Patent Publication Number: US-2003232389-A1

Title: Urokinase peptide structure mimetics

Description:
[0001] The present invention concerns the use of the NMR structure of cy-clo[21,29][D-Cys21Cys29]-uPA 21-30 for the design of inhibitors that interfere with the binding of urokinase to its receptor, and it concerns peptidomimetics that imitate the binding mode of cyclo[21,29][D-Cys21Cys29]-uPA 21-30  to its receptor and therefore interfere with the binding of urokinase to its receptor.  
       [0002] Urokinase-type plasminogen activator (uPA) is a serine protease that is secreted as a single chain proenzyme. Limited proteolysis leads to the generation of the mature, two chain form of the enzyme, that catalyzes the conversion of the zymogen plasminogen to plasmin. Plasmin directs the degradation of the extracellular matrix either directly or indirectly via the activation of matrix metalloproteinases. Therefore, uPA plays a major role in matrix degradation, both in physiological and pathophysiological processes. In metastasis, uPA is an important factor, because it helps tumors to invade the surrounding tissue.  
       [0003] uPAR (uPA receptor) is a glycosyl-phosphatidylinositol (GPI) linked cell surface protein, that binds uPA with subnanomolar affinity. It recruits uPA to the cell surface. The importance of the uPA binding to uPAR for tumor spread has been demonstrated in many cases. Conversely, the addition of a recombinant solubable form of the receptor reduced the invasive capacity of ovarian cancer cells (Wilhelm et al., FEBS Lett. 337 (1994), 131-134).  
       [0004] As a result, uPA antagonists that block the interaction of uPA with its receptor can be used for the treatment of invasive tumors. Other indications for uPA antagonists include conditions such as arthritis, inflammation and osteoporosis. uPA antagonists can also be used as contraceptives.  
       [0005] A successful strategy to design uPA antagonists has built on the modular organisation of uPA. The molecule consists of (a) a growth factor domain (GFD, amino acids 1-44 and 46, respectively), (b) a kringle domain (amino acids 45 and 47, respectively, to 135), that together form the amino terminal fragment (ATF), and (c) a serine protease domain. It was found that ATF, and in particular residues 20-30 of the so-called loop B of GFD, compete efficiently with uPA for binding to uPAR.  
       [0006] Wilhelm et al. have investigated cyclic disulfide peptides that mimick this loop. Their studies identified cyclo[21,29][D-Cys21Cys29]-uPA 21-30  with an IC50 of 78 nM as a particularly promising drug lead (German patent application 199 33 701.2). Residues in this cyclic peptide cyclo[1,9] D-Cys-Asn-Lys-Tyr-Phe-Ser-Asn-Ile-Cys-Trp will be numbered sequentially, assigning residue number 1 to D-Cys. Thus, residue 1,2,3 . . . of the cyclic peptide correspond to residues 21,22,23, . . . in the ATF of uPA.  
       [0007] Although replacement of the Lys residue abolishes the susceptibility of the Lys-Tyr bond to the proteolytic action of plasmin (German patent application 199 33 701.2), it is expected that the peptide still suffers from some of the disadvantages of peptide drugs. These include lability against proteolysis in the stomach/intestine, low resorption if administered perorally, fast elimination by the liver and kidney and the risk of allergic reactions. Due to their conformational flexibility, peptide drugs and/or their metabolic products may interact with molecules other than their target molecules, leading to side effects that are both unwanted and hard to predict.  
       [0008] It is therefore an object of the present invention to provide inhibitor molecules that do not suffer from the above-mentioned disadvantages of the peptide lead compound and still maintain the affinity for uPAR.  
       [0009] This object is solved with the determination of the NMR solution structure of the lead compound, cyclo[21,29][D-Cys21Cys29]-uPA 21-30.  The procedure for structure determination is described in detail in Example 1 and the result is presented as a stereo representation of the molecule in FIG. 3 and as a coordinate file in FIG. 6.  
       [0010] It is a further object of the present investigations to provide molecules that mimick the lead compound cyclo[21,29][D-Cys21Cys29]-uPA 21-30 .  
       [0011] In an embodiment of the invention, conformation stabilizing cycles such as  
                 
 
       [0012] are chosen for incorporation into the peptide, so that Ramachandran angles actually found in the lead peptide are enforced by the additional cycles (Gante, Angew.Chemie 1994, 106:1780-1802). In another preferred embodiment, conformationally constrained amino acid analogs are used to limit space (Gibson, S. E., Guillo, N., Toser, M. J., Tetrahedron 1999, 55:585-615) to regions actually used by the cyclic peptide and identified as part of this invention (see FIG. 4).  
       [0013] In another embodiment of the invention, β-turn mimetics  
                 
 
       [0014] (Gante, J., Angew.Chemie 1994, 106:1780-1802; Böhm, H. J., Klebe, G., Kubinyi H., Wirkstoffdesign, Spektrum Adamischer Verlage, Gannis, A., Kolter, T., Angew. Chemie 1993, 105:1303-1326) are chosen to replace the type Iβ-turn forming tetrapeptides Asn-Lys-Tyr-Phe and/or Phe-Ser-Asn-Ile.  
       [0015] In a preferred embodiment β-turn mimetics that allow the attachment of side chains in positions i+1 and i+2 are used. Such scaffolds are for example the β-D-glucose scaffold (Nicolaou et al., Pept. Chem. Struct. Biol. Proc. Am. Pept. Symp. 11th, 1989 (1990), 881) or the cyclohexane scaffold (Olson et al., Proc. Biotechnol (USA), Conference Management Corporation, Norwalk, Conn., 1989, p.348).  
       [0016] In another embodiment of the invention, two subsequent residues with Rarnachandran angles typical of residues in an α-helical arrangement are replaced with α-helix inducing mimetics such as  
                 
 
       [0017] As shown in FIG. 5, such subsequent residues in cyclo[21,29][D-Cys21Cys-29]-uPA 21-30  are Lys3/Tyr4 and/or Ser6/Asn7.  
       [0018] In another embodiment of the invention, the polypeptide backbone is altered in such a way that the orientation of side chains is not substantially altered. Modifications include replacement of a peptide amido group with a ketomethylene, hydroxyethylene or ethylene group, leading to the formation of carbapeptide moieties in the molecule. The converse strategy, replacement of an α-carbon with a substituted nitrogen atom is equally possible and leads to the formation of azapeptide moieties. Azapeptides can be formed conviniently by condensing carboxyterminally acitivated azaamino acids.  
