Hydantoins, triazolones, and imidazolones as selective non-hydroxamate inhibitors of tumor necrosis factor-alpha converting enzyme (TACE)

We have discovered selective and potent inhibitors of TACE that replace the common hydroxamate zinc binding group with a hydantoin, triazolone, and imidazolone heterocycle. These novel heterocyclic inhibitors of a zinc metalloprotease were designed using a pharmacophore model that we previously described while developing hydantoin and pyrimidinetrione (barbiturate) inhibitors of TACE. The potency and binding orientation of these inhibitors is discussed and they are modeled into the X-ray crystal structure of TACE and compared to hydroxamate and earlier hydantoin TACE inhibitors which share the same 4-[(2-methyl-4-quinolinyl)methoxy]benzoyl P1' group.     

Discovery and SAR of hydantoin TACE inhibitors

We disclose inhibitors of TNF-α converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first reported X-ray structures of TACE with a hydantoin zinc ligand.     

The discovery of novel tartrate-based TNF-α converting enzyme (TACE) inhibitors

A novel series of TNF-α convertase (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds are bis-amides of l-tartaric acid (tartrate) and coordinate to the active site zinc in a tridentate manner. They are selective for TACE over other MMP’s. We report the first X-ray crystal structure for a tartrate-based TACE inhibitor.     

Crystal structure of the complex between carboxypeptidase A and the biproduct analog inhibitor L-benzylsuccinate at 2.0 A resolution.

The X-ray crystal structure of the carboxypeptidase A-L-benzylsuccinate complex has been refined at 2.0 A resolution to a final R-factor of 0.166. One molecule of the inhibitor binds to the enzyme active site. The terminal carboxylate forms a salt link with the guanidinium group of Arg145 and hydrogen bonds with Tyr248 and Asn144. The second carboxylate group binds to the zinc ion in an asymmetric bidentate fashion replacing the water molecule of the native structure. The zinc ion moves 0.5 A from its position in the native structure to accommodate the inhibitor binding. The overall stereochemistry around the zinc can be considered a distorted tetrahedron, although six atoms of the co-ordinated groups lie within 3.0 A from the zinc ion. The key for the strong inhibitory properties of L-benzylsuccinate can be found in its ability both to co-ordinate the zinc and to form a short carboxyl-carboxylate-type hydrogen bond (2.5 A) with Glu270.     

Inhibition of the tumor necrosis factor-alpha-converting enzyme by its pro domain

Tumor necrosis factor-alpha-converting enzyme (TACE) is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen, and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. To study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that the TACE pro domain (TACE Pro) is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC(50) of 70 nm. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC(50) greater that 2 microm). The disintegrin/cysteine-rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine switch motif located in TACE Pro was not essential for inhibition of the enzymatic activity of TACE; the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys(184) residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. Despite this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteine switch ligation of the zinc ion within the active site of TACE.     

X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-beta-lactamase ImiS from Aeromonas veronii bv. sobria

X-ray absorption spectroscopy was used to investigate the metal-binding sites of ImiS from Aeromonas veronii bv. sobria in catalytically active (1-Zn), product-inhibited (1-Zn plus imipenem), and inactive (2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn(2) site. The reaction of 1-Zn ImiS with imipenem leads to a product-bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild-type ImiS with 1 and 2 equiv of bound zinc. 2-Zn ImiS extended X-ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [Garau et al. (2005) J. Mol. Biol. 345, 785-795], we propose that the inhibitory binding site is formed by M146, found on the B2-distinct alpha3 helix, and H118, a canonical Zn(1) ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn and establishes, for the first time, the location of the inhibitory metal site.     

