Inhibition of matrix metalloproteinase-2 by PARP inhibitors

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC₅₀ values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.     

Binding affinity of hydroxamate inhibitors of matrix metalloproteinase-2

Here we report molecular dynamics (MD) and free energy perturbation (FEP) simulations applied to hydroxamate-matrix metalloproteinase-2 (MMP-2) complex systems. We have developed some new force field parameters for the hydroxamate functional group that were not included in the AMBER94 force field but were necessary in our simulations. For the representation of the active zinc center, a bonded model was adopted in which restrained electrostatic potential fitting (RESP) charges were used as the electrostatic representation of this model. Using the resulted bonded model, FEP simulations predict the relative binding free energy in good agreement with the experimental value. By analyzing the molecular dynamics (MD) trajectories of the two complex systems, we can provide an explanation of why one of the two inhibitors is favored over the other. The results provide a chemical insight into the interactions between inhibitor and enzyme, and can indicate changes in the inhibitor that would enhance inhibitor–enzyme interactions.Figure The scheme of the binding site     

Estimation of the binding affinities of FKBP12 inhibitors using a linear response method.

A series of non-immunosuppressive inhibitors of FK506 binding protein (FKBP12) are investigated using Monte Carlo statistical mechanics simulations. These small molecules may serve as scaffolds for chemical inducers of protein dimerization, and have recently been found to have FKBP12-dependent neurotrophic activity. A linear response model was developed for estimation of absolute binding free energies based on changes in electrostatic and van der Waals energies and solvent-accessible surface areas, which are accumulated during simulations of bound and unbound ligands. With average errors of 0.5 kcal/mol, this method provides a relatively rapid way to screen the binding of ligands while retaining the structural information content of more rigorous free energy calculations.     

Identification,purification and partial characterization of tissue inhibitor of matrix metalloproteinase-2 in bovine pulmonary artery smooth muscle

Bovine pulmonary artery smooth muscle possesses the tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) as revealed by Western immunoblot study of its cytosol fraction with bovine polyclonal TIMP-2 antibody. This potent polypeptide inhibitor of matrix metalloproteinases (MMPs) was purified to homogeneity from cytosol fraction of bovine pulmonary artery smooth muscle. This inhibitor was purified by ammonium sulfate precipitation followed by gelatin sepharose and lentil lectin sepharose affinity chromatography and continuous elution electrophoresis by Prep Cell Model 491 (Bio-Rad, USA). SDS-PAGE revealed that the inhibitor has an apparent molecular mass of 21 kDa and was confirmed as TIMP-2 by (i) Western immunoblot assay using bovine polyclonal TIMP-2 antibody; and also by (ii) amino terminal amino acid sequence analysis of the purified inhibitor is found to be identical with TIMP-2 obtained from other sources. The purified 21 kDa inhibitor was found to be active against matrix metalloproteinase-2 (MMP-2, 72 kDa gelatinase) and matrix metalloproteinase-9 (MMP-9, 92 kDa gelatinase), the ambient MMPs in the pulmonary artery smooth muscle. The inhibitor was also found to be sensitive to the activated 72 kDa gelatinase-TIMP-2 complex and also active human interstitial collagenase. By contrast, it was found to be insensitive to the serine proteases: trypsin and plasmin. The inhibitor was heat and acid resistant and it had the sensitivity to trypsin degradation and reduction-alkylation. Treatment of the inhibitor with hydrogen peroxide, superoxide generating system (hypoxanthine plus xanthine oxidase) and peroxynitrite inactivated the inhibitor.     