       [0019] In another embodiment of the invention, the two strategies of the preceding paragraphs are combined to form peptoid (Simon et al., Proc. Nat. Acad. Sci. USA 89, 9367 (1992) moieties. Peptoids contain nitrogen atoms instead Cα-atoms and carbon atoms instead of the α-amino nitrogen atoms, such that an NR—CO peptide-like bonded chain of N-alkylated glycines is formed.  
       [0020] The present invention additionaly concerns a pharmaceutical composition which contains at least one peptide or polypeptide or analogue thereof as defined above as the active substance, optionally together with common pharmaceutical carriers, auxilliary agents or diluents. The peptides or polypeptides according to the invention are used especially to produce uPA antagonists which are suitable for treating diseases associated with the expression of uPAR and especially for treating tumors.  
       [0021] An additional subject matter of the present invention is the use of peptides derived from the uPA sequence and in particular of uPA antagonists such as the above mentioned peptides and polypeptides to produce targeting vehicles e.g. liposomes, viral vectors etc. for uPAR-expressing cells. The targeting can be used for diagnostic applications to steer the transport of marker groups e.g. radioactive or non-radioactive marker groups. On the other hand, the targeting can be for therapeutic applications e.g. to transport pharmaceutical agents and for example also to transport nucleic acids for gene therapy.  
       [0022] The pharmaceutical compositions according to the invention can be present in any form, for example as tablets, as coated tablets or in the form of solutions or suspensions in aqueous and non-aqueous solvents. The peptides are preferably administered orally or parenterally in a liquid or solid form. When they are administered in a liquid form, water is preferably used as the carrier medium which optionally contains stabilizers, solubilizers and/or buffers that are usually used for injection solutions. Such additives are for example tartrate of borate buffer, ethanol, dimethyl sulfoxide, complexing agents such as EDTA, polymers such as liquid polyethylene oxide etc.  
       [0023] If they are administered in a solid form, then solid carrier substances can be used such as starch, lactose, mannitol, methyl cellulose, talcum, highly dispersed silicon dioxide, high molecular weight fatty acids such as stearic acid, gelatin, agar, calcium phosphate, magnesium stearate, animal and vegetable fats or solid high molecular polymers such as polyethylene glycols. The formulations can also contain flavourings and sweeteners if desired for oral administration.  
       [0024] The therapeutic compositions according to the invention can also be present in the form of complexes e.g. with cyclodextrins such as γ-cyclodextrin.  
       [0025] The administered dose depends on the age, state of health and weight of the patient, on the type and severity of the disease, on the type of the treatment, the frequency of administration and the type of desired effect. The daily dose of the active compound is usually 0.1 to 50 mg/kilogramme body weight. Normally 0.5 to 40 and preferably 1.0 to 20 mg/kg/day in one or several doses are adequate to achieve the desired effects.  
       EXAMPLE 1  
       [0026] Abbreviations: SA, simulated annealing; MD, molecular dynamics; rMD, restrained molecular dynamics; fMD, free molecular dynamics; NOE, nuclear Overhauser enhancement; RMSD, root mean square deviation; uPA, urokinase-type plasminogen activator; ATF, amino-terminal fragment of uPA;  
       [0027] Materials and Methods  
       [0028] NMR Spectroscopy. All NMR spectra were acquired on a Bruker DMX600 spectrometer and processed using the X-WINNMR software. A set of 1D spectra was acquired at the following temperatures: 275 K, 276 K, 278 K, 280 K, 282 K, 284 K and 285 K. COSY and NOESY spectra were acquired in water with 1024 and 512 complex points in t2 and t1, respectively, performing 64 scans per increment. A mixing time of 80 ms was chosen for the NOESY. Water suppression was accomplished using WATERGATE. The E.COSY spectrum was recorded in D 2 O at a resolution of 4096(t2)*256(t1) complex points, with 48 scans per increment. All 2D spectra were recorded at 280 K.  
       [0029] NOE-Derived Distance Restraints. NOE crosspeaks were converted into distance restraints d NOE  according to their integrated volumes using the two-spin approximation. The lower and upper bound of each distance restraint was set to 0.9 d NOE  and 1.1 d NOE , respectively. The average intensity of NOEs between geminal methylen protons (corresponding to a distance of 1.8 Å) was used for calibration, Standard corrections for center averaging [1] were applied.  
       [0030] Coupling Constants.  3 J(H N H α ) were obtained from the COSY spectrum using the methodology pioneered by Kim and Prestegard  [2] .  3 J(H α H β ) were extracted from the E.COSY recorded in D 2 O.  
       [0031] Amide Proton Temperature Coefficients. Temperature dependencies of the backbone amide proton chemical shifts were calculated from the above temperature series of  1 H-1D experiments.  
       [0032] Structure Calculations. Structure calculations consisted of a two-step procedure involving conformational space sampling followed by refinement of the obtained three-dimensional structure. In vacuo conformational space sampling was performed with the X-PLOR 3.5 program [3]  employing a standard simulated annealing (SA) protocol.  [4,5]  A random conformation with optimized covalent bond geometries was used as the initial structure for all calculations. NOE-derived distances as well as  3 J(H N H α ) coupling constants were employed as restraints. Ten low-energy conformations out of a total of 20 generated structures were selected for analysis of the agreement with the NMR-derived restraints. A structural representative of the ensemble of low-energy structures was then chosen and refined in extensive molecular dynamics (MD) simulations. To this end, the representative was placed in a 35 Å cubic simulation cell soaked with water molecules. The simulation cell was then energy-minimized and slowly heated up to the target temperature of 280 K. After equilibration, 200 ps restrained MD (rMD) were performed. Solely NOE-derived distances were employed, acting as time averaged distance restraints  [6-9]  with a memory decay time of r=20 ps.  [9]  To obtain average properties, the above simulation protocol was carried out twice, starting from different initial velocities. Finally, one MD simulation was resumed in absence of restraints to probe the stability of the structure (free MD, fMD). All MD simulations were performed with the DISCOVER 98 program (Molecular Simulations Inc.) using a home-written C program handling the time averaging of distance restraints.  
       [0033] Results and Discussion  
       [0034] Nomenclature. For sake of clarity residues of cyclo[21,29][D-Cys 21 ,Cys 29 ] uPA 21-30  will be numbered from 1 through 10 in the following, while for the corresponding residues of the ATF of uPA the original numbering scheme is retained.  
