Proton Dissociation Energies of Zinc-Coordinated Hydroxamic Acids

and Research, 200 First Street SW, Rochester, Minnesota 55905. ReceiVed: April 3, 2000. Hydroxamic acids, known as iron chelators, have recently been ...
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J. Phys. Chem. B 2000, 104, 6499-6504

6499

Proton Dissociation Energies of Zinc-Coordinated Hydroxamic Acids and Their Relative Affinities for Zinc: Insight into Design of Inhibitors of Zinc-Containing Proteinases Jamal El Yazal and Yuan-Ping Pang* Mayo Clinic Cancer Center, Tumor Biology Program, Department of Molecular Pharmacology and Experimental Therapeutics, Molecular Neuroscience Program, Mayo Foundation for Medical Education and Research, 200 First Street SW, Rochester, Minnesota 55905 ReceiVed: April 3, 2000

Hydroxamic acids, known as iron chelators, have recently been widely used as a key functional group of potential therapeutics targeting at zinc proteinases such as matrix metalloproteinases involved in cancers and at other drug targets associated with cardiovascular diseases, AIDS, and Alzheimer’s disease. However, the protonation states of zinc-coordinated hydroxamic acids in proteins and relative affinities of hydroxamic acids for Zn2+ are still unclear due to the intricacy of the hydroxamic acid structures. Here, we report a comprehensive ab initio study of stable configurations and tautomers of neutral and deprotonated, Zn2+coordinated acetohydroxamic acid and its N-methyl analogue in the gas-phase employing the B3LYP/6311+G(2d,2p) method. The results suggest that both zinc-coordinated acetohydroxamic and N-methylacetohydroxamic acids exist in the oxygen-deprotonated Z-keto form with their two oxygen atoms coordinating to zinc in proteins in which the acidic amino acid side chains serve as a proton acceptor. This conclusion is consistent with a survey of experimentally determined protein 3D structures complexed with zinc-coordinated hydroxamic acids documented in the Protein Data Bank. The results also suggest that the zinc affinity of N-methylacetohydroxamic acid is 11 kcal/mol higher than that of acetohydroxamic acid and is up to 43 kcal/ mol higher than those of common zinc ligands in proteins. It thus cautions the use of N-methylacetohydroxamic acid as a functional group in rational design of inhibitors for zinc proteinases, since it may interact with other zinc proteins due to its high affinity for zinc.

Introduction Hydroxamic acids, best known as iron chelators,1 have recently been widely used as a key functional group of potential therapeutics targeted at zinc proteinases such as matrix metalloproteinases involved in cancers 2-22 and at other drug targets associated with cardiovascular diseases,23-25 AIDS,26-28 and Alzheimer’s disease.29 It has long been assumed that acetohydroxamic acid can exist in the keto and iminol forms and that each can be present in either the E or Z configuration about the C-N bond.30 Ab initio calculations suggest the E-keto form to be the most stable structure in the gas phase,31,32 while the X-ray crystallographic analysis of acetohydroxamic acid reveals the stable structure to be in the Z-keto form in the solid state.33 Recently, we have reported ab initio calculations of hydroxamic acids using the coupled-cluster method,34 suggesting that in the gas-phase acetohydroxamic and N-methylacetohydroxamic acids exist in the E- and Z-keto forms and the Z-iminol form that are in equilibrium, whereas the corresponding deprotonated acids exist in the nitrogen deprotonated Z-keto form and the Chydroxy oxygen deprotonated Z-iminol form that are in resonance. Due to the intricacy of acetohydroxamic acid structure, the protonation states of zinc-coordinated hydroxamic acids and relative affinities of different hydroxamic acids for zinc are still unclear. Most people reported that acetohydroxamic acid exists in the neutral form when coordinating to zinc in proteins3,12,35,22 except for one reasoned that it resembles carboxylic acid and exists in the deprotonated form when coordinating to zinc in proteins.36 It was thought that the more popular use of * To whom correspondence should be addressed. E-mail: [email protected].