Insulin like growth factor-1 selectively regulates the expression of matrix metalloproteinase-2 in malignant H-ras transformed cells

The present study is designed to investigate the effect of myocardial preconditioning with oxidative stress induced by pyrogallol or H₂O₂, on ischaemia-reperfusion induced myocardial injuiry. Isolated perfused rat heart was subjected to global ischaemia for 30 min followed by reperfusion for 120 min. Coronary effluent was analysed for LDH and CK release to assess the degree of cardiac injury. Myocardial infarct size was estimated macroscopically using TTC staining. Four episodes of preconditioning induced by pyrogallol or hydrogen peroxide (H₂O₂) or ischaemia markedly reduced LDH and CK release in coronary effluent and decreased myocardial infarct size. Administration of polymyxin B, a protein kinase C (PKC) inhibitor, during pyrogallol, H₂O₂ or ischaemic preconditioning markedly attenuated the cardioprotective effect of preconditioning produced with oxidative stress or ischaemia. These results suggest that preconditioning with oxidative stress may provide cardioprotection similar to ischaemic preconditioning, against ischaemia-reperfusion injury and this cardioprotective effect may be mediated through activation of PKC.     

Suppressive properties of ginsenoside Rb2, a protopanaxadiol-type ginseng saponin,on reactive oxygen species and matrix metalloproteinase-2 in UV-B-irradiated human dermal keratinocytes

Ginsenosides, also known as ginseng saponins, are the principal bioactive ingredients of ginseng, which are responsible for its diverse pharmacological activities. The present work aimed to assess skin anti-photoaging properties of ginsenoside Rb2 (Rb2), one of the predominant protopanaxadiol-type ginsenosides, in human epidermal keratinocyte HaCaT cells under UV-B irradiation. When the cultured keratinocytes were subjected to Rb2 prior to UV-B irradiation, Rb2 displayed suppressive activities on UV-B-induced reactive oxygen species elevation and matrix metalloproteinase-2 expression and secretion. However, Rb2 at the used concentrations was unable to modulate cellular survivals in the UV-B-irradiated keratinocytes. In brief, Rb2 possesses a protective role against the photoaging of human keratinocyte cells under UV-B irradiation.     

A single nucleotide polymorphism in the matrix metalloproteinase-2 promoter is associated with colorectal cancer

Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism C-->T transition at -1306, which disrupts an Sp1-type promoter site (CCACC box), displayed a strikingly lower promoter activity with T allele. Our study investigated whether the MMP-2 -1306 C-->T polymorphism contributed to the development and progression of colorectal cancer in the Chinese population. One hundred twenty-six colorectal cancer patients and 126 age- and sex-matched controls were included in this study. PCR-based denaturing high performance liquid chromatography analysis and sequencing were used to determine the MMP-2 genotypes. MMP-2 expression of each genotype was analyzed in four colorectal cancer cell lines by semi-quantitative RT-PCR. The correlation between the genotypes and clinicopathological parameters among colorectal cancer cases was investigated. The results showed that the levels of MMP-2 mRNA expression in cell lines containing CC genotype were much higher compared with cell with CT genotype. The frequency of MMP-2 CC genotype was significantly higher in colorectal cancer patients when compared with controls (OR, 1.959; 95% CI, 1.055-3.637). Colorectal cancers with CC genotype were more common with serosa/adventitia layer involvement compared with CT+TT genotypes. Our data suggest that MMP-2 -1306 C-->T polymorphism may be associated with colorectal cancer development and invasion in the Chinese population.     

Binding modes of Bcl-2 homology 3 (BH3) peptides with anti-apoptotic protein A1 and redesign of peptide inhibitors: a computational study

The interaction between protein and peptide ligand is a challenging problem in molecular biology and drug design. The binding of the Bcl-2 homology 3 (BH3) peptide to the anti-apoptotic protein A1 was revealed as a critical step in the regulation of apoptosis. These BH3 peptides hold high structural similarity, but are diverse in their regulation abilities. Based on molecular simulations and MM-P(G)BSA methods, this work presented a detailed analysis on binding mechanism of the BH3 peptides derived from PUMA and BMF. Residue-level energy decomposition showed that the core regions of BH3 peptides maintain in stable helical conformations and the four conserved hydrophobic residues together with an invariant aspartic acid contribute the major driving force for binding, whereas their two terminal segments exhibit obvious flexibility and various binding modes. Such kind of behavior was suggested as the reason for binding diversity and selectivity of BH3 peptides. As a further step, several BH3-mimetic peptides have been redesigned by computational mutation. Those new peptides showed not only stronger affinities when binding to protein A1, as well transferable binding patterns at some specific positions. A long-range coupling effect was disclosed for BH3 peptides, side-chain orientation and binding contribution of terminal residues were even affected by mutations at large sequence interval. Overall, this work reports that the binding modes of BH3 peptides are primarily dependent on its two terminal segments. The computational methods applied herein are also demonstrated to be of great assistance in the rational design of peptide inhibitors.     

