SELECTIVE INHIBITION OF MATRIX METALLOPROTEINASES BY NEW HYDROXAMATE COMPOUNDS

Transcription

1 ORIGINAL ARTICLES SELECTIVE INHIBITION OF MATRIX METALLOPROTEINASES BY NEW HYDROXAMATE COMPOUNDS ELENA GANEA 1*, CRISTINA LASLO 1, IOANA GOLDNER 1, GABRIELA PUTINA 2 1 Institute of Biochemistry of the Romanian Academy, Splaiul Independenţei 296, Bucharest, Romania 2 National Institute of Chemical Pharmaceutical Research and Development (NICPRD), Bucharest, Romania (Received September 18, 28) Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes that participate in the degradation of extracellular-matrix components. These enzymes play a key role in the regulation of the extracellular matrix integrity and composition. Other zinc-dependent peptidases include leucine aminopeptidase (LAP), an enzyme which catalyses the hydrolysis of leucine residues from the N- terminal end of polypeptides, and carboxypeptidase A (CxpA), which cleaves the peptide bond adjacent to C-terminal end of polypeptide chain. MMP activity is regulated by specific tissue inhibitors, and the lack of balance between enzymatic activity and inhibition may play an important role in the pathology of different diseases, such as HIV and cancer. As a consequence, the design and synthesis of various classes of metalloproteinase inhibitors are of a great importance for a possible use as therapeutical agents. The present paper investigates the inhibitory effect of a series of hydroxamate-based compounds (C1, C2, C3), newly synthesized at NICPRD, Bucharest, Romania, on MMPs. The selective inhibition induced by these compounds was demonstrated on various enzymes (MMP, LAP, Cxp A), suggesting that these compounds could be considered for clinical trial. Key words: selective inhibition, matrix metalloproteinases, hydroxamate compounds. INTRODUCTION Matrix metalloproteinases are a family of Zn-dependent enzymes that participate in the degradation process of the extracellular matrix components, such as elastin, collagen, laminin, fibronectin and the protein part of proteoglycans. These enzymes play a key role in the regulation of the extracellular matrix integrity and composition, MMP function being much more complex than the simple degradation of the extracellular matrix. Moreover, the number of substrates not * Corresponding author ( eganea24@yahoo.com, Tel.: ) ROM. J. BIOCHEM., 46, 1, 3 12 (29)

2 4 Elena Ganea et al. 2 originating from the extracellular matrix is continuously growing, underlining the importance of MMPs in human physiology and pathology (1). MMP activity is regulated by protease inhibitors, such as α 2 -macroglobulin, and by a group of specific tissue inhibitors of metalloproteinases (TIMPs). TIMPs are kda proteins that contain three highly conserved disulfide bonds. They inhibit MMP activity by forming high affinity non-covalent complexes (2, 3). Four types of endogenous inhibitors are known in vertebrates (TIMP 1 4), their expression being regulated during tissue development and remodeling. Alterations of TIMP values are very important under pathological conditions associated with abnormal MMP activity. A lack of balance between activity and inhibition can play an important role in the pathology of different diseases such as HIV, cancer, etc. Although TIMPs inhibit the activity of all MMPs tested so far, their use as therapeutical agents in cardiovascular diseases and cancer is still at an early stage of research. Lately, there has been a growing interest in testing different types of synthetic Zn-dependent metalloproteinase inhibitors, the final goal being their possible use as therapeutical agents. Of the inhibitors synthesized so far (carboxylates, organoborates, dithiolates, hydroxamates), hydroxamate-based inhibitors seem to be the most promising. In this paper we have studied the inhibitory effect of three new heterocyclic hydroxamate compounds on the enzymatic activity of matrix metalloproteinases from bovine brain, in comparison to the activity of two Zn-dependent peptidases: leucine aminopeptidase and carboxypeptidase A (LAP and CxpA). MATERIALS AND METHODS MATERIALS The matrix metalloproteinases MMP2 and MMP12 were purchased from Calbiochem. Other chemicals, including leucine aminopeptidase, L-leucine amide, carboxypeptidase and hyppuril L-phenylalanine, were obtained from Sigma-Aldrich. METHODS Matrix metalloproteinase separation from bovine brain The bovine brain homogenate was obtained by manual homogenization, as a first step, and then with the help of a mechanical homogenizer, with.25 M saccharose (1/3, w/v). After 3 minutes of centrifugation at 15 rpm, the supernatant was intensively dialysed against water and centrifuged again at 15 rpm for 3 minutes. The protein concentration was determined by the Bradford method.