acetohydroxamic acid as a functional group in drug design than N-methylacetohydroxamic acid was due to the higher affinity of acetohydroxamic acid for zinc.37 However, this is contradictory to the common knowledge that the electron donating effect of a methyl group should confer a higher affinity of the N-methyl analogue for zinc. Ab initio calculations of proton dissociation energies of hydroxamic acids and their affinities for zinc can help to resolve these controversies. Nonetheless, probably due to convergence problems that often occurred in ab initio calculations of zinc-coordinated hydroxamic acids, to the best of our knowledge, to date there has been no report of ab initio calculations of zinc-coordinated hydroxamic acids. As a prelude to our rational design of zinc proteinase inhibitors as potential antiangiogenic agents for treating cancers, we set out to investigate the energetically stable configurations and tautomers of neutral and deprotonated, zinc-coordinated acetohydroxamic acid and its N-methyl analogue in the gas phase in order to correctly estimate the proton dissociation energies of the two hydroxamic acids and their relative affinities for zinc. In comparison with our recently reported proton dissociation energies of common zinc ligands, some of which are known to be deprotonated in proteins,38 the calculated proton dissociation energies of hydroxamic acids will provide insights into their protonation states in proteins. The relative affinities of hydroxamic acids for zinc will provide a guide to structural optimizations of hydroxamic acid-containing drug leads. Here, we report a comprehensive ab initio study of stable configurations and tautomers of zinc-coordinated acetohydroxamic acid and its N-methyl analogue in the gas-phase employing the B3LYP/6-311+G(2d,2p) method.

10.1021/jp0012707 CCC: $19.00 © 2000 American Chemical Society Published on Web 06/17/2000

6500 J. Phys. Chem. B, Vol. 104, No. 27, 2000

El Yazal and Pang

TABLE 1: Energies (kcal/mol) of Zinc-Coordinated Hydroxamic Acids in Different Configurations and Tautomers method Aa

method Bb

method Cc

method Dd

structure

complex energy

relative energy

complex energy

relative energy

complex energy

relative energy

1 2 3 4 5 6 7 8 9 10

-219137 -219090 -219155 -219137 -219141 -219138 -219121 -219132 -219137 -219153

19 65 0 18 15 18 34 23 18 3

-1294590 -1294546 -1294607 -1294592 -1294599 -1294584 -1294584 -1294580 -1294596 -1294603

17 60 0 15 8 22 23 26 10 4

-1294597 -1294552 -1294613 -1294598 -1294606 -1294590 -1294591 -1294587 -1294604 -1294610

16 62 0 15 7 23 22 26 10 3

11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

-219017 -219021 -219061 -219016 -219007 -219028 -219042 -219042 -219005 -219009 -219009 -219019 -219010 -219010 -219013 -219037

44 40 0 45 53 33 19 19 56 52 52 42 51 51 48 24

-1294473 -1294486 -1294519 -1294477 -1294476 -1294487 -1294498 -1294498 -1294474 -1294478 -1294478 -1294476 -1294465 -1294465 -1294480 -1294500

46 33 0 42 43 32 21 21 45 41 41 43 54 54 39 19

-1294480 -1294492 -1294527 -1294483 -1294483 -1294494 -1294505 -1294505 -1294480 -1294485 -1294485 -1294484 -1294471 -1294471 -1294486 -1294507

47 35 0 44 44 33 23 23 47 42 42 43 56 56 41 20

27 28 29 30 31 32 33 34

-243810 -243766 -243830 -243762 -243815 -243828 -243830 -243810

20 64 0 68 16 2 0 21

-1319276 -1319234 -1319291 -1319235 -1319283 -1319289 -1319291 -1319280

16 57 0 56 8 3 0 12

-1319284 -1319241 -1319299 -1319242 -1319292 -1319297 -1319299 -1319288

16 59 0 57 8 3 0 11

35 36 37

-243688 -243694 -243728

40 35 0

-1319162 -1319170 -1319196

34 27 0

-1319169 -1319177 -1319205

37 29 0

a

b

c

complex energy

relative energy

-1294617

0

-1294609

8

-1294607 -1294613

10 4

-1294530

0

-1319302

0

-1319295 -1319299 -1319302 -1319292

7 3 0 10

-1319209

0 d

B3LYP/LANL2DZ//B3LYP/LANL2DZ. B3LYP/6-311+G(d,p)//B3LYP/LANL2DZ. B3LYP/6-311+G(2d,2p)//B3LYP/LANL2DZ. B3LYP/ 6-311+G(2d,2p)//B3LYP/6-311+G(2d,2p).

