Towards third generation matrix metalloproteinase inhibitors for cancer therapy

The failure of matrix metalloproteinase (MMP) inhibitor drug clinical trials in cancer was partly due to the inadvertent inhibition of MMP antitargets that counterbalanced the benefits of MMP target inhibition. We explore how MMP inhibitor drugs might be developed to achieve potent selectivity for validated MMP targets yet therapeutically spare MMP antitargets that are critical in host protection

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

Type IV collagen remodeling plays a critical role in inflammatory responses, angiogenesis and metastasis. Its remodeling is executed by a family of matrix metalloproteinases (MMPs), of which the constitutive gelatinase A (MMP2) and the inducible gelatinase B (MMP9) are key examples. Thus, in many pathological conditions, both gelatinases act together. Kinetic data are reported for the enzymatic processing at 37 degrees C of type IV collagen from human placenta by MMP9 and its modulation by the fibronectin-like collagen binding domain (CBD) of MMP2. The alpha l and alpha 2 chain components of type IV collagen were cleaved by gelatinases and identified by mass spectrometry as well as Edman sequencing. Surface plasmon resonance interaction assays showed that CBD bound type IV collagen at two topologically distinct sites. On the basis of linked-function analysis, we demonstrated that CBD of MMP2 tuned the cleavage of collagen IV by MMP9, presumably by inducing a ligand-linked structural change on the type IV collagen. At low, concentrations, the CBD bound the first site and thereby allosterically modulated the binding of MMP9 to collagen IV, thus enhancing the collagenolytic activity of MMP9. At high concentrations, CBD binding to the second site interfered with MMP9 binding to collagen IV, acting as a competitive inhibitor. Interestingly, modulation of collagen IV degradation by inactive forms of MMP2 also occurred in a cell-based system, revealing that this interrelationship affected neutrophil migration across a collagen IV membrane. The regulation of the proteolytic processing by a catalytically inactive domain (i.e., CBD) suggests that the two gelatinases might cooperate in degrading substrates even when either one is inactive. This observation reinforces the idea of exosite targets for MMP inhibitors, which should include all macromolecular substrate recognition site

Direct Visualization of Protease Action on Collagen Triple Helical Structure

Enzymatic processing of extracellular matrix (ECM) macromolecules by matrix metalloproteases (MMPs) is crucial in mediating physiological and pathological cell processes. However, the molecular mechanisms leading to effective physiological enzyme-ECM interactions remain elusive. Only scant information is available on the mode by which matrix proteases degrade ECM substrates. An example is the enzymatic degradation of triple helical collagen II fragments, generated by the collagenase MMP-8 cleavage, during the course of acute inflammatory conditions by gelatinase B/MMP-9. As is the case for many other matrix proteases, it is not clear how MMP-9 recognizes, binds and digests collagen in this important physiological process. We used single molecule imaging to directly visualize this protease during its interaction with collagen fragments. We show that the initial binding is mediated by the diffusion of the protease along the ordered helix on the collagen ¾ fragment, with preferential binding of the collagen tail. As the reaction progressed and prior to collagen degradation, gelatin-like morphologies resulting from the denaturation of the triple helical collagen were observed. Remarkably, this activity was independent of enzyme proteolysis and was accompanied by significant conformational changes of the working protease. Here we provide the first direct visualization of highly complex mechanisms of macromolecular interactions governing the enzymatic processing of ECM substrates by physiological protease

An investigation into the prevalence of dog bites to primary school children in Trinidad