       [0035] NMR Assignments. The  1 H chemical shifts (Table 1) were assigned from analysis of the COSY and NOESY spectra. In the first step of the assignment procedure, frequencies of non-aromatic protons of each of the amino acid spin systems were determined using the COSY spectrum. Next, frequencies of aromatic protons were obtained from the NOESY spectrum. To this end, the chain of strong NOEs between adjacent protons in each aromatic side chain was traced, starting from the H β  protons. Finally, the sequential order of the amino acid spin systems was determined using characteristic H α   i -H N   i+1 -NOEs as well as interresidue side-chain NOEs. A comparison of the obtained  1 H chemical shifts with the corresponding random coil values (Wüthrich, K., NMR of Proteins and Nucleic Acids, Wiley, N.Y., 1996) reveals a considerable upfield shift for Lys 3  (random coil chemical shifts are given in parentheses; H β : 1.33, 1.45 (1.76, 1.85); H γ : 0.54, 0.79 (1.45); H δ : 1.24 (1.70)) and Ile 8  (γCH 3 : 0.42 (0.95), δCH 3 : 0.48 (0.89)) side-chain protons, which is due to aromatic ring systems adjacent in space (see Structure section).  
       [0036] NMR-Derived Structure Parameters. A total of 110 unambiguous NOE-derived distance restraints was obtained from analysis of the NOESY spectrum, including 30 nontrivial intraresidue, 40 sequential, 25 short-range (|i−j|&lt;5, where i and j are residue numbers), and 15 long-range (|i−j|≧5) NOEs. Due to signal overlap in the 2D NOESY spectrum, a considerable amount of structural information is lost (see similarity of chemical shift values given in Table 1). A histogram of the NOE restraints for each residue is shown in FIG. 1. Aside from NOE-derived distances, nine  3 J(H N H α ) (Table 2) and an almost complete set of  3 J(H α H β ) (Table 3) coupling constants were obtained from analysis of the COSY and E.COSY spectra. NOESY signal overlap and/or averaged  3 J(H α H β ) coupling constants due to side-chain rotation (Table 3) did not allow for diastereotopic assignment of H β . In addition to NOE distances and vicinal coupling constants, temperature dependencies of the chemical shifts from six out of a total of nine backbone amide protons were obtained from the temperature series of 1D spectra.  
       [0037] Conformational Space Sampling. Only one family of backbone conformations was observed during conformational space sampling in vacuo using X-Plor (average backbone RMSD 0.6 Å from the family representative for residues 2 through 8). As already mentioned in the above paragraph, a considerable amount of signals in the 2D NOESY spectrum overlap, giving rise to ambiguous distance restraints. However, ambiguous distance restraints cannot be treated in the current version of the DISCOVER program which is used for subsequent refinement. To probe whether the set of ambiguous distance restraints influences the convergence of the X-Plor runs, three-dimensional structures were generated with and without incorporation of ambiguous distance restraints. The results are virtually identical (backbone RMSD between structural representatives 0.5 Å for residues 2 through 8). Thus, the set of unambiguous distance restraints already contains the principal structural information. Therefore, only unambiguous distance restraints were employed in the refinement stage.  
       [0038] Structural Refinement. The single structural representative obtained during conformational space sampling was refined in the course of 200 ps rMD simulations. To obtain average properties, two simulations were performed, starting from the same system configuration but different initial velocities. Both rMD simulations lead to similar results (backbone RMSD between energy-minimized average structures 0.3 Å for residues 2 through 8). To probe the stability of the rMD structure, one simulation was resumed in absence of restraints for another 200 ps (fMD). An inspection of the Ramachandran plots of the fMD trajectory (not shown) reveals that the rMD conformation is retained, a finding which is confirmed by the backbone RMSD between the energy-minimized average structures of both simulations (0.9 Å for residues 2 through 8).  
       [0039] According to analysis of the joint rMD trajectories (in the following denoted as rMD trajectory), the average violation of NOE-derived distance restraints is 0.1 Å with no single distance restraint violated by more than 0.5 Å. Although coupling constants were not employed as restraints in the refinement stage,  3 J(H N H α ) calculated from the rMD trajectory are close to their experimental values (Table 2). Deviations by more than 2 Hz can be explained in terms of the steep gradient of the corresponding Karplus curve at φ=−80±30° (curve not shown). Similar considerations apply for  3 J(H α H β ). Despite the fact that no diastereotopic assignment of H β  was possible, a comparison of calculated versus experimental values of  3 J(H α H β ) yields similar pairings (Table 3), suggesting that the side-chain rotamer distribution is correctly reproduced by the rMD trajectory. Deviations occur for Tyr 4 , Ser 6 , Asn 7  and Trp 10 . In case of Ser 6 , no NOE-derived distance restraints are available due to signal overlap. Therefore, the calculated rotamer distribution merely reflects the force-field preferences. This is also true for Asn 7 , where NOEs to the H β  are present, but, due to the fact that the DISCOVER program cannot handle pseudo atoms under periodic boundary conditions, act on the C β  atom, thereby eliminating their influence on the X 1  rotamer distribution. Deviations of the experimental  3 J(H α H β ) values of Tyr 4  and Trp 10  will be discussed in conjunction with the three-dimensional structure of the molecule (see Section Structure and Dynamics). Temperature dependancies of backbone amide proton chemical shifts are in good agreement with the corresponding amide proton solvent accessibilities calculated from the rMD trajectory (FIG. 2).  
       [0040] Structure and Dynamics of cyclo[21,29][D-Cys 21 ,Cys 29 ]uPA 21-30 . The three-dimensional structure of the molecule is characterized by a hydrophobic cluster on one side of the ring, involving residues Tyr 4 , Phe 5 , Ile 8  and Trp 10 , and two type βI turns centered at Lys 3 , Tyr 4  and Ser 6 , Asn 7 , respectively (FIG. 3).  