Methods The DFT calculations were carried out by using the Gaussian 98 program (revision A.7)39 running on one SGI Origin 2000 (8 × 195 MHz, 2.0 GB memory, and 40 GB disk) and four SGI Origin 200s (8 × 180 MHz, 1.2 GB memory, and 16 GB disk). Different structures of zinc-coordinated hydroxamic acids were generated with the Quanta program and optimized by energy minimizations with the CHARMm molecular mechanics force field.40 Further geometry optimizations and harmonic frequencies used to identify minimal energy geometries were computed by using Becke’s three-parameter formulation (B3LYP) density functional method41,42 with the LANL2DZ basis set.43 The relative single-point energies of different structures of zinccoordinated hydroxamic acids were obtained from the LANL2DZminimized geometries using the LANL2DZ, 6-311+G(d,p), and 6-311+G(2d,2p) basis sets.44 For the relatively energetically stable structures revealed by the calculations with the B3LYP/ 6-311+G(2d,2p)//B3LYP/LANL2DZ method, the relative energies of such structures were further calculated with the B3LYP/ 6-311+G(2d,2p)//B3LYP/6-311+G(2d,2p) method. The LANL2DZ basis set is developed to handle heavy atoms after the third row of the periodic table and treat the electrons near the nucleus in an approximate way via effective core potentials.45 The 6-311+G(d,p) basis set adds the d functions

as polarization functions to heavy atoms (labeled with “d”) and the p functions as polarization functions to hydrogen atoms (marked with “p”) in order to allow orbitals to change size and shape. The p functions for the hydrogen atoms are important for calculations when the hydrogen atoms are the sites of interest, as in our study of proton dissociation. It also adds the large-size versions of the s- and p-type functions as diffuse functions (noted with “+”) to heavy atoms to allow orbitals to occupy a larger region of space. The diffuse functions are necessary for molecules in our study with electrons relatively far from the nucleus, molecules with lone electron pairs and molecules with significant negative charges. Furthermore, it adds three extra sizes of s and p functions to heavy atoms in order to model heavy atoms such as zinc. The 6-311+G(2d,2p) basis set is the same as the 6-311+G(d,p) basis set except that it adds 2d functions to the heavy atoms and 2p functions to the hydrogen atoms to account for the polarization effect. All the three basis sets have been reported to be successful in computing the hydration energy of the zinc divalent cation.46 The quadratic convergence method (SCF ) QC)47 was used to solve numerous convergence problems encountered in energy minimizations. Proton dissociation energy was computed as the energy difference between the molecule of interest and the same molecule that was deprotonated. The zinc affinity was calculated

Zinc-Containing Proteinase Inhibitor Design

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Figure 1. Different tautomers and configurations of zinc-coordinated, neutral acetohydroxamic acid.

as the energy of the zinc complex minus the energies of the two partners in their free state. Both singlet- and triplet-state energies of each molecule in Table 1 were taken into account. Only the lower energy of the two was reported and used in the calculations of proton dissociation energies and the zinc affinities. The energy of each molecule was corrected with the thermal energy that was calculated by running a frequency job at the optimized geometry using the same method and basis set. Results and Discussion Stable Configurations and Tautomers. Structures of neutral and deprotonated, zinc-coordinated acetohydroxamic and Nmethylacetohydroxamic acids in the keto and iminol tautomers that each exists in either Z or E configuration (Figures 1-4) were optimized with the B3LYP/LANL2DZ method. Singlepoint energy calculations with the B3LYP method were then carried out on all the optimized structures. The energies of these structures, calculated with the three basis sets described above, are listed in Table 1. The use of the B3LYP/6-311+G(2d,2p)// B3LYP/LANL2DZ method is justified by the insignificant energy differences of 3, 5, 9, 10, 13, 29, 31, 32, 34, and 37 due to the calculations using the B3LYP/6-311+G(2d,2p)//B3LYP/ 6-311+G(2d,2p) and B3LYP/6-311+G(2d,2p)//B3LYP/ LANL2DZ methods. It is also justified by our recent report that, with both MP2 and B3LYP methods, single point energy calculations with the 6-311+G(2d,2p) basis set using the structures that were optimized with the LANL2DZ basis set