A computational analysis of the binding affinities of FKBP12 inhibitors using the MM-PB/SA method

The FK506-binding proteins have been targets of pharmaceutical interests over years. We have studied the binding of a set of 12 nonimmunosuppressive small-molecule inhibitors to FKBP12 through molecular dynamics simulations. Each complex was subjected to 1-ns MD simulation conducted in an explicit solvent environment under constant temperature and pressure. The binding free energy of each complex was then computed by the MM-PB/SA method in the AMBER program. Our MM-PB/SA computation produced a good correlation between the experimentally determined and the computed binding free energies with a correlation coefficient (R(2)) of 0.93 and a standard deviation as low as 0.30 kcal/mol. The vibrational entropy term given by the normal mode analysis was found to be helpful for achieving this correlation. Moreover, an adjustment to one weight factor in the PB/SA model was essential to correct the absolute values of the final binding free energies to a reasonable range. A head-to-head comparison of our MM-PB/SA model with a previously reported Linear Response Approximation (LRA) model suggested that the MM-PB/SA method is more robust in binding affinity prediction for this class of compounds.     

Binding of Clostridium difficile to Caco-2 epithelial cell line and to extracellular matrix proteins

Adhesion of Clostridium difficile to Caco-2 was examined as a function of monolayers polarization and differentiation. The number of adherent C. difficile C253 bacteria per cell strongly decreased when postconfluent 15-day-old monolayers were used (1.7 bacteria per cell versus 17.3 with 3-day-old monolayers). Following disruption of intercellular junctions by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid, a significant rise in the level of bacterial adhesion was observed, above all in postconfluent monolayers. Immunofluorescence studies of bacteria and transferrin receptor, a marker of basolateral pole of polarized monolayers, showed that C. difficile C253 adheres mainly to the basolateral surface of differentiated and undifferentiated polarized Caco-2 cells. Furthermore, binding of C. difficile C253 to several extracellular matrix proteins in vitro was demonstrated by an ELISA-based assay.     

A tale of two paralogs: human Transformer2 proteins with differential RNA-binding affinities

The Transformer2 (Tra2) proteins in humans are homologues of the Drosophila Tra2 protein. One of the two RNA-binding paralogs, Tra2β, has been very well-studied over the past decade, but not much is known about Tra2α. It was very recently shown that the two proteins demonstrate the phenomenon of paralog compensation. Here, we provide a structural basis for this genetic backup circuit, using molecular modelling and dynamics studies. We show that the two proteins display similar binding specificities, but differential affinities to a short GAA-rich RNA stretch. Starting from the 6-nucleotide RNA in the solution structure, close to 4000 virtual mutations were modelled on RNA and the domain–RNA interactions were studied after energy minimisation to convergence. Separately, another known 13-nucleotide stretch was docked and the domain–RNA interactions were observed through a 100-ns dynamics trajectory. We have also demonstrated the ‘compensatory’ mechanism at the level of domains in one of the domain repeat-containing RNA-binding proteins.     

Concomitant reduction of matrix metalloproteinase-2 secretion and intracellular reactive oxygen species following anti-sense inhibition of telomerase activity in PC-3 prostate carcinoma cells