       [0041] All hydrophobic residues (Tyr 4 , Phe 5 , Ile 8  and Trp 10 ) participate in the formation of a hydrophobic cluster. Ile 8  is found at the core of the cluster, with its side chain being shielded from the aqueous environment by the phenyl ring of Phe 5  and the indole moiety of Trp 10 . This finding is consistent with the distinct upfield shift observed for the chemical shifts of the methyl groups of the isoleucine side chain, suggesting these methyls to be located above the plane of aromatic ring systems (see section NMR Assignment). However, the nature of the hydrophobic cluster is not as static as FIG. 3 might suggest. As can be seen in FIG. 4, Ile 8  displays remarkable flexibility around X 1 . According to one larger and one smaller  3 J(H α H β ) value (Table 3), Tyr 4  partially adopts the g −  and t rotamer, while in the rMD simulation only the g −  rotamer is populated (FIG. 4), allowing for the formation of a hydrophobic cluster with Phe 5  (FIG. 3). In contrast, the g −  rotamer enables a hydrophobic interaction with the methylens of the lysine side chain, a feature also found in the corresponding ω loop in the NMR solution structure of the ATF of uPA. [10]  The resulting spatial arrangement would still be consistent with the observed NOEs between the side chains of Lys 3  and Tyr 4  and could also account for the distinct upfield shift of the β, γ and δ protons of the lysine side chain (see section NMR Assignment). In case of Trp 10 , the experimental evidence (both  3 J(H α H β ) around 7.0 Hz, upper bound of H α -H 2  distance restraint violated) also indicates side-chain rotation, albeit not reproduced in the rMD simulation (FIG. 4). Rotation around X 1  would bring the indole ring of Trp 10  in a position comparable to that observed for its counterpart in the solution structure of the ATF. Obviously, the chosen time averaging regime for NOE-derived distance restraints using a memory decay time T of 20 ps [9]  does not allow for side-chain rotational fluctuations large enough to correctly reproduce the experimental  3 J(H α H β ) values.  
       [0042] In addition to a hydrophobic cluster, the molecule also displays regular secondary structure. A type βI turn (ideal φ,ψ dihedral values: −60°, −30° (i+1 position) and −90°, 0° (i+2 position)) [11,12]  is centered at Lys 3  and Tyr 4  (FIG. 5, FIG. 3). The corresponding (i,i+3) hydrogen bond is not populated to an appreciable extent, a phenomenon also encountered in 25% of the β-turns found in protein structures. [13]  The turn is stabilized by a sidechain-backbone hydrogen bond between Asn 2 O δ1  and the amide proton of Tyr 4 , forming another turn-like structure known as “Asx turn”. [14]  In addition, Asn 2 O δ1  hydrogen-bonds to Phe 5 H N , providing a rationale for the weakly populated (i,i+3) hydrogen bond of this βI turn (Table 4). Another type βI turn is centered at Ser 6  and Asn 7 , with the corresponding (i,i+3) hydrogen bond between Phe 5 CO and Ile 8 H N  populated in more than half of the rMD simulation time (Table 4). An equally populated hydrogen bond between Ser 6 O γ  and Asn 7 H N  stabilizes the ψ i+1  angle of this turn (Table 4). In the course of the rMD simulation, the Phe5-Ser 6  amide bond rotates (FIG. 5), giving rise to a weakly populated type γ turn centered at Ser 6  (Table 4) with the φ i+1  angle stabilized by an additional sidechain-backbone hydrogen bond between Phe 5 CO and Ser 6 H γ  (Table 4). The φ,ψ pairs of this turn are close to their ideal values (70°, −70°). [11,12]  The observed arrangement of two consecutive type βI turns is additionally stabilized by a strongly populated hydrogen bond between Asn 2 H N  and Ile 8 CO (Table 4).  
       [0043] Agreement with statistically determined β-turn positional preferences. The large body of experimental information on the three-dimensional structure of proteins available in the Brookhaven Protein Data Bank [15]  has enabled conformational and positional preferences of residues to be statistically determined. [16-20]  Using a nonhomologous dataset of 205 protein chains, Hutchinson and Thornton derived β-turn positional potentials for the 20 naturally occuring amino acids. [20]  For position i of type βI turns, they found a strong preference for side chains that can act as hydrogen bond acceptors (Asn, Asp, Cys, Ser, His). These stabilize the turn by the formation of a hydrogen bond with the main-chain nitrogen of the i+2 residue. Thereby another turn-like structure known as “Asx turn”  [14]  arises, made up of the side chain and main chain of residue i, together with the main chains of residues i+1 and i+2. For the remaining positions of type βI turns, Hutchinson and Thornton found significant positional preferences for the following residues: i+1: Pro, Ser, Glu; i+2: Thr, Ser, Asn, Asp; i+3: Gly. Indeed, an “Asx turn” is observed for the type βI turn centered at Lys 3  and Tyr 4  of cyclo[21,29][D-Cys 21 ,Cys 29 ]uPA 21-30 , bearing Asn 2  in position i (see section Structure and Dynamics). However, none of the other residues of this βI turn (Lys 3  in i+1, Tyr 4  in i+2, and Phe 5  in i+3 position) displays significant propensity to appear in its respective position. In contrast, Ser 6  and Asn 7  in i+1 and i+2 position, respectively, of the second βI turn are in perfect agreement with the statistically derived preferences (see above). Ser 6 O γ  hydrogen-bonds to Asn 7 H N , thereby stabilizing the ψ i+1  angle. As for position i+2, an analysis of high-resolution protein structures shows that Asn, along with Asp, Ser and Thr, is more likely to adopt the backbone conformation required for this position (φ=−90°, ψ=0°). [21]   
       [0044] Comparison with Solution Structure of Amino-Terminal Fragment of uPA. Cyclo[21,29][D-Cys 21 ,Cys 29 ]uPA 21-30  and the ATF of uPA display similar binding characteristics with respect to the uPA receptor (uPAR). Thus, similar orientations of residues critical for receptor binding can be expected. These residues comprise Tyr 24 , Phe 25 , Ile 28 , and Trp 30  within the ω loop of ATF [22] and the corresponding residues Tyr 4 , Phe 5 , Ile 8 , and Trp 10  in our cyclic peptide, as determined by alanine replacements. Superposition with the solution structure of ATF  [10]  reveals that residues Tyr 24  (Tyr 4  in the cyclic peptide), Phe 25  (Phe 5 ), and Ile 28  (Ile 8 ) indeed adopt indentical positions and orientations relativ to each other (RMSD between C α -C β  vectors of corresponding tyrosine, phenylalanine and isoleucine residues 0.6 Å, see also FIG. 6). Trp 30  (Trp 10 ), however, is found in different orientations in both uPAR ligands. In the cyclic peptide, Trp 10  is located outside the cyclic backbone of the peptide, which confers considerable conformational flexibility to this C-terminal residue. Thus, Trp 10  can participate in the formation of the observed hydrophobic cluster, together with Tyr 4 , Phe 5  and Ile 8 . Upon receptor binding, however, its conformational flexibility enables Trp 10  to bring its indole in a position comparable to that found in the ATF. Interestingly, the presence of Phe and Trp seperated by five residues in sequence is among the essential features of uPAR binding peptide antagonists identified by phage display technology. [23]  The consensus sequence derived from these linear peptides is XFXXYLW. The importance of proper spacing is further corroborated by the experimental finding that insertion of either Gly or β-Ala between Phe and Trp results in loss of antagonist function. [24]  Furthermore, a manual alignment of our peptide and the above consensus sequence reveals the hydrophobic residues Ile 8  and the consensus Tyr to be located in equivalent positions. Thus, formation of a hydrophobic cluster between Phe and Ile (Tyr), as observed for our peptide, as well as an appropriately spaced Trp seem to constitute preconditions for high affinity binding to uPAR.  
