitors. Replacement of the charged guanidine function with the uncharged isosteric urea group (Cit) and acetylation of the N-terminus lead to ca. 36-fold and 3.8-fold potency reductions, respectively. The importance of the P1 charged side chain is less pronounced in the octapeptide inhibitor series (McInnes et al., 2003), where the loss of the charge-charge interaction is partially offset by other favourable interactions not available in the pentapeptides. A second charged residue present at P2 in CBMs can be eliminated more readily, due to the lesser contribution to binding. Although the side chain of Arg2 does not form direct contacts with the cyclin groove, it clearly participates in long-range electrostatic interactions. Arg at P2 can be effectively replaced e.g. with Cit or Gln, and with other residues capable of both donating and accepting H-bonds. The Leu residue at P3 is invariant in all known CBMs and our results confirm that the Leu side chain optimally occupies the hydrophobic pocket at that site. The P4 residue serves predominantly as a linker and a steric constraint. As it does not form any direct interactions with the protein, it is a potential candidate for modifications designed to improve the physicochemical characteristics of future inhibitors. Finally, P5 is preferably an aromatic residue with a halogen-substituted phenyl group. The crystal structures and potency data indicate that occupying the volume filled by the halogen atoms makes important contributions to binding and hence should be accounted for in ligand design. Molecular modelling shows that there is sufficient space around this ring in the hydrophobic groove pocket to accommodate additional substituents, which could further increase the buried surface and hence the potency.
Overall it has been shown that peptide potency can be regained after truncation of the p21WAF1 octapeptide and that a molecule with a lower net charge can be obtained through incorporation of unnatural amino acids. The structural basis for these improvements was rationalised and suggests that the development of small molecule inhibitors of the CDK2/CA via the substrate-binding site is feasible. The important features of both the binding site and bound peptides incorporating non-natural residues found during this work should facilitate the design of more potent and less peptidic inhibitors.
The present invention is further described by way of Example and with reference to the following Figures, wherein:
Expression and Purification of CDK2, Cyclin A, and CDK2/CA Complex
Human recombinant CDK2 was expressed and purified as described (Wu et al., 2003). Human recombinant cyclin A2 (fragment encompassing residues 173-432) was expressed in Escherichia coli BL21 (DE3) using PET expression vectors. BL21 (DE3) was grown at 37° C. with shaking (200 r.p.m) to mid-log phase (A600nm˜0.6). Expression was induced by the addition of IPTG at a final concentration of 1 mM and the culture was incubated for a further 3 h. Bacteria were harvested by centrifugation, and the cell pellet was resuspended in buffer A (25 mM Tris pH 8.0, 5 mM DTT, 1 mM PMSF, 1 mM EDTA, 1 mM benzamidine, and protease inhibitor cocktail). After sonication the lysate was clarified by centrifugation for 30 min at 15,000 g and 4° C. The supernatant was passed through a DEAE-Sepharose column (pre-equilibrated with buffer B (25 mM Tris pH 8.0, 2 mM DTT, 1 mM PMSF, 1 mM EDTA, 1 mM benzamidine, and 10 mM NaCl). After washing, bound protein was eluted with an ascending concentration gradient of NaCl in buffer B. The fractions containing cyclin A (typically eluted at 300-400 mM NaCl) were pooled, diluted (5 times in buffer A without protease inhibitor cocktail), and loaded on to a pre-equilibrated (with buffer B) SP-Sepharose column. After washing, bound protein was eluted with a NaCl gradient in buffer B. The fractions containing cyclin A (typically eluted at 300-400 cmM NaCl) were pooled, diluted (5 times in buffer A without protease inhibitor cocktail) and loaded on to a pre-equilibrated (with buffer B) High-Trap Q column. After washing, bound protein was eluted with a NaCl gradient in buffer B. Pooled fractions containing pure cyclin A were mixed in a 1:1 molar ratio with CDK2 and were concentrated before loading on to a pre-equilibrated (with 50 mM Tris pH 8.2, 100 mM NaCl, 1 mM EDTA, 1 mM DTT) Superdex 75 SE column. Eluted fractions containing stoichiometric mixtures of CDK2 and cyclin A were used for crystallisation after concentration by ultrafiltration (Amicon concentration unit).
Crystallisation and Structure Determination
The CDK2/CA/peptide crystals were grown by vapour diffusion using the hanging drop method. The peptides H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID No: 2) and H-Arg-Arg-Leu-Ile-(p-F-Phe)-NH2 (SEQ. ID No: 4) were co-crystallised with CDK2/CA.