3 3 Matrix metalloproteinases inhibition 5 Protein purification from brain homogenate by gel filtration and SDS-PAGE characterisation Protein homogenate (2 mg protein) was separated on a Sephadex G-1 column (7 1.2 cm), and eluted by.1m Na phosphate buffer ph 7.4. Fractions of 1.5 ml were collected. Absorption values at 28 nm indicated two major protein peaks, followed by two minor fractions. The protein peaks were characterised by SDS-PAGE separation and Coomassie Brilliant Blue R25 staining. Bovine brain matrix metalloproteinases enzymatic activity assay by gelatin zymography The proteins from the bovine brain homogenate were separated by SDS-PAGE under non-reducing conditions, on 7.5% polyacrylamide gel containing 1 mg/ml gelatin (4). After electrophoresis, the polyacrylamide gel was incubated for 1 hour in 2.5% Triton X-1 for SDS removal. The gel was then incubated at 37 C for 24 hours in 5 mm Tris-HCl buffer ph 7.5, containing 5 mm CaCl 2 and.5% Triton X-1, so as to allow the matrix metalloproteinases from the bovine brain homogenate to break down the gelatin contained by the gel. Lytic bands were visualised by staining the gels with Coomassie Brilliant Blue R25 and destaining in 1% acetic acid. The quantitative analysis of the bands was performed using Gel Doc/Quantity One (Bio Rad). Enzymatic activity assay of Zn-dependent peptidases Leucine aminopeptidase (LAP) activity was determined spectrophotometrically, by measuring the absorbance at λ=238 nm for 1 minutes, at 25 C. The decrease in absorbance is proportional to the rate of the substrate (L-leucine amide) hydrolysis. Carboxypeptidase A (CxpA) activity was assayed using the substrate hyppuril L-phenylalanine in.25 M Tris-HCl buffer ph 7.5, containing.5 M NaCl, and measuring the absorbance at 254 nm for 3 5 minutes. The enzymatic activity is calculated using the formula: Units/mg = A 254 / min.36* mg enzyme/ml reaction mixture where *= millimolar extinction coefficient of hippuric acid at 254 nm. ELISA analysis of bovine brain MMP2 and MMP12 MMP2 and MMP12 were estimated in the bovine brain homogenate, in the presence and absence of the C1, C2, C3 hydroxamate compounds by ELISA

4 6 Elena Ganea et al. 4 method (6). For this purpose, the plates were sensitized by incubation of aliquots of the peaks separated by gel filtration for 1 hour at 37 C. The non-specific sites were blocked by incubation with.5% gelatin for 1 hour at 37 C; rabbit anti-mmp2 and rabbit anti-mmp12 (1/1 dilution) were used as primary antibodies, and rabbit anti-igg conjugated with alkaline phosphatase as secondary antibodies. Quantitative estimation was achieved by reading the absorbance at λ=45 nm, which is the specific wavelength for PNPP. Estimation of the inhibitory effect of newly synthesized compounds The inhibitory effect of three heterocyclic, hydroxamate-based compounds, C1 (MW = ), C2 (MW= ) and C3 (MW = ), synthesized at NICPRD, was tested at various concentrations (.2,.5, 1, and 2 mm), obtained from a stock solution of 1 mm inhibitor in DMSO. The inhibitory activity of the above mentioned compounds was estimated for various proteases: enzymes from bovine brain homogenate (6 mg/ml total protein), pure MMPs (MMP2 and MMP12), and Zn-dependent peptidases (LAP, CxpA). RESULTS AND DISCUSSION Recent data indicate the overexpression of matrix metalloproteinases in human cerebral tissue following ischemic and hemorrhagic shock, suggesting that some MMPs may contribute to cerebral lesions produced under these conditions (7). Other studies indicate that MMP9 contributes to the alteration of the hematoencephalic barrier and to the formation of edema caused by brain traumatisms (8). Such results suggest the importance of synthesizing selective inhibitors of central nervous system MMPs, these inhibitors having the potential to become valuable therapeutic agents Fig. 1. PAGE analysis of protein fractions separated from bovine brain homogenate by Sephadex G-1 gel filtration. 1 Brain homogenate; 2 Fraction I; 3 Fraction II.