Figure 2. Different tautomers and configurations of zinc-coordinated, deprotonated acetohydroxamic acid.

were nearly as good as the calculations using the structures optimized with the 6-311+G(2d,2p) basis set.38 As apparent from Table 1, the most energetically stable, neutral, zinc-coordinated acetohydroxamic acid exists in the Z-keto form (3) with its two oxygen atoms coordinating to the zinc divalent cation in the gas phase. Interestingly, the zwitterionic, Z-iminol form (10) is only 3 kcal/mol higher in energy than 3, suggesting that 10 and 3 are in equilibrium. Similarly,

6502 J. Phys. Chem. B, Vol. 104, No. 27, 2000

El Yazal and Pang TABLE 2: Bond Lengths (Å), Angles (deg. of arc), and Torsions (deg. of arc) of the Most Energetically Stable Structures of Neutral and Deprotonated, Zinc-Coordinated Acetohydroxamic and N-methylacetohydroxamic Acids ZnO′ ZnO C′O′ C′C C′N NC(H) NO OH O′ZnO NOZn O′C′N O′C′C C(H)NO NOH C(H)NC′ C(H)NC′C O′C′NO C′NOH O′C′CH ZnO′C′N

Figure 3. Different tautomers and configurations of zinc-coordinated, neutral N-methylacetohydroxamic acid.

Figure 4. Different tautomers and configurations of zinc-coordinated, deprotonated N-methylacetohydroxamic acid.

the most energetically stable, deprotonated, zinc-coordinated acetohydroxamic acid exists in the oxygen-deprotonated Z-keto form (13) with its two oxygen atoms coordinating to the zinc divalent cation in the gas phase. In contrast, the oxygendeprotonated Z-iminol from (16) is 33 kcal/mol higher in energy than 13. All other deprotonated structures are at least 20 kcal/ mol higher in energy than 13, suggesting that acetohydroxamic acid exists in the oxygen-deprotonated Z-keto form exclusively when coordinating to zinc in the presence of a proton acceptor. Like acetohydroxamic acid, the most energetically stable, neutral, zinc-coordinated N-methylacetohydroxamic acid exists in the Z-keto form (29) with its two oxygen atoms coordinating to the zinc divalent cation in the gas phase; the zwitterionic, Z-iminol form (32) is only 3 kcal/mol higher in energy than 29, suggesting that 32 and 29 are in equilibrium as well. Similarly, the most energetically stable, deprotonated, zinccoordinated N-methylacetohydroxamic acid exists in the oxygendeprotonated Z-keto form (37) with its two oxygen atoms

3

13

29

37

1.873 2.089 1.279 1.447 1.337 1.018 1.416 0.979 83.0 104.3 119.9 120.8 113.7 107.0 123.8 -22.7 7.5 -138.9 178.1 -6.7

1.901 1.873 1.294 1.487 1.317 1.011 1.378 91.7 100.9 119.6 120.0 113.8 123.5 0 0

1.861 2.037 1.292 1.489 1.325 1.469 1.433 0.975 82.0 107.9 120.2 118.5 115.4 108.8 131.6 0 0 -180.0 0 0