Background: The level of activity of the telomerase has been shown to correlate with the degree of invasiveness in several tumor types. In addition, cellular redox state is believed to regulate the secretion of matrix metalloproteinase-2 (MMP-2).Aims: To determine the effect of anti-sense telomerase treatment of prostate cancer cells on MMP-2 activity, and the reactive oxygen and nitrogen species (two effectors of cellular redox state).Methods: Anti-sense oligonucleotide against RNA component of human telomerase (hTR) was introduced into the cells using Fugene-6 transfection reagent. The activity of telomerase was assessed using Telomere Repeat Amplification Protocol (TRAP assay). Activity of matrix metalloproteinase-2 (MMP-2) was determined by zymography. Levels of intracellular reactive oxygen species (ROS) and nitric oxide metabolites were measured by dichlorofluorescein diacetate (DCFH-DA) staining and Griess reagent, respectively. The level of apoptosis was determined using TUNEL assay.Results: TRAP assay showed more than 90% inhibition of telomerase activity after 72 h of transfection. Pro-MMP-2 activity was decreased down to 50% of the control levels. Intracellular reactive oxygen species were also significantly decreased. Neither apoptosis rate nor the level of nitric oxide metabolites was significantly different between anti-sense treated and control cells.Conclusions: Concomitant reduction of the pro-MMP-2 secretion and ROS in PC-3 cells following hTR inhibition suggests that over-activity of telomerase in cancer cells might increase the level of matrix metalloproteinase-2 and thus, be directly involved in the invasion process through enhancement of intracellular oxidative stress.     

The discovery of reverse tricyclic pyridone JAK2 inhibitors. Part 2: Lead optimization

This communication discusses the discovery of novel reverse tricyclic pyridones as inhibitors of Janus kinase 2 (JAK2). By using a kinase cross screening approach coupled with molecular modeling, a unique inhibitor–water interaction was discovered to impart excellent broad kinase selectivity. Improvements in intrinsic potency were achieved by utilizing a rapid library approach, while targeted structural changes to lower lipophilicity led to improved rat pharmacokinetics. This multi-pronged approach led to the identification of 31, which demonstrated encouraging rat pharmacokinetics, in vivo potency, and excellent off-target kinase selectivity.     

A platform for designing HIV integrase inhibitors. Part 2: a two-metal binding model as a potential mechanism of HIV integrase inhibitors

We propose a two-metal binding model as a potential mechanism of chelating inhibitors against HIV integrase (HIV IN) represented by 2-hydroxy-3-heteroaryl acrylic acids (HHAAs). Potential inhibitors would bind to two metal ions in the active site of HIV IN to prevent human DNA from undergoing the integration reaction. Correlation of the results of metal (Mg²⁺ and Mn²⁺) titration studies with HIV IN inhibition for a series of active and inactive compounds provides support for the model. Results suggest Mg²⁺ is an essential cofactor for chelating inhibitors.     

In Silico prediction of the molecular basis of ClTx and AaCTx interaction with matrix metalloproteinase-2 (MMP-2) to inhibit glioma cell invasion

Glioblastoma is the deadliest type of brain cancer. Treatment could target the Matrix metalloproteinase-2 (MMP-2), which is known to be involved in the invasion process of glioblastoma cells. But current available inhibitors are not selective to MMP-2 due to their interaction with the catalytic binding site, which is highly conserved in all MMPs structures. Interestingly, members of the chloride channel blocker scorpion toxins, such as chlorotoxin (ClTx) and AaCTx, inhibit glioblastoma cell invasion and show a promising therapeutic potential. Indeed, it has been shown that CITx inhibits selectively MMP-2 and was also able to cross the blood brain and tissue barriers. Although ClTx and AaCTx show high sequence similarity, AaCTx is ten times less active than ClTx. By using molecular modeling, molecular dynamics and MM-PB(GB)SA free energy estimation, we present the first computational study reporting the interaction mode of ClTx/AaCTx with MMP-2. We found that the two peptides probably act on an exosite of MMP-2 comprising mainly residues from the collagen binding domain, a feature that could be exploited to enhance the selectivity toward MMP-2. van der Waals and hydrophobic forces are the primary mediators of this interaction. The N- and C-termini of the two peptides harbor the key residues of the interaction spread across a conserved amino acid patch. In particular, F6 contributes mostly to the binding free energy in ClTx. We also suggest that the lack of the C-terminal arginine and the residues P10 and R24, might be responsible for altering the activity of AaCTx toward glioblastoma cells compared to ClTx.