       [0045] Besides the above hydrophobic residues, substitution of Ser 6  by Ala also results in weaker binding to uPAR. This observation can be explained in terms of the structure-stabilizing effect of the serine residue by sidechain-backbone hydrogen bonds, as described in section Structure and Dynamics.  
               TABLE 1                            1 H chemical shifts [ppm] of cyclo[21,29][D-Cys 21 ,Cys 29 ]-uPA 21-30  in water at 280 K. a                                               Residue   H N     H α     H β     H γ     H δ     H ε     misc.               D-Cys 1     —   3.81   2.62/3.26   —   —   —   —       Asn 2     8.57   4.51   2.62/2.79   —   6.99/7.38   —   —                                                     Lys 3     8.74   3.73   1.33/1.45   0.54/0.79       1.24   2.57   7.32   (HN ε )                                                 Tyr 4     8.03   4.16   2.52/2.62   —   —   —   6.86   (H 2,6 )                                   6.57   (H 3,5 )       Phe 5     7.59   4.61   2.46/3.12   —   —   —   6.99   (H 2,6 )                                   7.06   (H 3,5 )                                             Ser 6     8.40   3.91   3.70/3.79   —   —   —   —       Asn 7     8.00   3.97   2.34/2.86   —   6.79/7.48   —   —                                                 Ile 8     7.42   3.76   1.56   0.91/1.16   (CH 2 )   0.48                               0.42   (CH 3 )                                             Cys 9     8.31   4.58   2.74/3.01   —   —   —   —                                                 Trp 10     7.97   4.47   2.98/3.08   —   —   —   9.76   (H 1 )                                   6.91   (H 2 )                                   7.27   (H 4 )                                   6.77   (H 5 )                                   6.77   (H 6 )                                   7.05   (H 7 )                          
 
       [0046]               TABLE 2                            3 J(H N H α ) of cyclo[21,29][D-Cys 21 ,Cys 29 ]-uPA 21-30  in water       at 280 K. NMR-derived values and the corresponding values       calculated from the rMD trajectory are given.  3 J(H N H α )       were not employed as restraints during the rMD simulation.                                 Residue     3 J(H N H α ) exp       3 J(H N H α ) calc                                               Asn 2     9.1   7.1 ± 2.3           Lys 3     7.1   5.3 ± 2.0           Tyr 4     11.3   8.0 ± 1.9           Phe 5     11.9   9.7 ± 1.3           Ser 6     8.7   3.9 ± 3.2           Asn 7     9.1   6.5 ± 2.5           Ile 8     8.6   5.6 ± 2.4           Cys 9     8.7   9.6 ± 1.1           Trp 10     9.4   8.8 ± 1.7                        
       [0047]               TABLE 3                            3 J(H α H β ) of cyclo[21,29][D-Cys 21 ,Cys 29 ]-uPA 21-30  in water       at 280 K. NMR-derived values and the corresponding values       calculated from the rMD trajectory are given. Due to side-       chain rotation or NOESY signal overlap no diastereotopic       assignment could be made.  3 J(H α H β ) were not employed as       restraints during the rMD simulation.                                 Residue     3 J(H α H β ) exp       3 J(H α H β )calc                                                     D-Cys 1     4.5,   10.2   9.2 ± 4.3   (proS)                       4.6 ± 1.7   (proR)           Asn 2     4.6,   9.2   12.1 ± 1.6   (proS)                       3.1 ± 0.9   (proR)           Lys 3     6.3,   6.4   7.8 ± 5.0   (proS)                       4.6 ± 2.4   (proR)           Tyr 4     6.0,   10.3   3.8 ± 1.2   (proS)                       3.5 ± 1.2   (proR)           Phe 5     6.4,   9.0   3.1 ± 1.7   (proS)                       11.8 ± 2.5   (proR)                                     Ser 6     both ca. 7.0 (overlapped)   2.6 ± 0.7   (proS)                                                     5.1 ± 1.3   (proR)           Asn 7     7.4,   7.8   12.0 ± 1.1   (proS)                       2.4 ± 0.7   (proR)           Ile 8     6.8        6.9 ± 4.5           Cys 9     5.3,   9.5   8.8 ± 4.2   (proS)                       5.1 ± 4.6   (proR)           Trp 10     6.5,   7.5   3.0 ± 1.0   (proS)                       6.0 ± 3.5   (proR)                        
       [0048]               TABLE 4                          Populations of hydrogen bonds of cyclo[21,29][D-Cys 21 ,Cys 29 ]-       uPA 21-30  in water at 280 K calculated from the rMD trajectory a                                   donor   acceptor   population                       Asn 2 H N     Ile 8 CO   76           Asn 2 H N     Ser 6 CO   23           Lys 3 H N     Asn 2 O δ1     42           Tyr 4 H N     Asn 2 O δ1     60           Phe 5 H N     Asn 2 O δ1     52           Ser 6 H N     Tyr 4 CO   14           Ser 6 HO γ     Phe 5 CO   10           Asn 7 H N     Ser 6 O γ     49           Asn 7 H N     Phe 5 CO   14           Ile 8 H N     Phe 5 CO   59           Trp 10 H N     Ile 8 CO   13                                    