1.889 1.867 1.301 1.497 1.319 1.459 1.389 91.2 102.5 120.2 117.8 111.5 128.1 0 0

180.0 0

0.1 0

coordinating to the zinc divalent cation in the gas phase. All other deprotonated structures are at least 29 kcal/mol higher in energy than 37, suggesting that N-methylacetohydroxamic acid exists in the oxygen-deprotonated Z-keto form exclusively when coordinating to zinc in the presence of a proton acceptor. The bond lengths, angles, and torsions of the four most energetically stable structures of zinc-coordinated hydroxamic acids (3, 13, 29, and 37) are listed in Table 2. The relative energies calculated with the 6-311+G(2d,2p) basis set are nearly identical to those with the 6-311+G(d,p) basis set, indicating the convergence of the relative energies calculated with the 6-311+G(2d,2p) basis set. Although the relative energies calculated with the LANL2DZ basis set are significantly different from those with the 6-311+G(d,p) and 6-311+G(2d,2p) basis sets, according to the chemical accuracy (ca. 2 kcal/mol),48 the rank order of the relative energy for each class of structures calculated with the LANL2DZ is nearly identical to those with the 6-311+G(d,p) and 6-311+G(2d,2p) basis sets. This suggests that the LANL2DZ basis set is valuable in obtaining qualitative information regarding the relative energies of zinc-coordinated molecules. Experimental Confirmations. To confirm our prediction that in proteins the most energetically stable structures of zinccoordinated acetohydroxamic acid and its N-methyl analogue are in the Z configuration with their two oxygen atoms coordinating to zinc, we visually examined 28 experimentally determined protein 3D structures complexed with zinccoordinated hydroxamic acids documented in the Protein Data Bank (PDB) on October 3, 1999.49 Such structures were retrieved from the PDB by using keywords of “hydroxamic”, “acetohydroxamic”, “hydroxamate”, “acetohydroxamate”, “matrix metalloproteinase”, and “MMP” with the SearchLite browser provided by the PDB World Wide Web site. Consistent with our prediction, as apparent in Table 3, all of the zinc-coordinated hydroxamic acids are in the Z configuration with their two oxygen atoms coordinating to zinc in proteins with one exception. In the human carbonic anhydrase II complex, acetohydroxamic acid was exceptionally found to be in the Z-configuration but with its nitrogen atom coordinating to zinc.50 According to the calculated energies listed in Table 1, the association energy of the zinc divalent cation coordinating to the nitrogen atom of Z-acetohydroxamic acid is 42 to 47 kcal/

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TABLE 3: Coordination Modes of Hydroxamic Acids Found in 28 Experimentally Determined Protein 3D Structures Complexed with Zinc-Coordinated Hydroxamic Acids PDB codea

resolution (Å)

coordination mode

1A85 1A86 1IGB 456C 1AM6 4FUA 830C 966C 1C3R 1C3S 7TLN 1HFC 1IGB 1JAN 1JAP 1JAQ 1MMB 1MMQ 1MNC 1BIW 1BQO 1FBL 1KBC 3AYK 1 µB6 4AYK 1UMS 1UMT

2.00 2.00 2.30 2.40 2.10 2.43 1.60 1.90 2.00 2.50 2.30 1.56 2.30 2.50 1.82 2.55 2.10 1.90 2.10 2.50 2.30 2.50 1.81 NMR NMR NMR NMR NMR

Ab A A A Bc A A A A A A A A A A A A A A A A A A A A A A A

a References for the protein structures are available in the PDB coordinate files; b Hydroxamic acid is in the Z configuration with its oxygen atoms coordinating to zinc. c Hydroxamic acid is in the Z configuration with its nitrogen atom coordinating to zinc.

mol higher than that of the zinc divalent cation coordinating to two oxygen atoms of Z-acetohydroxamic acid. Further examining the X-ray structure of the human carbonic anhydrase II complex, we found that acetohydroxamic acid gained favorable interaction energies, which may not be sufficient to compensate the energy loss (42-47 kcal/mol), through two hydrogen bonds and hydrophobic interactions with residues of carbonic anhydrase when the nitrogen atom of the Z-acetohydramic acid was coordinating to the zinc ion. However, it was conceivable from the crystal structure that the methyl group of acetohydroxamic acid would have close van der Waals contacts with Leu198 and Thr199 if two oxygen atoms of Z-acetohydroxamic acid were coordinating to the zinc ion. This may explain the preference of the nitrogen atom coordination, since the repulsion resulted from the close van der Waals’ contacts is governed approximately by the 12-6 Lennard-Jones potential and greatly increases with the decrease of the interatomic distance. It thus offers a caveat that substituted groups of acetohydroxamic acid makes an important contribution to zinc enzyme affinity as well. Proton Dissociation Energies. Given the most energetically stable configurations and tautomers of neutral and deprotonated, zinc-coordinated acetohydroxamic and N-methylacetohydroxamic acids, the proton dissociation energies of the two acids in the presence of zinc were computed and compared with our recently reported proton dissociation energies of common zinc ligands in proteins (Table 4).38 The proton dissociation energies of both hydroxamic acids are lower than that of N-methylacetamide, suggesting the oxygen protons of the two hydroxamic acids are more acidic than the amide proton. NMR and X-ray crystallographic experiments have revealed that the amide group is deprotonated when it coordinates to zinc.51,52 Therefore, the

