Development of a K562 cell-based assay for screening anticancer agents.

AIM: To develop a leukemia cell line K562-based assay for high-throughput screening. METHODS: The screening was carried out on 96-well plates with monitoring cell proliferation by a combined 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium (MTS)/phenazine methosulfate (PMS) method. Conditions for evaluating effects on the proliferation of K562 cells by individual compounds on the 96-well plates were optimized. RESULTS: A set of 800 small organic compounds was screened for anticancer activity by this cell-based assay, with consumption of each compound at 500 ng. Eleven compounds were identified with >80 % inhibitory activity at 5 mg/L, among which 9 compounds were confirmed by subsequent testing at multiple concentrations. The most potent compound showed an IC50 at 170 nmol/L, and there were total of 7 compounds showed IC50 less than 10 micromol/L. CONCLUSION: The high-throughput method using K562 cell line is fast, economical, effective, and practical in identifying inhibitors as potential therapeutic agents for cancer.

High-throughput screening for human collagenase 1 inhibitors.

AIM: To establish a high-throughput method for inhibitor screening using a recombinant collagenase catalytic domain. METHODS: Human collagenase 1 catalytic domain protein was expressed in E coli and used for screening a set of 2720 compounds in a high-throughput fashion. RESULTS: The screening was accomplished within 2 h and 10 min with consumption of each compound at 4 micrograms. Sixty-six compounds were identified with > 60% inhibitory activity at 20 mg/L, among which 44 compounds were confirmed by subsequent testing at multiple concentrations. The most potent compound showed an IC50 at 4.3 mumol/L, and there were total 15 compounds with IC50 less than 20 mumol/L. CONCLUSION: The high-throughput method using the recombinant collagenase is fast, effective and practical in identifying inhibitors.

Catalytic activities and substrate specificity of the human membrane type 4 matrix metalloproteinase catalytic domain.

Membrane type (MT) matrix metalloproteinases (MMPs) are recently recognized members of the family of Zn(2+)- and Ca(2+)-dependent MMPs. To investigate the proteolytic capabilities of human MT4-MMP (i.e. MMP-17), we have cloned DNA encoding its catalytic domain (CD) from a breast carcinoma cDNA library. Human membrane type 4 MMP CD (MT4-MMPCD) protein, expressed as inclusion bodies in Escherichia coli, was purified to homogeneity and refolded in the presence of Zn(2+) and Ca(2+). While MT4-MMPCD cleaved synthetic MMP substrates Ac-PLG-[2-mercapto-4-methylpentanoyl]-LG-OEt and Mca-PLGL-Dpa-AR-NH(2) with modest efficiency, it catalyzed with much higher efficiency the hydrolysis of a pro-tumor necrosis factor-alpha converting enzyme synthetic substrate, Mca-PLAQAV-Dpa-RSSSR-NH(2). Catalytic efficiency with the pro-tumor necrosis factor-alpha converting enzyme substrate was maximal at pH 7.4 and was modulated by three ionizable enzyme groups (pK(a3) = 6.2, pK(a2) = 8.3, and pK(a1) = 10.6). MT4-MMPCD cleaved gelatin but was inactive toward type I collagen, type IV collagen, fibronectin, and laminin. Like all known MT-MMPs, MT4-MMPCD was also able to activate 72-kDa progelatinase A to its 68-kDa form. EDTA, 1,10-phenanthroline, reference hydroxamic acid MMP inhibitors, tissue inhibitor of metalloproteinases-1, and tissue inhibitor of metalloproteinases-2 all potently blocked MT4-MMPCD enzymatic activity. MT4-MMP is, therefore, a competent Zn(2+)-dependent MMP with unique specificity among synthetic substrates and the capability to both degrade gelatin and activate progelatinase A.

X-ray structure of human stromelysin catalytic domain complexed with nonpeptide inhibitors: implications for inhibitor selectivity.

Effective inhibitors of matrix metalloproteinases (MMPs), a family of connective tissue-degrading enzymes, could be useful for the treatment of diseases such as cancer, multiple sclerosis, and arthritis. Many of the known MMP inhibitors are derived from peptide substrates, with high potency in vitro but little selectivity among MMPs and poor bioavailability. We have discovered nonpeptidic MMP inhibitors with improved properties, and report here the crystal structures of human stromelysin-1 catalytic domain (SCD) complexed with four of these inhibitors. The structures were determined and refined at resolutions ranging from 1.64 to 2.0 A. Each inhibitor binds in the active site of SCD such that a bulky diphenyl piperidine moiety penetrates a deep, predominantly hydrophobic S'1 pocket. The active site structure of the SCD is similar in all four inhibitor complexes, but differs substantially from the peptide hydroxamate complex, which has a smaller side chain bound in the S'1 pocket. The largest differences occur in the loop forming the "top" of this pocket. The occupation of these nonpeptidic inhibitors in the S'1 pocket provides a structural basis to explain their selectivity among MMPs. An analysis of the unique binding mode predicts structural modifications to design improved MMP inhibitors.