Structural consequences of the active site substitution Cys181 ==> Ser in metallo-beta-lactamase from Bacteroides fragilis

The metallo-beta-lactamases require divalent cations such as zinc or cadmium for hydrolyzing the amide bond of beta-lactam antibiotics. The crystal structure of the Zn2+ -bound enzyme from Bacteroides fragilis contains a binuclear zinc center in the active site. A hydroxide, coordinated to both zinc atoms, is proposed as the moiety that mounts the nucleophilic attack on the carbonyl carbon atom of the beta-lactam bond of the substrate. It was previously reported that the replacement of the active site Cys181 by a serine residue severely impaired catalysis while atomic absorption measurements indicated that binding of the two zinc ions remained intact. Contradicting data emerge from recent mass spectrometry results, which show that only a single zinc ion binds to the C181S metallo-beta-lactamase. In the current study, the C181S mutant enzyme was examined at the atomic level by determining the crystal structure at 2.6 A resolution. The overall structure of the mutant enzyme is the same as that of the wild-type enzyme. At the mutation site, the side chain of Ser181 occupies the same position as that of the side chain of Cys181 in the wild-type protein. One zinc ion, Zn1, is present in the crystal structure; however, the site of the second zinc ion, Zn2 is unoccupied. A water molecule is associated with Zn1, reminiscent of the hydroxide seen in the structure of the wild-type enzyme but farther from the metal. The position of the water molecule is off the plane of the carboxylate group of Asp103; therefore, the water molecule may be less nucleophilic than a water molecule which is coplanar with the carboxylate group.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK(a) of the inhibitor carboxylate group is lowered by at least 1.5 pK(a) units when it binds to either enzyme. The signal at ~183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-(13)C]-L-tryptophan is ≤3.71? from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-(13)C]-L-tryptophan is not bound in the S(2)' subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.     

Pronounced diversity in electronic and chemical properties between the catalytic zinc sites of tumor necrosis factor-alpha-converting enzyme and matrix metalloproteinases despite their high structural similarity

The metalloproteinase tumor necrosis factor-alpha-converting enzyme (TACE) is involved in the regulation of several key physiological and pathological processes. Therefore, potent and selective synthetic inhibitors are highly sought for the study of the physiological roles of TACE as well as for therapeutic purposes. Because of the high structural similarities between the active site of TACE and those of other related zinc endopeptidases such as disintegrin (ADAMs) and matrix metalloproteinases (MMPs), the design of such tailor-made inhibitors is not trivial. To obtain new insights into this problem, we have used a selective MMP inhibitor as a probe to examine the structural and kinetic effects occurring at the active site of TACE upon inhibition. Specifically, we used the selective MMP mechanism-based inhibitor SB-3CT to characterize the fine structural and electronic differences between the catalytic zinc ions within the active sites of TACE and MMP-2. We show that SB-3CT directly binds the metal ion of TACE as observed before with MMP-2. However, in contrast to MMP-2, the binding mode of SB-3CT to the catalytic zinc ion of TACE is different in the length of the Zn-S(SB-3CT) bond distance and the total effective charge of the catalytic zinc ion. In addition, SB-3CT inhibits TACE in a non-competitive fashion by inducing significant conformational changes in the structure. For MMP-2, SB-3CT behaved as a competitive inhibitor and no significant conformational changes were observed. An examination of the second shell amino acids surrounding the catalytic zinc ion of these enzymes indicated that the active site of TACE is more polar than that of MMP-2 and of other MMPs. On the basis of these results, we propose that although there is a seemingly high structural similarity between TACE and MMP-2, these enzymes are significantly diverse in the electronic and chemical properties within their active sites.     

Amphipathic benzoic acid derivatives: synthesis and binding in the hydrophobic tunnel of the zinc deacetylase LpxC