TABLE 4: Proton Dissociation Energies (PDE) in the Presence of Zn2+ Calculated at the B3LYP/6-311+G(2d,2p)// B3LYP/6-311+G(2d,2p) Level structures

PDE (kcal/mol)

HOH MeOH MeSH MeCONHOH MeCON(Me)OH Imidazole(NH) MeCONHMe

5538 6838 7038 80 86 11838 12238

TABLE 5: Zinc Affinities of Acetohydroxamic Acid, N-Methylacetohydroxamic Acid, and Common Zinc Ligands in Proteins in the Neutral and Deprotonated Forms Calculated at the B3LYP/6-311+G(2d,2p)//B3LYP/ 6-311+G(2d,2p) Level structures

affinity (kcal/mol)

H2O MeOH MeSH MeCONHMe MeCONHOH imidazole MeCON(Me)OH imidazolate MeCON-Me OHMeOMeSMeCONHOMeCON(Me)O-

-102 -123 -151 -177 -181 -181 -191 -412 -417 -437 -438 -439 -444 -455

two hydroxamic acids will be deprotonated if they coordinate to zinc in the presence of a suitable proton acceptor. It should be noted that our conclusion of the deprotonated hydroxamic acids is not made on the basis of the absolute acidities of hydroxamic acids and amide proton in a four-ligand zinc complex of a specific protein. For the absolute acidities, it is necessary to compute the influences on proton dissociation energies by the presence of other zinc ligands, the residues in the second and third zinc coordination shells, and the electrostatic field of the entire protein. Instead, our conclusion is based on the calculated proton dissociation energy difference between two test ligands that does not require calculations of the influences on proton dissociation energies by other factors in proteins, since such influences can be canceled out as demonstrated in our previous report.38 Furthermore, according to our recent report that the acidic amino acid side chains in proteins can serve as a proton acceptor for zinc-coordinated imidazole whose protonation dissociation energy is at least 38 kcal/mol higher than those of the two hydroxamic acids,38 suggesting that zinc-coordinated hydroxamic acids are more acidic than zinc-coordinated imidazole. It is therefore conceivable that acetohydroxamic and N-methylacetohydroxamic acids are deprotonated when coordinating to zinc in proteins in which the acidic amino acid sides can serve as a proton acceptor. Relative Affinities for Zinc. Given the most energetically stable configurations and tautomers of neutral and deprotonated, zinc-coordinated hydroxamic acids, the zinc affinities of acetohydroxamic, N-methylacetohydroxamic acids, and common zinc ligands in proteins were also evaluated (Table 5). Like the common zinc ligands in proteins, both deprotonated acetohydroxamic and N-methylacetohydroxamic acids have higher affinities for zinc than the neutral ones. The zinc affinity of N-methylacetohydroxamic acid is 11 kcal/mol higher than that of acetohydroxamic acid, which can be attributed to the electrondonating effect of the methyl group. Compared to the affinities of other common zinc ligands (Table 5), N-methylacetohydrox-