Designing inhibitors of the metalloproteinase superfamily: comparative analysis of representative structures.

Structural comparisons of representative members of the zinc metalloproteinase superfamily show that the key secondary structural elements are conserved, in spite of major variations in the sequences including insertions and deletions of functional domains. Major differences between the matrix metalloproteinases (matrixins) are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. A comparison of the structures of matrixin-inhibitor complexes shows that there are significant differences even among the closely related matrixins, not only in the peripheral regions but also in the specificity pockets; these differences offer an excellent opportunity for the design of specific inhibitors targetted to individual members.

X-ray structure of a hydroxamate inhibitor complex of stromelysin catalytic domain and its comparison with members of the zinc metalloproteinase superfamily.

BACKGROUND: Stromelysin belongs to a family of zinc-dependent endopeptidases referred to as matrix metalloproteinases (MMPs, matrixins) because of their capacity for selective degradation of various components of the extracellular matrix. Matrixins play key roles in diseases as diverse as arthritis and cancer and hence are important targets for therapeutic intervention. RESULTS: The crystal structure of the stromelysin catalytic domain (SCD) with bound hydroxamate inhibitor, solved by multiple isomorphous replacement, shows deep S1' specificity pocket which explains differences in inhibitors binding between the collagenases and stromelysin. The binding of calcium ions by loops at the two ends of a beta-strand which marks the boundary of the active site provides a structural rationale for the importance of these cations for stability and catalytic activity. Major differences between the matrixins are clustered in two regions forming the entrance to the active site and hence may be determinants of substrate selectivity. CONCLUSIONS: Structural comparisons of SCD with representative members of the metalloproteinase superfamily clearly highlight the conservation of key secondary structural elements, in spite of major variations in the sequences including insertions and deletions of functional domains. However, the three-dimensional structure of SCD, which is generally closely related to the collagenases, shows significant differences not only in the peripheral regions but also in the specificity pockets; these latter differences should facilitate the rational design of specific inhibitors.

The structural basis for the elastolytic activity of the 92-kDa and 72-kDa gelatinases. Role of the fibronectin type II-like repeats.

Several matrix metalloproteinases, including the 92-kDa and 72-kDa gelatinases, macrophage metalloelastase (MME), and matrilysin degrade insoluble elastin. Because elastolytically active MME and matrilysin consist only of a catalytic domain (CD), we speculated that the homologous CDs of the 92-kDa and 72-kDa gelatinases would confer their elastolytic activities. In contrast to the MME CD, the 92 and 72 CDs expressed in Escherichia coli (lacking the internal fibronectin type II-like repeats) had no elastase activity, although both were gelatinolytic and cleaved a thiopeptolide substrate at rates comparable to the full-length gelatinases. To test the role of the fibronectin type II-like repeats in elastolytic activity, we expressed the 92-kDa gelatinase CD with its fibronectin type II-like repeats (92 CD/FN) in yeast. 92 CD/FN degraded insoluble elastin with activity comparable to full-length 92-kDa gelatinase. 92 and 72 CDs lacking the fibronectin type II-like repeats did not bind elastin, whereas the parent enzymes and 92 CD/FN did bind elastin. Furthermore, recombinant 92-kDa fibronectin type II-like repeats inhibited binding of the 92-kDa gelatinase to elastin. We conclude that the 92- and 72-kDa gelatinases require the fibronectin type II-like repeats for elastase activity.

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

Reconstructed 19 kDa catalytic domain of gelatinase A is an active proteinase.

Matrix metalloproteinases share high protein sequence homology and have defined domain structures. Gelatinases have a unique 19 kDa fibronectin-like insert in the catalytic domain. A synthetic gene was made to express the catalytic domain of human gelatinase A (GCD), in which two polypeptide fragments of the catalytic domain were joined with deletion of the insert. The synthetic gene was highly expressed in Escherichia coli, and the 19 kDa GCD was purified to homogeneity after in vitro refolding. The GCD showed activity at a pH range of 5.5-9 in cleavage of the thiopeptolide Ac-Pro-Leu-Gly-thioester-Leu-Leu-Gly-OEt with optimal activity at neutral pH (Km = 134 microM and kcat = 16 s-1 at pH 7.0). The activity required both zinc and calcium ions, but high concentration of zinc ion showed inhibition. Several stromelysin catalytic domain inhibitors inhibited the GCD with similar specificity. The GCD cleaved the fluorogenic peptides Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 and Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 with catalytic efficiency close to full length human gelatinase A. The reconstructed GCD cleaves not only thiopeptolide and peptide substrates but also protein substrates such as gelatin. These results are consistent with the notion that gelatinases have the same structure for the catalytic domain as other matrix metalloproteinases like stromelysins and collagenases.