Typically, a 2-μL solution of CDK2/cyclin A (7-8 mg/mL in 40 mM HEPES pH 7.0, 200 mM NaCl, 5 mM DTT) and containing a 5-fold molar excess of peptide was mixed with 2 μL of well solution. The precipitant solution contained 18% PEG-3,350 and 100 mM trisodium citrate. Crystals were obtained after 1-2 weeks at 17° C. CDK2/CA complexes with Ac-Arg-Arg-Leu-Asn-(m-Cl-Phe)-NH2, (SEQ. ID No: 3) H-Cit-Cit-Leu-Ile-(p-F-Phe)-NH2, (SEQ. ID No: 5) and H-Ala-Ala-Abu-Arg-Ser-Leu-Ile-(p-F-Phe)-NH2 (SEQ. ID No: 1) were prepared by a ligand exchange technique using CDK2/CA/H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID No: 2) complex crystals. Initially the exchange technique was carried out in two steps. First, H-Arg-Arg-Leu-Ile-Phe-NH2 peptide (SEQ. ID No: 2) was eluted from the crystal after soaking in a solution containing 18% PEG-3,350 and 100 mM trisodium citrate. The unliganded crystal was then soaked in 18% PEG-3,350, 100 vmM trisodium citrate and 1 mM of one of the above peptide. Later experiments showed that elution of H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID No: 2) and soaking of a new peptide could be performed simultaneously. The final protocol consisted of soaking of CDK2/ CA/peptide A crystals in mother liquor plus 1 mM peptide B for three days. The soaking solution (˜10 μL) was replaced and the mixture stirred regularly during the soaking period. Under the experimental conditions used here, one crystal was soaked in a 10-μL drop of a 1 mM exchange peptide solution. This corresponds to less than 5 pmol of CDK2/CA mixed with 10 nmol of exchange peptide. Assuming a simple bimolecular equilibrium, the dissociation constant (Kc) between the ligand L and the protein P crystal is given by: Kc=([Punbound] [Lunbound])/[PL]. This can be simply related to crystallographic (Wu et al., 2001) occupancy (Q) by Kc=(1−Q)×[Lfree]/Q. Thus for Kc values between 1 and 100 μM, and assuming unrestricted access to the cyclin ligand binding sites in the crystal, crystallographic occupancies would be expected to be greater than 90%.
Crystals of about 0.05×0.1×0.1 mm were mounted in 0.05-0.1 mm cryo-loops (Hampton Research). The crystals were immersed briefly in a cryoprotectant (28% PEG-3,350 and 0.1 M trisodium citrate) and then flash-frozen in liquid nitrogen. Data were collected at the Daresbury (UK) and Grenoble (France) synchrotron facilities. Data processing was carried out using the program DENZO and SCALEPACK (Otwinowski and Minor, 1997), or MOSFLM (Leslie, 1992) and SCALA (Evans, 1993) from the CCP4 program suite (1994). Data often showed signs of crystal radiation damage. The structures were solved by molecular replacement using MOLREP (Vagin and Teplyakov, 1997) and PDB entries 1 HCL or 1 CKP as the search model. ARP/wARP (Lamzin and Wilson, 1997) was used for initial density interpretation and the addition of water molecules. REFMAC (Murshudov et al., 1997) was used for structural refinement. A number of rounds of refinement and model building with the program Quanta (Accelrys, San Diego, USA) were carried out.
Computational Chemistry
Calculation of protein ligand interaction energies for the cyclin A inhibitors was performed using the program CDISCOVER after addition of hydrogens and steepest-descent minimisation, using the modelling package InsightII (Accelrys, San Diego, USA).
Peptide Synthesis
Peptides were prepared, purified, and characterised using procedures as previously described (Zheleva et al., 2002).
Biochemical Assays
These were carried out as previously described (Atkinson et al., 2002; McInnes et al., 2003; Zheleva et al., 2002).
Accessions Numbers
Structures have been deposited in the Protein Data Bank under ID codes 1OKU, 1OKV, 1OKW, 1OL2 and 1OL1 for the CDK2/CA complexes with peptides 1-5.
Various modifications and variations of the described aspects of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes of carrying out the invention which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.
Zheleva, D. I., McInnes, C., Gavine, A.-L., Zhelev, N. Z., Fischer, P. M., and Lane, D. P. (2002). Highly potent p21WAF1 derived peptide inhibitors of CDK-mediated pRb phosphorylation: delineation and structural insight into their interactions with cyclin A. J. Peptide Res. 60, 257-270.
* Peptides with CDK2/CA complex structures.
Peptides:
1, H-Arg-Arg-Leu-Ile-Phe-NH2 (SEQ. ID No: 2);
2, H-Arg-Asn-Leu-Phe-Gly-OH (SEQ. ID No: 9);
3, H-Arg-Arg-Leu-Phe-NH2 (SEQ. ID No: 20).
*Water-bridged H-bond between the peptide Leu3 (CO) and Gln254 side chain (
| Number | Date | Country | Kind |
|---|---|---|---|
| 0324465.4 | Oct 2003 | GB | national |