<p>Abstract</p> <p>Background</p> <p>To estimate the prevalence of dog bites to primary school children between the ages of 8–12 years using a semi-structured interview process. With the increase in the pet population and popularity of dangerous breeds of dog and a high stray dog population combined with a dearth of information on the risk of dog attacks to children in Trinidad, a semi-structured interview process was used to determine risk factors associated with dog attacks.</p> <p>Methods</p> <p>A questionnaire survey of 1109 primary school children between the ages of 8–12 years was conducted in Trinidad from November 2002 to September 2003. The survey was conducted to determine the risk factors such as age, gender, size of dog and relationship of dog and victim, in dog bite incidents. The chi-square statistic and odds ratios were used to estimate risk factors for a bite incident.</p> <p>Results</p> <p>Twenty-eight percent of children were bitten at least once by a dog. Gender (male) and owning a dog were statistically significant risk factors (p = 0.003 and 0.008 respectively, χ²<it>df</it>, 95% confidence). Most attacks occurred outside of the home (58.0%) followed by the victims' home (42.0%) and were by a dog known but not owned (54.6%) by the victim. Many victims (33.0%) were bitten without having any interaction with the dog and the majority (61.9%) of victims did not receive professional medical assistance. Overall, the lower leg or foot was most often injured (39.3%).</p> <p>Conclusion</p> <p>A public educational campaign is needed on responsible pet ownership. In addition, children must be taught effective ways of avoiding attacks or reducing injury in the event of a dog attack. The Dangerous dogs Act 2000 must be proclaimed in parliament by the Government of Trinidad and Tobago to exert more pressure on pet owners to safeguard the public from the menace of dog attacks.</p

Pleiotropic functions of the tumor- and metastasis-suppressing Matrix Metalloproteinase-8 in mammary cancer in MMTV-PyMT transgenic mice

Matrix metalloproteinase-8 (MMP-8; neutrophil collagenase) is an important regulator of innate immunity which has onco-suppressive actions in numerous tumor types

Anti-dsDNA Antibodies Promote Initiation, and Acquired Loss of Renal Dnase1 Promotes Progression of Lupus Nephritis in Autoimmune (NZBxNZW)F1 Mice

BACKGROUND:Lupus nephritis is characterized by deposition of chromatin fragment-IgG complexes in the mesangial matrix and glomerular basement membranes (GBM). The latter defines end-stage disease. METHODOLOGY/PRINCIPALS: In the present study we determined the impact of antibodies to dsDNA, renal Dnase1 and matrix metalloprotease (MMP) mRNA levels and enzyme activities on early and late events in murine lupus nephritis. The major focus was to analyse if these factors were interrelated, and if changes in their expression explain basic processes accounting for lupus nephritis. FINDINGS:Early phases of nephritis were associated with chromatin-IgG complex deposition in the mesangial matrix. A striking observation was that this event correlated with appearance of anti-dsDNA antibodies and mild or clinically silent nephritis. These events preceded down-regulation of renal Dnase1. Later, renal Dnase1 mRNA level and enzyme activity were reduced, while MMP2 mRNA level and enzyme activity increased. Reduced levels of renal Dnase1 were associated in time with deficient fragmentation of chromatin from dead cells. Large fragments were retained and accumulated in GBM. Also, since chromatin fragments are prone to stimulate Toll-like receptors in e.g. dendritic cells, this may in fact explain increased expression of MMPs. SIGNIFICANCE:These scenarios may explain the basis for deposition of chromatin-IgG complexes in glomeruli in early and late stages of nephritis, loss of glomerular integrity and finally renal failure

Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates

Maintenance of response with atypical antipsychotics in the treatment of schizophrenia: a post-hoc analysis of 5 double-blind, randomized clinical trials

<p>Abstract</p> <p>Background</p> <p>How long an antipsychotic is effective in maintaining response is important in choosing the correct treatment for people with schizophrenia. This post-hoc analysis describes maintenance of response over 24 or 28 weeks in people treated for schizophrenia with olanzapine, risperidone, quetiapine, ziprasidone, or aripiprazole.</p> <p>Methods</p> <p>This was a post-hoc analysis using data from 5 double-blind, randomized, comparative trials of 24 or 28 weeks duration in which olanzapine was compared to risperidone (1 study; N = 339), quetiapine (1 study; N = 346), ziprasidone (2 studies; N = 548 and 394) or aripiprazole (1 study; N = 566) for treatment of schizophrenia. For each study, time to loss of response in patients who met criteria for response at Week 8 and the proportion of patients who lost response following Week 8 were compared by treatment group. The number needed to treat (NNT) with olanzapine rather than comparator to avoid loss of one additional responder over 24 or 28 weeks of treatment was calculated for each study.</p> <p>Results</p> <p>Time maintained in response was significantly longer (p < .05) for olanzapine compared to risperidone, quetiapine, and ziprasidone. Olanzapine did not significantly differ from aripiprazole. The proportion of patients who lost response was significantly lower for olanzapine versus risperidone, quetiapine, and ziprasidone (p < .05). NNTs to avoid one additional patient with loss of response with olanzapine versus risperidone, quetiapine and ziprasidone were favourable, ranging from 5 to 9.</p> <p>Conclusion</p> <p>During 24 and 28 weeks of treatment, the antipsychotics studied differed in the time that treated patients with schizophrenia remained in response and the proportion of patients who lost response. Olanzapine treatment resulted in a consistent and statistically significant advantage in maintenance of response compared to treatment with risperidone, quetiapine and ziprasidone; but not compared to treatment with aripiprazole.</p