Calcium stoichiometry determination for calcium binding proteins by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry was used for the determination of calcium-binding stoichiometry for calcium-binding proteins. Bovine calmodulin, bovine alpha-lactalbumin, and rabbit parvalbumin were found to bind specifically to 4, 1, and 2 Ca(2+) ions, respectively, in agreement with previously reported results obtained by using other physical methods. This mass spectrometry method could also be applied to proteins that bind more than one type of metal ion. The Zn(2+)- and Ca(2+)-binding stoichiometries for human stromelysin catalytic domain were determined to be 3 and 2, respectively.

Zinc content of promatrilysin, matrilysin and the stromelysin catalytic domain.

Promatrilysin expressed in Escherichia coli and Chinese hamster ovary cells contains 2.36 +/- 0.19 and 2.13 +/- 0.39 moles of zinc per mole of protein, respectively, while the activated enzyme contains 2.22 +/- 0.21. The catalytic domain of stromelysin-1 expressed in E. coli contains 2.22 +/- 0.11. Thus these matrix metalloproteinases contain two metal binding sites at which zinc is bound firmly and possibly a third site at which it is bound weakly. Promatrilysin and matrilysin do not contain significant amounts of Fe, Cu, Mn, or Ni. All known matrix metalloproteinases have a sequence homologous to the zinc binding site of astacin, HExxHxxGxxH, suggesting that one of the zinc sites is catalytic in agreement with the known inhibition of these enzymes by chelators.

Contribution of the C-terminal domain of metalloproteinases to binding by tissue inhibitor of metalloproteinases. C-terminal truncated stromelysin and matrilysin exhibit equally compromised binding affinities as compared to full-length stromelysin.

In this study, we have used high resolution gel-filtration chromatography and measurements of Ki to compare the capacity of full-length native stromelysin, C-terminal truncated stromelysin (Phe100-Pro273), and matrilysin (the only metalloproteinase spontaneously lacking a C-terminal hemopexin-like domain) to bind to the tissue inhibitor of metalloproteinases (TIMP). While prostromelysin failed to bind TIMP, active stromelysin bound to the inhibitor avidly, exhibiting an affinity for TIMP (Ki = 8.3 x 10(-10) M) essentially identical to that of active interstitial collagenase as determined by competition experiments. C-terminal truncated stromelysin also formed a higher M(r) complex with TIMP which survived gel filtration. However, when truncated stromelysin was forced to compete with its full-length parent molecule for limiting amounts of TIMP, the full-length enzyme preferentially bound to the inhibitor. Indeed, binding studies indicated a Ki of 5.95 x 10(-9) M for the truncated variant's interaction with TIMP, only 14% as tight as that of full-length stromelysin. We also examined the interaction between TIMP and matrilysin, the only metalloproteinase which naturally lacks a C-terminal domain. Promatrilysin failed to bind the inhibitor. However, active matrilysin readily bound TIMP, forming a complex that resisted separation by gel filtration. When active matrilysin was forced to compete with truncated stromelysin for limiting amounts of TIMP, both enzymes appeared to complex the inhibitor with nearly equivalent efficacy. Indeed, active matrilysin exhibited a Ki for TIMP of 4.5 x 10(-9) M, essentially identical to that of truncated stromelysin. These data indicate that, as is true for collagenase, the C-terminal domain of stromelysin contributes significantly to its capacity to bind the physiologic inhibitor, TIMP. Furthermore, since stromelysin readily processes in vitro to a C-terminal truncated form, this enzyme species, as well as the full-length metalloproteinase matrilysin, may resist inhibition by TIMP in areas of active inflammation in vivo.

A recombinant human stromelysin catalytic domain identifying tryptophan derivatives as human stromelysin inhibitors.