The first committed step in lipid A biosynthesis is catalyzed by uridine diphosphate-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase, and inhibitors of LpxC may be useful in the development of antibacterial agents targeting a broad spectrum of Gram-negative bacteria. Here, we report the design of amphipathic benzoic acid derivatives that bind in the hydrophobic tunnel in the active site of LpxC. The hydrophobic tunnel accounts for the specificity of LpxC toward substrates and substrate analogues bearing a 3-O-myristoyl substituent. Simple benzoic acid derivatives bearing an aliphatic 'tail' bind in the hydrophobic tunnel with micromolar affinity despite the lack of a glucosamine ring like that of the substrate. However, although these benzoic acid derivatives each contain a negatively charged carboxylate 'warhead' intended to coordinate to the active site zinc ion, the 2.25A resolution X-ray crystal structure of LpxC complexed with 3-(heptyloxy)benzoate reveals 'backward' binding in the hydrophobic tunnel, such that the benzoate moiety does not coordinate to zinc. Instead, it binds at the outer end of the hydrophobic tunnel. Interestingly, these ligands bind with affinities comparable to those measured for more complicated substrate analogue inhibitors containing glucosamine ring analogues and hydroxamate 'warheads' that coordinate to the active site zinc ion. We conclude that the intermolecular interactions in the hydrophobic tunnel dominate enzyme affinity in this series of benzoic acid derivatives.     

The relation of conformation and association of insulin to receptor binding; x-ray and circular-dichroism studies on bovine and hystricomorph insulins.

Crystal and solution structure studies on insulins of different sequences and of widely different receptor binding affinities are reported. Bovine insulin, studied as a control, has a circular dichroism spectrum which is dependent both on protein concentration and zinc concentration. The spectrum appears to be related to the level of association of the insulin molecules. This implies that when using circular dichroism to compare solution structures of insulin derivatives or species variants one must make the comparison at equivalent levels of association and not merely at the same concentration. Changes in circular dichroism are related to the known crystal structure of zinc insulin hexamers. The chinchilla insulin spectrum shows a reduced zinc dependence in low-salt conditions which correlates with the inability to form crystals in similar conditions. This is attributed to an amino acid substitution at position B4. Crystals are obtained in high-salt conditions and X-ray diffraction patterns show these to be isomorphous with bovine 4Zn insulin crystals. Guinea pig insulin failed to crystallise under conditions which are normally conducive to the formation of crystals of zinc insulin hexamers and the circular dichroism showed no zinc dependence. This is consistent with a monomeric structure. The significance of the association behaviour of chinchilla and guinea pig insulins may be in the storage of the hormone in vivo. Whereas the monomeric form of chinchilla insulin has a structure closely related to bovine insulin, the circular dichroism indicates a gross structural difference for guinea pig insulin. This may be similar to that in des-A21, des-B30-insulin, as both lack the Arg-B22--Asn-A21 carboxylate ion pair. The similarity of structure of chinchilla and bovine insulins is reflected in their receptor binding whereas the low receptor binding of guinea pig insulin probably results from the changes in its conformation rather than an alteration in residues of a receptor binding region.     

Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA

The zinc finger domain of the Wilms tumor suppressor protein (WT1) contains four canonical Cys(2)His(2) zinc fingers. WT1 binds preferentially to DNA sequences that are closely related to the EGR-1 consensus site. We report the structure determination by both X-ray crystallography and NMR spectroscopy of the WT1 zinc finger domain in complex with DNA. The X-ray structure was determined for the complex with a cognate 14 base-pair oligonucleotide, and composite X-ray/NMR structures were determined for complexes with both the 14 base-pair and an extended 17 base-pair DNA. This combined approach allowed unambiguous determination of the position of the first zinc finger, which is influenced by lattice contacts in the crystal structure. The crystal structure shows the second, third and fourth zinc finger domains inserted deep into the major groove of the DNA where they make base-specific interactions. The DNA duplex is distorted in the vicinity of the first zinc finger, with a cytidine twisted and tilted out of the base stack to pack against finger 1 and the tip of finger 2. By contrast, the composite X-ray/NMR structures show that finger 1 continues to follow the major groove in the solution complexes. However, the orientation of the helix is non-canonical, and the fingertip and the N terminus of the helix project out of the major groove; as a consequence, the zinc finger side-chains that are commonly involved in base recognition make no contact with the DNA. We conclude that finger 1 helps to anchor WT1 to the DNA by amplifying the binding affinity although it does not contribute significantly to binding specificity. The structures provide molecular level insights into the potential consequences of mutations in zinc fingers 2 and 3 that are associated with Denys-Drash syndrome and nephritic syndrome. The mutations are of two types, and either destabilize the zinc finger structure or replace key base contact residues.     