MMP28 (epilysin) as a novel promoter of invasion and metastasis in gastric cancer

Background\ud The purpose of this study was to investigate invasion and metastasis related genes in gastric cancer.\ud \ud Methods\ud The transwell migration assay was used to select a highly invasive sub-line from minimally invasive parent gastric cancer cells, and gene expression was compared using a microarray. MMP28 upregulation was confirmed using qRT-PCR. MMP28 immunohistochemistry was performed in normal and gastric cancer specimens. Invasiveness and tumor formation of stable cells overexpressing MMP28 were tested in vitro and in vivo.\ud \ud Results\ud MMP28 was overexpressed in the highly invasive sub-cell line. Immunohistochemistry revealed MMP28 expression was markedly increased in gastric carcinoma relative to normal epithelia, and was significantly associated with depth of tumor invasion, lymph node metastasis and poorer overall survival. Ectopic expression of MMP28 indicated MMP28 promoted tumor cell invasion in vitro and increased gastric carcinoma metastasis in vivo.\ud \ud Conclusions\ud This study indicates MMP28 is frequently overexpressed during progression of gastric carcinoma, and contributes to tumor cell invasion and metastasis. MMP28 may be a novel therapeutic target for prevention and treatment of metastases in gastric cancer

The Role of MMP7 and Its Cross-Talk with the FAS/FASL System during the Acquisition of Chemoresistance to Oxaliplatin

Background: The efficacy of oxaliplatin in cancer chemotherapy is limited by the development of drug resistance. MMP7 has been related to the loss of tumor cell response to cytotoxic agents although the exact mechanism is not fully understood. Moreover, MMP7 is an independent prognosis factor for survival in patients with colorectal cancer. The aim of the present study was to analyze the role of MMP7 and its cross-talk with the Fas/FasL system during the acquisition of oxaliplatin resistance in colon cancer cells. Principal Findings: For this purpose we have developed three different oxaliplatin-resistant cell lines (RHT29, RHCT116 p53+/+, RHCT116 p53−/−) from the parental HT29, HCT116 p53+/+ and HCT116 p53−/− colon cancer cells. MMP7 basal expression was higher in the resistant compared to the parental cell lines. MMP7 was also upregulated by oxaliplatin in both HT29 (p53 mutant) and RHCT116 p53−/− but not in the RHCT116 p53+/+. Inhibition of MMP by 1,10-phenantroline monohydrate or siRNA of MMP7 restores cell sensitivity to oxaliplatin-induced apoptosis in both HT29 and RHCT116 p53−/− but not in the RHCT116 p53+/+. Some of these effects are caused by alterations in Fas receptor. Fas is upregulated by oxaliplatin in colon cancer cells, however the RHT29 cells treated with oxaliplatin showed a 3.8-fold lower Fas expression at the cell surface than the HT29 cells. Decrease of Fas at the plasma membrane seems to be caused by MMP7 since its inhibition restores Fas levels. Moreover, functional analysis of Fas demonstrates that this receptor was less potent in inducing apoptosis in RHT29 cells and that its activation induces MAPK signaling in resistant cells. Conclusions: Taking together, these results suggest that MMP7 is related to the acquisition of oxaliplatin-resistance and that its inhibition restores drug sensitivity by increasing Fas receptor. Furthermore, Fas undergoes a change in its functionality in oxaliplatin-resistant cells inducing survival pathways instead of apoptotic signals