       [0049]                                               TABLE 5                          ATOM   1   N   CYS   1   23.523   11.953   18.425   N       ATOM   2   CA   CYS   1   23.062   13.252   18.958   C       ATOM   3   C   CYS   1   21.585   13.483   18.552   C       ATOM   4   O   CYS   1   20.678   12.784   19.019   O       ATOM   5   CB   CYS   1   23.252   13.289   20.488   C       ATOM   6   SG   CYS   1   22.725   14.883   21.147   S       ATOM   7   1H   CYS   1   23.021   11.171   18.860   H       ATOM   8   2H   CYS   1   24.524   11.803   18.593   H       ATOM   9   3H   CYS   1   23.374   11.885   17.413   H       ATOM   10   HA   CYS   1   23.713   14.040   18.528   H       ATOM   11   1HB   CYS   1   24.309   13.117   20.767   H       ATOM   12   2HB   CYS   1   22.664   12.494   20.985   H       ATOM   13   N   ASN   2   21.356   14.487   17.688   N       ATOM   14   CA   ASN   2   19.992   14.928   17.286   C       ATOM   15   C   ASN   2   19.459   14.098   16.077   C       ATOM   16   O   ASN   2   20.213   13.751   15.160   O       ATOM   17   CB   ASN   2   20.072   16.450   16.982   C       ATOM   18   CG   ASN   2   18.746   17.206   16.792   C       ATOM   19   OD1   ASN   2   17.679   16.832   17.284   O       ATOM   20   ND2   ASN   2   18.801   18.316   16.078   N       ATOM   21   H   ASN   2   22.201   14.959   17.348   H       ATOM   22   HA   ASN   2   19.316   14.803   18.158   H       ATOM   23   1HB   ASN   2   20.612   16.974   17.794   H       ATOM   24   2HB   ASN   2   20.709   16.604   16.093   H       ATOM   25   1HD2   ASN   2   17.920   18.827   15.955   H       ATOM   26   2HD2   ASN   2   19.714   18.549   15.668   H       ATOM   27   N   LYS   3   18.143   13.809   16.086   N       ATOM   28   CA   LYS   3   17.468   12.989   15.036   C       ATOM   29   C   LYS   3   17.537   13.619   13.608   C       ATOM   30   O   LYS   3   18.126   13.016   12.707   O       ATOM   31   CB   LYS   3   16.015   12.678   15.502   C       ATOM   32   CG   LYS   3   15.273   11.590   14.686   C       ATOM   33   CD   LYS   3   13.783   11.403   15.047   C       ATOM   34   CE   LYS   3   13.507   10.961   16.499   C       ATOM   35   NZ   LYS   3   12.077   10.675   16.708   N       ATOM   36   H   LYS   3   17.625   14.200   16.880   H       ATOM   37   HA   LYS   3   18.005   12.020   14.997   H       ATOM   38   1HB   LYS   3   16.029   12.352   16.559   H       ATOM   39   2HB   LYS   3   15.416   13.608   15.501   H       ATOM   40   1HG   LYS   3   15.322   11.843   13.609   H       ATOM   41   2HG   LYS   3   15.806   10.625   14.784   H       ATOM   42   1HD   LYS   3   13.239   12.343   14.836   H       ATOM   43   2HD   LYS   3   13.359   10.657   14.349   H       ATOM   44   1HE   LYS   3   14.101   10.063   16.752   H       ATOM   45   2HE   LYS   3   13.818   11.750   17.208   H       ATOM   46   1HZ   LYS   3   11.769   9.875   16.145   H       ATOM   47   2HZ   LYS   3   11.872   10.459   17.689   H       ATOM   48   3HZ   LYS   3   11.491   11.474   16.443   H       ATOM   49   N   TYR   4   16.958   14.821   13.423   N       ATOM   50   CA   TYR   4   16.972   15.552   12.126   C       ATOM   51   C   TYR   4   18.303   16.239   11.688   C       ATOM   52   O   TYR   4   18.450   16.486   10.488   O       ATOM   53   CB   TYR   4   15.732   16.489   12.011   C       ATOM   54   CG   TYR   4   15.605   17.804   12.830   C       ATOM   55   CD1   TYR   4   15.897   17.873   14.199   C       ATOM   56   CD2   TYR   4   15.027   18.917   12.206   C       ATOM   57   CE1   TYR   4   15.599   19.021   14.929   C       ATOM   58   CE2   TYR   4   14.728   20.064   12.939   C       ATOM   59   CZ   TYR   4   15.010   20.111   14.301   C       ATOM   60   OH   TYR   4   14.677   21.218   15.035   O       ATOM   61   H   TYR   4   16.517   15.217   14.261   H       ATOM   62   HA   TYR   4   16.792   14.782   11.349   H       ATOM   63   1HB   TYR   4   15.629   16.730   10.935   H       ATOM   64   2HB   TYR   4   14.822   15.888   12.212   H       ATOM   65   HD1   TYR   4   16.336   17.041   14.723   H       ATOM   66   HD2   TYR   4   14.773   18.898   11.154   H       ATOM   67   HE1   TYR   4   15.817   19.054   15.988   H       ATOM   68   HE2   TYR   4   14.256   20.901   12.448   H       ATOM   69   HH   TYR   4   14.748   21.000   15.967   H       ATOM   70   N   PHE   5   19.255   16.535   12.601   N       ATOM   71   CA   PHE   5   20.570   17.136   12.237   C       ATOM   72   C   PHE   5   21.699   16.228   12.809   C       ATOM   73   O   PHE   5   21.830   16.066   14.025   O       ATOM   74   CB   PHE   5   20.683   18.606   12.731   C       ATOM   75   CG   PHE   5   19.648   19.636   12.221   C       ATOM   76   CD1   PHE   5   19.300   19.710   10.864   C       ATOM   77   CD2   PHE   5   19.051   20.526   13.123   C       ATOM   78   CE1   PHE   5   18.352   20.629   10.427   C       ATOM   79   CE2   PHE   5   18.115   21.456   12.680   C       ATOM   80   CZ   PHE   5   17.762   21.504   11.334   C       ATOM   81   H   PHE   5   19.024   16.289   13.570   H       ATOM   82   HA   PHE   5   20.681   17.175   11.134   H       ATOM   83   1HB   PHE   5   20.685   18.599   13.838   H       ATOM   84   2HB   PHE   5   21.683   18.987   12.451   H       ATOM   85   HD1   PHE   5   19.753   19.045   10.142   H       ATOM   86   HD2   PHE   5   19.314   20.508   14.172   H       ATOM   87   HE1   PHE   5   18.077   20.662   9.382   H       ATOM   88   HE2   PHE   5   17.656   22.137   13.381   H       ATOM   89   HZ   PHE   5   17.028   