5 5 Matrix metalloproteinases inhibition 7 In this paper, matrix metalloproteinases were partially purified from bovine brain (Fig. 1) and used, along with other proteases, as enzymatic substrates for testing the activity of newly synthesized hydroxamate compounds. Inhibitory effect of the heterocyclic compounds on bovine brain MMPs enzymatic activity Brain homogenate proteins separated by Sephadex G-1 gel filtration and incubated with the hydroxamate compounds C1, C2 and C3 induced a differential inhibition of enzymatic activity, as seen in figure 2. The zymograms illustrated in figure 2 (A1 and B1) show a decrease of the lytic bands intensity corresponding to the enzymes incubated with C1, C2 and C3, as compared to the native enzymes. Quantification of the lytic bands was performed with a Gel-Doc densitometer (Bio-Rad) and illustrated in figure 2 (A2 and B2). The results show that all of the hydroxamate compounds inhibit the matrix metalloproteinases from the protein peaks 1 and 2, including MMP2 and P1 P1+C1 P1+C2 P1+C3 A1 P2 P2+C1 P2+C2 P2+C3 Band area (CNTx mm) P1 P1+C1 P1+C2 P1+C3 A2 Band area (CNTx mm) P2 P2+C1 P2+C2 P2+C3 P1+C3 B1 Fig. 2. The inhibitory effect of C1, C2 and C3 on enzymatic activity of matrix metalloproteinases separated from bovine brain homogenate by Sephadex G-1. B2

6 8 Elena Ganea et al. 6 MMP12. The quantitative estimation of the lytic bands corresponding to the gelatinolytic activity of the matrix metalloproteinases incubated in the presence of each of the three hydroxamate compounds shows a selective inhibitory effect on the enzymes of the two protein peaks: compound C1 provides the strongest inhibition of the enzymes in peak 1, while compound C3 shows the strongest inhibition of the proteinases in peak 2 (Fig. 2). ELISA analysis of the C1, C2 and C3 effect on MMP2 and MMP12 The quantitative estimation of C1, C2 and C3 effect on MMP2 and MMP12 is illustrated in figures 3 and 4. A 45 nm (% of control) MMP2 MMP2+C1 MMP2+C2 MMP2+C3 MMP12 MMP12+C1 MMP12+C2 MMP12+C3 MMP2 MMP12 Fr. 1 2 Fig. 3. ELISA assay of MMP2 and MMP12 in fraction I of the bovine brain homogenate, and the effect of C1, C2, and C3 hydroxamate compounds (.1 mm). The results show that the binding of metalloproteinases from fraction 1 and fraction 2 of the brain homogenate to anti-mmp2 and anti-mmp12 antibodies, respectively, was disturbed by the presence of C1, C2, and C3 compounds. As shown in figures 3 and 4, MMP2 is much stronger affected by the presence of inhibitors than MMP12, both in the first and second fraction of the homogenate. Although the differences between the effects of the three compounds on the same enzyme are not always significant, C3 displays a significantly more intense inhibition of the enzyme binding to specific antiserum than the other two compounds.

7 7 Matrix metalloproteinases inhibition 9 12 A 45 nm.(% of control) MMP2 MMP2+C1 MMP2+C2 MMP2+C3 MMP12 MMP12+C1 MMP12+C2 MMP12+C3 MMP2 MMP12 Fr. 2 2 Fig. 4. ELISA assay of MMP2 and MMP12 in fraction II of the bovine brain homogenate, and the effect of C1, C2, and C3 hydroxamate compounds (.1 mm). C1, C2 and C3 hydroxamate compounds selectively inhibit Zn-dependent peptidases Leucine aminopeptidase (LAP) is a Zn-dependent exopeptidase which is generally localized in the cytosol, but it is also found in microsomes. It catalyses the hydrolysis of amino acids, especially substrates containing leucine from the amino-terminal end of polypeptide chains (9). Carboxypeptidase A (CxpA) is also a Zn-dependent metallopeptidase, an exopeptidase that cleaves carboxyterminal amino acids from polypeptides, preferably those containing aromatic ramifications. The enzyme is involved in certain types of cancers (1) and it is resistant to the usual protease inhibitors, such as PMSF (phenylmethanesulphonyl fluoride), facts which only underline the importance of testing the inhibitory effect of hydroxamate compounds. LAP was incubated for 1 hour at 4ºC, in the presence and absence of C1-C3 heterocyclic compounds; subsequently, the enzymatic activity was estimated by monitoring the absorbance at 238 nm wavelength, for 1 minutes at 25 C. The results showed that LAP incubation in the presence of C1 induced different amounts of enzyme inactivation, depending on the inhibitor concentration (Fig. 5). Thus, incubation of LAP with.2 mm,.6 mm and.1 mm C1 leads to 43.4%, 62.2% and 77.4% inhibition, respectively. When incubated with C2 at the same three concentrations used for C1, LAP enzymatic activity was inhibited by