Insight into the stereochemistry in the inhibition of carboxypeptidase A with N-(hydroxyaminocarbonyl)phenylalanine: binding modes of an enantiomeric pair of the inhibitor to carboxypeptidase A

Both D- and L-isomers of N-(hydroxyaminocarbonyl)phenylalanine () were shown to have strong binding affinity towards carboxypeptidase A (CPA) with D- being more potent than its enantiomer by 3-fold (Chung, S. J.; Kim, D. H. Bioorg. Med. Chem. 2001, 9, 185.). In order to understand the reversed stereochemical preference shown in the CPA inhibition, we have solved the crystal structures of CPA complexed with each enantiometer of up to 1.75 A resolution. Inhibitor L- whose stereochemistry belongs to the stereochemical series of substrate binds CPA like substrate does with its carbonyl oxygen coordinating to the active site zinc ion. Its hydroxyl is engaged in hydrogen bonding with the carboxylate of Glu-270. On the other hand, in binding of D- to CPA, its terminal hydroxyl group is involved in interactions with the active site zinc ion and the carboxylate of Glu-270. In both CPA small middle dot complexes, the phenyl ring in is fitted in the substrate recognition pocket at the S(1)' subsite, and the carboxylate of the inhibitors forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and a hydrogen bond with the guanidinium of Arg-127. In the complex of CPA small middle dotD-, the carboxylate of the inhibitor is engaged in hydrogen bonding with the phenolic hydroxyl of the down-positioned Tyr-248. While the L- binding induces a concerted movement of the backbone amino acid residues at the active site, only the downward movement of Tyr-248 was noted when D- binds to CPA.     

Inhibition of adamalysin II and MMPs by phosphonate analogues of snake venom peptides

Phosphonate analogues of the peptidomimetic N-(Furan-2-yl)carbonyl-Leu-Trp-OH were prepared with the goal of evaluating the effect of phosphonate for carboxylate replacement on binding with snake venom metalloproteinases and MMPs. N-(Furan-2-yl)carbonyl-Leu-L-Trp(P)-(OH)2 showed a 75-fold increase of the inhibiting activity against adamalysin II, a snake venom metalloproteinase structurally related to MMPs and TACE. Both the phosphonate and carboxylate peptidomimetics fit into the active site adopting a retrobinding mode and provide the structural base for a new class of metalloproteinases inhibitors.     

Synthesis and structure-activity relationship of a novel, non-hydroxamate series of TNF-alpha converting enzyme inhibitors

A novel series of TNF-alpha converting enzyme (TACE) inhibitors which are non-hydroxamate have been discovered. These compounds use a triazolethione moiety as the zinc binding ligand and exhibit IC50 values from 1.5 to 100 nM in a porcine TACE assay. They also have excellent selectivities over other MMPs.     

IK682, a tight binding inhibitor of TACE

TNFalpha converting enzyme (TACE) is the major metalloproteinase for the processing of TNFalpha, a key inflammatory cytokine. IK682, a hydroxamate compound, was reported to be a potent and specific TACE inhibitor [J.J. Duan, L. Chen, Z.R. Wasserman, Z. Lu, R.Q. Liu, M.B. Covington, M. Qian, K.D. Hardman, R.L. Magolda, R.C. Newton, D.D. Christ, R.R. Wexler, C.P. Decicco, J. Med. Chem. 45 (2002) 4954-4957]. The binding kinetics of IK682 and the ectodomain of human TACE was examined. The k(on) of IK682 was determined as 1.1+/-0.3 x 10(8) M(-1) min(-1). No detectable dissociation of IK682 from TACE was observed following dialysis, dilution, and extensive washing over a maximum of 72 h. This was in contrast to the rapid dissociation of IK682 from ADAM10. LC/MS analysis of the TACE-IK682 complex after dissociation under denaturing conditions indicated that the tight binding is not due to covalent interaction. The X-ray crystal structure of TACE-IK682 complex revealed multiple binding points at the S1' and S3' sites and the movement of a loop (from Ala349 to Gly442) to accommodate the binding of the quinolinyl group of IK682 at the S3' pocket. The conformational changes of TACE may contribute significantly to the high affinity binding as a result of a more stable TACE-inhibitor complex.     