22.218   10.991   H       ATOM   90   N   SER   6   22.500   15.622   11.912   N       ATOM   91   CA   SER   6   23.473   14.549   12.270   C       ATOM   92   C   SER   6   24.681   14.973   13.162   C       ATOM   93   O   SER   6   24.844   14.411   14.248   O       ATOM   94   CB   SER   6   23.898   13.794   10.987   C       ATOM   95   OG   SER   6   24.543   14.644   10.042   O       ATOM   96   H   SER   6   22.276   15.833   10.934   H       ATOM   97   HA   SER   6   22.909   13.802   12.863   H       ATOM   98   1HB   SER   6   24.574   12.955   11.238   H       ATOM   99   2HB   SER   6   23.018   13.327   10.503   H       ATOM   100   HG   SER   6   23.863   15.240   9.717   H       ATOM   101   N   ASN   7   25.501   15.956   12.731   N       ATOM   102   CA   ASN   7   26.610   16.522   13.562   C       ATOM   103   C   ASN   7   26.149   17.380   14.792   C       ATOM   104   O   ASN   7   26.812   17.346   15.834   O       ATOM   105   CB   ASN   7   27.587   17.286   12.617   C       ATOM   106   CG   ASN   7   28.971   17.655   13.200   C       ATOM   107   OD1   ASN   7   29.544   16.946   14.027   O       ATOM   108   ND2   ASN   7   29.555   18.758   12.754   N       ATOM   109   H   ASN   7   25.235   16.372   11.831   H       ATOM   110   HA   ASN   7   27.175   15.659   13.969   H       ATOM   111   1HB   ASN   7   27.787   16.669   11.718   H       ATOM   112   2HB   ASN   7   27.082   18.193   12.226   H       ATOM   113   1HD2   ASN   7   30.478   18.976   13.145   H       ATOM   114   2HD2   ASN   7   29.042   19.302   12.052   H       ATOM   115   N   ILE   8   25.018   18.109   14.682   N       ATOM   116   CA   ILE   8   24.388   18.875   15.799   C       ATOM   117   C   ILE   8   23.851   17.907   16.913   C       ATOM   118   O   ILE   8   23.318   16.832   16.618   O       ATOM   119   CB   ILE   8   23.300   19.830   15.170   C       ATOM   120   CG1   ILE   8   23.944   20.995   14.350   C       ATOM   121   CG2   ILE   8   22.286   20.404   16.187   C       ATOM   122   CD1   ILE   8   23.000   21.854   13.490   C       ATOM   123   H   ILE   8   24.569   18.049   13.762   H       ATOM   124   HA   ILE   8   25.170   19.522   16.245   H       ATOM   125   HB   ILE   8   22.699   19.224   14.473   H       ATOM   126   1HG1   ILE   8   24.511   21.656   15.032   H       ATOM   127   2HG1   ILE   8   24.705   20.583   13.661   H       ATOM   128   1HG2   ILE   8   22.792   21.032   16.942   H       ATOM   129   2HG2   ILE   8   21.507   21.016   15.701   H       ATOM   130   3HG2   ILE   8   21.738   19.610   16.729   H       ATOM   131   1HD1   ILE   8   22.260   22.403   14.099   H       ATOM   132   2HD1   ILE   8   23.572   22.612   12.924   H       ATOM   133   3HD1   ILE   8   22.443   21.249   12.756   H       ATOM   134   N   CYS   9   23.995   18.332   18.186   N       ATOM   135   CA   CYS   9   23.537   17.555   19.367   C       ATOM   136   C   CYS   9   22.612   18.431   20.257   C       ATOM   137   O   CYS   9   23.085   19.261   21.041   O       ATOM   138   CB   CYS   9   24.777   17.030   20.126   C       ATOM   139   SG   CYS   9   24.310   16.032   21.558   S       ATOM   140   H   CYS   9   24.448   19.248   18.285   H       ATOM   141   HA   CYS   9   22.977   16.653   19.045   H       ATOM   142   2HB   CYS   9   25.424   17.860   20.475   H       ATOM   143   1HB   CYS   9   25.404   16.402   19.463   H       ATOM   144   N   TRP   10   21.287   18.212   20.147   N       ATOM   145   CA   TRP   10   20.289   18.723   21.125   C       ATOM   146   CB   TRP   10   19.858   20.208   20.932   C       ATOM   147   CG   TRP   10   19.268   20.673   19.587   C       ATOM   148   CD1   TRP   10   17.996   20.331   19.072   C       ATOM   149   CD2   TRP   10   19.770   21.626   18.709   C       ATOM   150   NE1   TRP   10   17.707   21.016   17.880   N       ATOM   151   CE2   TRP   10   18.815   21.818   17.675   C       ATOM   152   CE3   TRP   10   20.961   22.400   18.735   C       ATOM   153   CZ2   TRP   10   19.049   22.773   16.657   C       ATOM   154   CZ3   TRP   10   21.170   23.334   17.717   C       ATOM   155   CH2   TRP   10   20.230   23.514   16.693   C       ATOM   156   C   TRP   10   19.102   17.737   21.221   C       ATOM   157   OT1   TRP   10   18.533   17.327   20.183   O       ATOM   158   OT2   TRP   10   18.722   17.377   22.358   O       ATOM   159   HN   TRP   10   21.026   17.528   19.428   H       ATOM   160   HA   TRP   10   20.763   18.692   22.126   H       ATOM   161   1HB   TRP   10   20.732   20.840   21.169   H       ATOM   162   2HB   TRP   10   19.134   20.474   21.727   H       ATOM   163   HD1   TRP   10   17.297   19.666   19.558   H       ATOM   164   HE1   TRP   10   16.867   20.943   17.297   H       ATOM   165   HE3   TRP   10   21.702   22.270   19.511   H       ATOM   166   HZ2   TRP   10   18.325   22.939   15.875   H       ATOM   167   HZ3   TRP   10   22.081   23.915   17.709   H       ATOM   168   HH2   TRP   10   20.425   24.240   15.918   H                    
       [0050] List of atomic coordinates in units of 0.1 nm. Column 2 indicates atom number, column 3 atom name, column 4 residue type, column 5 residue number, column 6,7,8 the x,y,z coordinates and column 9 indicates atom type.  