The human stromelysin catalytic domain (SCD) has been expressed in Escherichia coli and purified to homogeneity (Ye et al. Biochemistry 1992, 31, 11231). We have used this recombinant SCD for inhibitor screening and identified tryptophan derivatives as competitive inhibitors of SCD. Both Cbz-L-Trp-OH (1, IC50 2.5 microM, Ki 2.1 microM) and Boc-L-Trp-OH (3, IC50 10 microM, Ki 8 microM) showed good inhibitory activity. Modification at the indole nitrogen with formyl or mesitylene-2-sulfonyl group (16, IC50 34 microM, Ki 28 microM; 17, IC50 63 microM, Ki 52 microM) showed reduced activity. The amide Cbz-L-Trp-NH2 (13) was not active, but esters Cbz-L-Trp-OSu (14, IC50 13 microM, Ki 11 microM) and Boc-L-Trp-OSu (15, IC50 102 microM, Ki 84 microM) showed activity. Aromatic amino acid derivatives Cbz-L-Tyr-OH (18, IC50 24 microM, Ki 20 microM) and Cbz-L-Phe-OH (26, IC50 40 microM, Ki 33 microM) were also active, but other amino acid derivatives had no activity. Although Cbz-D-Trp-OH (2, IC50 86 microM, Ki 71 microM) was active, the L-configuration is consistently preferred for inhibitory activity. Some of the SCD inhibitors were tested on full-length human stromelysin purified from cultured human cells, and they showed the same potency rank order. These results demonstrate the usefulness of recombinant DNA technology in generating the authentic human protein with improved properties for drug discovery.

Assignments for the main-chain nuclear magnetic resonances and delineation of the secondary structure of the catalytic domain of human stromelysin-1 as obtained from triple-resonance 3D NMR experiments.

We report the NMR assignments for the main-chain 13C, 15N, and 1H resonances (1HN, 1H alpha, 15N alpha, 13C alpha, 13CO) for the 19.5-kDa catalytic domain of human stromelysin-1, a zinc endoproteinase thought to be involved in pathologic tissue degradation. The assignments were predominantly obtained from triple-resonance three-dimensional NMR experiments using double-labeled (15N/13C) samples. The secondary structure of the molecule was determined from analysis of 3D 15N-resolved NOESY experiments. It was found to consist of a five-stranded mixed beta-sheet with four parallel and one antiparallel strand and three helices. The topological arrangement of the secondary structure elements of stromelysin catalytic domain is remarkably similar to that found for astacin, a Zn proteinase for which the tertiary structure was recently determined from X-ray diffraction data [Bode et al. (1992) Nature 358, 164-167].

HIV1 integrase expressed in Escherichia coli from a synthetic gene.

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Purification and characterization of the human stromelysin catalytic domain expressed in Escherichia coli.

Human stromelysin is a member of the matrix metalloproteinase family involved in connective tissue degradation. The stromelysin catalytic domain (SCD) lacking both propeptide and C-terminal fragment was expressed in Escherichia coli in soluble and insoluble forms. The insoluble SCD was refolded to the active form in high yield. The protein showed remarkable thermal stability and was able to cleave a thiopeptolide substrate and its natural substrate proteoglycan. The stable and active 20-kDa protein provides an opportunity to elucidate the structure as well as the mechanism of catalysis and inhibition for matrix metalloproteinases.

Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase.

The gene for PUMP (putative metalloproteinase), a human matrix metalloproteinase, was synthesized by a PCR-based method. The DNA fragment of 546 bases containing the PUMP gene was generated by overlap extension of six long oligonucleotides (length ranging from 101 to 116 bases) and subsequent amplification by two short terminal oligonucleotide primers (length from 20 to 48 bases) in one pot without using restriction and ligation enzymes. The synthetic gene was cloned into a T7 expression vector in two ways to express PUMP as a non-fusion protein. Both constructs showed high level expression in E. coli.

Magnesium levels in middle ear fluids and sera in otitis media with effusion.

Middle ear fluids (MEE) and matched sera (S) were obtained from 50 patients with serous otitis media and magnesium levels were measured to determine if magnesium concentration was distinctly varied in otitis media with effusion (OME). The MEE/S ratio was considerably raised along with transient sensory hearing loss in chronic OME when compared with acute OME. The higher magnesium level found in the MEE implies that it is probably produced locally by the middle ear mucosa and may contribute to the hearing loss found. We also regard the MEE/S ratio as a prognostic factor in OME.

p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase.

The Escherichia coli pabA and pabB genes have been overexpressed separately and in tandem. Using purified PabB, we have confirmed the recent suggestion that PabB needs an additional protein, enzyme X, to convert chorismate and NH3 to p-aminobenzoate (PABA). With chorismate and NH3, pure PabB generates an intermediate presumed to be 4-amino-4-deoxychorismate based upon UV/visible spectroscopy and enzymatic and nonenzymatic transformations. The PabB-catalyzed interconversion of chorismate and isolated aminodeoxychorismate is readily reversible. With pure PabB as a stoichiometric assay reagent, enzyme X was purified approximately 800-fold to near homogeneity as an apparent homodimer of 50 kDa from E. coli. Enzyme X shows no activity on chorismate but quantitatively converts the preformed aminodeoxychorismate into p-aminobenzoate and pyruvate, acting thereby as an aminodeoxychorismate lyase.