8 1 Elena Ganea et al %, 87.4% and 87%, respectively. Unlike the previous two compounds, C3 inhibited LAP activity almost completely, i.e. 79.3%, 94.2% and 92.5% inhibition, respectively, even at the smallest concentration tested (Fig. 5). 1 Enzymic activity (% of control) LAP+C1 LAP+C2 LAP+C Heterocyclic compound concentration (mm) Fig. 5. The effect of various concentrations of C1, C2 and C3 compounds on enzymatic activity of leucine aminopeptidase. Figure 6 illustrates CxpA enzymatic activity after incubation in the absence and presence of C1, C2, C3 at concentrations identical to those used for LAP inhibition. The results showed different percentages of enzyme inhibition, according to inhibitor concentration (Fig. 6). Incubation of CxpA in the presence of.2 mm,.6 mm and.1 mm C1 induced 4.72%, 78.24% and 81.77% inhibition, respectively. Unlike C1, the effect of C2 on carboxypeptidase A activity did not depend on inhibitor concentration, incubation with all three C2 concentrations leading to 58.83%, 62.4% and 55.83% inhibition, respectively. C3 compound proved to be a stronger inhibitor than C1 and C2, as it was shown to provide 55.89%, 83.53% and 88.6% inhibition, respectively. Similarly with C1, C3 inhibitory effect also depends on the inhibitor concentration (Fig. 6). In conclusion, the heterocyclic hydroxamate compounds (C1, C2, C3) studied in this work display an inhibitory effect on the protease activity of both matrix metalloproteinases from the brain and Zn-dependent peptidases, leucine aminopeptidase and carboxypeptidase A.

9 9 Matrix metalloproteinases inhibition 11 1 Enzymic activity (%of control) CxpA + C1 CxpA + C2 CxpA + C Heterocyclic compound concentration (mm) Fig. 6. The effect of various concentrations of C1, C2 and C3 compounds on enzymatic activity of carboxypeptidase A. The inhibitory effect of the above mentioned compounds is selective for the type of enzyme they inhibit and the amount of inhibition. REFERENCES 1. Nagase H., Visse R., Murphy G., Structure and function of matrix metalloproteinases and TIMPs, Cardiovascular Res., 69, (26). 2. Visse R., Nagase H., Matrix metalloproteinases and tissue inhibitors of metalloproteinases, structure, function, and biochemistry, Circ. Res., 92, (23). 3. Bokarewa M., Dahlberg L., Tarkowski A., Expression and functional properties of antibodies to tissue inhibitors of metalloproteinases (TIMPs) in rheumatoid arthritis, Arthritis Research & Therapy, 7, (25). 4. Li B.H., Zhao P., Liu S.Z., Yu Y.M., Han M., Wen J.K., Matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in colorectal carcinoma invasion and metastasis, World J. Gastroenterol., 11, (25). 5. Binkley F., Torres C., Spectrophotometric assay of peptidase activity, Arch. Biochem. Biophys., 86, (196). 6. Harlow E., Lane D., in: Antibodies: A laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1988). 7. Rosell A., Ortega-Aznar A., Alvarez-Sabin J., Fernandez-Cadenas I., Ribo M., Molina C.A., Lo E.H., Montaner J., Increased Brain Expression of Matrix Metalloproteinase-9 After Ischemic and Hemorrhagic Human Stroke, Stroke, 37, (26).

10 12 Elena Ganea et al Shigemori Y., Katayama Y., Mori T., Maeda T., Kawamata T., Matrix metalloproteinase-9 is associated with blood-brain barrier opening and brain edema formation after cortical contusion in rats, Acta Neurochir. Suppl., 96, (26). 9. Himmelhoch S., Leucine Aminopeptidase: A Zinc Metalloenzyme, Arch. Biochem. Biophys., 134, (1969). 1. Hao X.K., Liu J.Y., Yue Q.H., Wu G.J., Bai Y.J., Yin Y., In vitro and in vivo prodrug therapy of prostate cancer using anti-gamma-sm-scfv/hcpa fusion protein, Prostate, 66, (26).