       [0051] 1 K. Wüthrich, M. Billeter, W. Braun,  J. Mol. Biol.  1983, 169, 949-961.  
       [0052] 2 Y. Kim, J. H. Prestegard,  J. Magn. Reson.  1989, 84, 9-13.  
       [0053] 3 A. T. Brünger  X - PLOR Manual.  Version 3.1.; Yale University Press: Cambridge, Mass., 1992.  
       [0054] 4 M. Nilges, G. M. Clore, A. M. Gronenborn,  FEBS Lett.  1988, 239, 129-136.  
       [0055] 5 M. Nilges, J. Kuszewski, A. T. Brünger, Sampling properties of simulated annealing and distance geometry. In  Computational aspects of the study of biological macromolecules by nuclear magnetic resonance spectroscopy,  J. C. Hoch, F. M. Poulsen and C. Redfield (Hrsg.), Plenum Press, New York, N.Y., 1991, 451-61.  
       [0056] 6 Torda, R. M. Scheek, W. F. van Gunsteren,  Chem. Phys. Lett.  1989, 157, 289-294.  
       [0057] 7 Torda, R. M. Scheek, W. F. van Gunsteren,  J. Mol. Biol.  1990, 214, 223-230.  
       [0058] 8 D. A. Pearlman, P. A. Kollmann,  J. Mol. Biol.  1991, 220, 457-79.  
       [0059] 9 A. P. Nanzer, W. F. van Gunsteren, A. E. Torda,  J. Biomol. NMR  1995, 6, 313-320.  
       [0060] 10 A. P. Hansen, A. M. Petros, R. P. Meadows, D. G. Nettesheim, A. P. Mazar, E. T. Olejnizak, R. X. Xu, T. M. Perderson, J. Henkin, S. W. Fesik,  Biochemistry  1994, 33, 4847-4864.  
       [0061] 11 G. Mueller, M. Gurrath, M. Kurz, H. Kessler,  Proteins  1993,. 15, 235-51.  
       [0062] 12 J. S. Richardson,  Adv. Protein Chem.  1981, 34, 167-339.  
       [0063] 13 P. N. Lewis, F. A. Momany, H. A. Scheraga,  Biochem. Biophys. Acta  1973, 303, 211-29.  
       [0064] 14 D. C. Rees, M. Lewis, W. N. Lipscomb,  J. Mol. Biol.  1983, 168, 367-87.  
       [0065] 15 F. C. Bernstein, T. F. Koetzle, G. J. Williams, E. J. Meyer, M. D. Brice, J. R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi,  J. Mol. Biol.  1977, 112, 535-42.  
       [0066] 16 M. Levitt,  J. Am. Chem. Soc.  1978, 17, 4277-85.  
       [0067] 17 T. M. Gray, B. W. Matthews,  J. Mol. Biol.  1984, 175, 75-81.  
       [0068] 18 J. M. Thornton, B. L. Sibanda, M. S. Edwards, D. J. Barlow,  Bioessays  1988, 8, 63-9.  
       [0069] 19 C. M. Wilmot, J. M. Thornton,  J. Mol. Biol.  1988, 203, 221-32.  
       [0070] 20 E. G. Hutchinson, J. M. Thornton,  Protein Sci.  1994, 3, 2207-16.  
       [0071] 21 R. A. Laskowski, M. W. Macarthur, D. S. Moss, J. M. Thornton,  Appl. Crystallogr.  1993, 26, 283-91.  
       [0072] 22 M. Bürgle, M. Koppitz, C. Riemer, H. Kessler, B. König, U. H. Weidle, J. Kellermann, F. Lottspeich, H. Graeff, M. Schmitt, L. Goretzki, U. Reuning, O. Wilhelm, V. Magdolen.  Biol. Chem.  1997, 378:231-237  
       [0073] 23 R. J. Goodson, M. V. Doyle, S. E. Kaufman, S. Rosenberg,  Proc. Natl. Acad. Sci. U.S.A.  1994, 91, 7129-33.  
       [0074] 24 M. Ploug, S. Ostergaard, L. B. L. Hansen, A. Holm, K. Dano,  Biochemistry  1998, 37, 3612-22.  
     
    
    
     [0075]FIG. 1: Histogram of NOE-derived distance restraints per residue. Intraresidue (black), short-range (gray; |i−j|&lt;5, where i and j are residue numbers of participating residues) and long-range (white; |i−j|&gt;5) NOEs are given.  
     [0076]FIG. 2: Radial distribution functions g(r) of water oxygens around backbone amide protons. A steep rise of g(r) at r=2.0 Å, as observed for Lys 3 , Ser 6 , and Cys 9 , indicates solvent exposition of the respective amide proton, allowing for the formation of hydrogen bonds with the solvent The gradual rise of g(r) seen in the plots for Asn 2 , Phe 5 , and Ile 8  results from shielding of the respective amide proton from solvent, accomplished by intramolecular hydrogen bonds or vicinity of side chains. Experimentally determined temperature dependances of the amide proton chemical shifts (Δδ/ΔT [−ppb/K], see plots) correlate well with the calculated radial distribution functions.  
     [0077]FIG. 3: Stereoview of cyclo[21,29][D-Cys 21 ,Cys 29 ]UPA 21-30 . Different atom types are shown in the following manner hydrogen (small white spheres), carbon (large white spheres), nitrogen (black spheres), oxygen (gray spheres). The three-dimensional structure is characterized by a hydrophobic cluster involving Tyr 4 , Phe 5 , Ile 8 , and Trp 10 , and two type βI turns centered at Lys 3 , Tyr 4  and Ser 6 , Asn 7 , respectively.  
     [0078]FIG. 4: χ 1  angles in the course of the two 200 ps rMD simulations starting from different initial velocities. Each plot is split by a vertical line, displaying the data of simulation 1 and simulation 2 on the left-hand and the right-hand side, respectively.  
     [0079]FIG. 5: Ramachandran plots generated from the two 200 ps rMD simulations starting from different initial velocities.  
     [0080]FIG. 6: Comparison of the NMR solution structures of the ATF of uPA and cyclo[21,29][D-Cys 21 ,Cys 29 ]uPA 21-30 . C α -C β  vectors of Tyr 4 , Phe 5  and Ile 8  of the peptide were superimposed on the corresponding protein residues (RMSD of C α ,C β  atoms after superposition: 0.6 Å).