Crystal structure of auxin-binding protein 1 in complex with auxin

The structure of auxin-binding protein 1 (ABP1) from maize has been determined at 1.9 A resolution, revealing its auxin-binding site. The structure confirms that ABP1 belongs to the ancient and functionally diverse germin/seed storage 7S protein superfamily. The binding pocket of ABP1 is predominantly hydrophobic with a metal ion deep inside the pocket coordinated by three histidines and a glutamate. Auxin binds within this pocket, with its carboxylate binding the zinc and its aromatic ring binding hydrophobic residues including Trp151. There is a single disulfide between Cys2 and Cys155. No conformational rearrangement of ABP1 was observed when auxin bound to the protein in the crystal, but examination of the structure reveals a possible mechanism of signal transduction.     

Model for calcium binding to gamma-carboxyglutamic acid residues of proteins: crystal structure of calcium alpha-ethylmalonate

The crystal structure of a Ca2+ salt of alpha-ethylmalonic acid was determined from three-dimensional X-ray diffraction data. The dicarboxylate anion represents the functional side chain of gamma-carboxyglutamic acid (Gla) residues, which are implicated as essential calcium-binding ligands in a variety of proteins. The alpha-ethylmalonate ion chelates the Ca2+ ion in a bidentate manner that involves an O atom from each of the two malonate carboxylate groups. This type of binding arises from the constrained arrangement of carboxylate ligands in the malonate group and may be of significance to the calcium-binding properties of Gla-containing sites in proteins. The Ca2+-malonate chelation forms a six-membered ring, which is stabilized by interactions that are consistent with the preferred stereochemistries of both calcium-carboxylate and metal-malonate complexes. No other interactions are observed between Ca2+ ions and alpha-ethylmalonate ions that depend upon the malonate juxtaposition of two carboxylate groups. The potential for this type of binding distinguishes Gla residues from the monocarboxylate residues, aspartate and glutamate, and confers a novel calcium-chelation ability upon Gla-containing sites in proteins.     

Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.     

Mechanism of the binding of Z-L-tryptophan and Z-L-phenylalanine to thermolysin and stromelysin-1 in aqueous solutions

The chemical shift of the carboxylate carbon of Z-tryptophan is increased from 179.85 to 182.82 ppm and 182.87 ppm on binding to thermolysin and stromelysin-1 respectively. The chemical shift of Z-phenylalanine is also increased from 179.5 ppm to 182.9 ppm on binding to thermolysin. From pH studies we conclude that the pK_a of the inhibitor carboxylate group is lowered by at least 1.5 pK_a units when it binds to either enzyme. The signal at ~ 183 ppm is no longer observed when the active site zinc atom of thermolysin or stromelysin-1 is replaced by cobalt. We estimate that the distance of the carboxylate carbon of Z-[1-¹³C]-L-tryptophan is ≤ 3.71 Å from the active site cobalt atom of thermolysin. We conclude that the side chain of Z-[1-¹³C]-L-tryptophan is not bound in the S₂′ subsite of thermolysin. As the chemical shifts of the carboxylate carbons of the bound inhibitors are all ~ 183 ppm we conclude that they are all bound in a similar way most probably with the inhibitor carboxylate group directly coordinated to the active site zinc atom. Our spectrophotometric results confirm that the active site zinc atom is tetrahedrally coordinated when the inhibitors Z-tryptophan or Z-phenylalanine are bound to thermolysin.