6504 J. Phys. Chem. B, Vol. 104, No. 27, 2000 amic acid has the highest affinity for zinc and its affinity is up to 43 kcal/mol higher than those of common zinc ligands in proteins. The estimated relative affinities thus caution the use of N-methylacetohydroxamic acid as a functional group of inhibitors designed for zinc proteinases, since it may interact with other zinc proteins due to its high affinity for zinc. Conclusions The present study suggests that in the gas-phase both zinccoordinated acetohydroxamic and N-methylacetohydroxamic acids exist in the oxygen-deprotonated Z-keto form with their two oxygen atoms coordinating to zinc in proteins and that the zinc affinity of deprotonated N-methylacetohydroxamic acid is 11 kcal/mol stronger than that of deprotonated acetohydroxamic acid and up to 43 kcal/mol stronger than those of common, deprotonated, zinc ligands in proteins. The result thus cautions the use of N-methylacetohydroxamic acid as a functional group in rational design of inhibitors for zinc proteinases, since it may interact with other zinc proteins due to its high affinity for zinc. Acknowledgment. This work was supported by the Mayo Foundation for Medical Education and Research. References and Notes (1) Miller, M. J. Chem. ReV. 1989, 89, 1563-1579. (2) Bode, W.; Reinemer, P.; Huber, R.; Kleine, T.; Schnierer, S.; Tschesche, H. EMBO J. 1994, 13, 1263-9. (3) Brandstetter, H.; Engh, R. A.; Von Roedern, E. G.; Moroder, L.; Huber, R.; Bode, W.; Grams, F. Protein Sci. 1998, 7, 1303-9. (4) DiMartino, M.; Wolff, C.; High, W.; Stroup, G.; Hoffman, S.; Laydon, J.; Lee, J. C.; Bertolini, D.; Galloway, W. A.; Crimmin, M. J.; Davis, M.; Davies, S. Inflamm. Res. 1997, 46, 211-5. (5) Ferry, G.; Boutin, J. A.; Hennig, P.; Genton, A.; Desmet, C.; Fauchere, J. L.; Atassi, G.; Tucker, G. C. Eur. J. Pharmacol. 1998, 351, 225-233. (6) Groneberg, R. D.; Burns, C. J.; Morrissette, M. M.; Ullrich, J. W.; Morris, R. L.; Darnbrough, S.; Djuric, S. W.; Condon, S. M.; McGeehan, G. M.; Labaudiniere, R.; Neuenschwander, K.; Scotese, A. C.; Kline, J. A. J. Med. Chem. 1999, 42, 541-4. (7) Holleran, W. M.; Galardy, R. E.; Gao, W. N.; Levy, D.; Tang, P. C.; Elias, P. M. Arch. Dermatol. Res. 1997, 289, 138-44. (8) Jacobson, I. C.; Reddy, P. G.; Wasserman, Z. R.; Hardman, K. D.; Covington, M. B.; Arner, E. C.; Copeland, R. A.; Decicco, C. P.; Magolda, R. L. Bioorg. Med. Chem. Lett. 1998, 8, 837-42. (9) Maskos, K.; Fernandez-Catalan, C.; Huber, R.; Bourenkov, G. P.; Bartunik, H.; Ellestad, G. A.; Reddy, P.; Wolfson, M. F.; Rauch, C. T.; Castner, B. J.; Davis, R.; Clarke, H. R.; Petersen, M.; Fitzner, J. N.; Cerretti, D. P.; March, C. J.; Paxton, R. J.; Black, R. A.; Bode, W. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3408-12. (10) Nixon, J. S.; Bottomley, K. M.; Broadhurst, M. J.; Brown, P. A.; Johnson, W. H.; Lawton, G.; Marley, J.; Sedgwick, A. D.; Wilkinson, S. E. Int. J. Tissue React. 1991, 13, 237-41. (11) Odake, S.; Morita, Y.; Morikawa, T.; Yoshida, N.; Hori, H.; Nagai, Y. Biochem. Biophys. Res. Commun. 1994, 199, 1442-6. (12) Pikul, S.; McDow Dunham, K. L.; Almstead, N. G.; De, B.; Natchus, M. G.; Anastasio, M. V.; McPhail, S. J.; Snider, C. E.; Taiwo, Y. O.; Chen, L.; Dunaway, C. M.; Gu, F.; Mieling, G. E. J. Med. Chem. 1999, 42, 87-94. (13) Preece, G.; Murphy, G.; Ager, A. J. Biol. Chem. 1996, 271, 1163411640. (14) Steinman, D. H.; Curtin, M. L.; Garland, R. B.; Davidsen, S. K.; Heyman, H. R.; Holms, J. H.; Albert, D. H.; Magoc, T. J.; Nagy, I. B.; Marcotte, P. A.; Li, J.; Morgan, D. W.; Hutchins, C.; Summers, J. B. Bioorg. Med. Chem. Lett. 1998, 8, 2087-92. (15) Steinmann-Niggli, K.; Lukes, M.; Marti, H. P. J. Am. Soc. Nephrol. 1997, 8, 395-405. (16) Steinmeyer, J.; Daufeldt, S.; Kalbhen, D. A. Pharmacology 1997, 55, 95-108. (17) Vandyk, D. E.; Marchand, P.; Bruckner, R. C.; Fox, J. W.; Jaffee, B. D.; Gunyuzlu, P. L.; Davis, G. L.; Nurnberg, S.; Covington, M.; Decicco, C. P.; Trzaskos, J. M.; Magolda, R. L.; Copeland, R. A. Bioorg. Med. Chem. Lett. 1997, 7, 1219-1224.

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