Inhibitory antibodies designed for matrix metalloproteinase modulation

The family of matrix metalloproteinases (MMPs) consists of a set of biological targets that are involved in a multitude of severe pathogenic events such as different forms of cancers or arthritis. Modulation of the target class with small molecule drugs has not led to the anticipated success until present, as all clinical trials failed due to unacceptable side effects or a lack of therapeutic outcome. Monoclonal antibodies offer a tremendous therapeutic potential given their high target selectivity and good pharmacokinetic profiles. For the treatment of a variety of diseases there are already antibody therapies available and the number is increasing. Recently, several antibodies were developed for the selective inhibition of single MMPs that showed high potency and were therefore investigated in in vivo studies with promising results. In this review, we highlight the progress that has been achieved toward the design of inhibitory antibodies that successfully modulate MMP-9 and MMP-14

Carborane‐Containing Matrix Metalloprotease (MMP) Ligands as Candidates for Boron Neutron‐Capture Therapy (BNCT)

Based on the previously reported potent and selective sulfone hydroxamate inhibitors SC‐76276, SC‐78080 (SD‐2590), and SC‐77964, potent MMP inhibitors have been designed and synthesized to append a boron‐rich carborane cluster by employing click chemistry to target tumor cells that are known to upregulate gelatinases. Docking against MMP‐2 suggests binding involving the hydroxamate zinc‐binding group, key H‐bonds by the sulfone moiety with the peptide backbone residues Leu82 and Leu83, and a hydrophobic interaction with the deep P1’ pocket. The more potent of the two triazole regioisomers exhibits an IC50 of 3.7 nM versus MMP‐2 and IC50 of 46 nM versus MMP‐9

Peroxisome Proliferator-Activated Receptor-γ Ligands: Potential Pharmacological Agents for Targeting the Angiogenesis Signaling Cascade in Cancer

Peroxisome proliferator-activated receptor-γ (PPAR-γ) has currently been considered as molecular target for the treatment of human metabolic disorders. Experimental data from in vitro cultures, animal models, and clinical trials have shown that PPAR-γ ligand activation regulates differentiation and induces cell growth arrest and apoptosis in a variety of cancer types. Tumor angiogenesis constitutes a multifaceted process implicated in complex downstream signaling pathways that triggers tumor growth, invasion, and metastasis. In this aspect, accumulating in vitro and in vivo studies have provided extensive evidence that PPAR-γ ligands can function as modulators of the angiogenic signaling cascade. In the current review, the crucial role of PPAR-γ ligands and the underlying mechanisms participating in tumor angiogenesis are summarized. Targeting PPAR-γ may prove to be a potential therapeutic strategy in combined treatments with conventional chemotherapy; however, special attention should be taken as there is also substantial evidence to support that PPAR-γ ligands can enhance angiogenic phenotype in tumoral cells

Molecular imaging of tissue repair after myocardial infarction : preclinical evaluation of novel 68Ga-labeled PET tracers

Congestive heart failure (HF) develops soon after acute myocardial infarction (AMI) in almost 25% of initial survivors. Modern cardiac imaging methods are useful for HF diagnostics and, possibly, the detection of underlying molecular mechanisms involved in myocardial repair. CD44, a cell-surface glycoprotein, is involved in various cellular functions, including cell proliferation, adhesion, migration and lymphocyte activation. Integrins are transmembrane proteins involved in various signaling pathways related to inflammation, angiogenesis and fibrosis. Expression of proteolytic matrix metalloproteinases 2 and 9 (MMP-2/9) also associates with extracellular matrix remodeling. The purpose of this thesis was to evaluate novel Gallium-68 labeled imaging agents targeting αvβ3 integrin, MMP-2/9, or CD44, for positron emission tomography (PET) imaging of post-MI repair in a surgical rat model. The MMP- 2/9 targeting tracer watarkias also evaluated for imaging of atherosclerotic lesions in a hypercholesterolemic mouse model. In vivo PET imaging, ex vivo biodistribution, ex vivo autoradiography, and immunohistochemistry were utilized to assess tracer stability, uptake in various tissues, as well as uptake correlation with various cellular level processes. Of the studied tracers, αvβ3 integrin targeting tracer showed the most optimal characteristics for imaging of myocardial healing processes. Tracer uptake in the damaged myocardium was clearly visible in vivo, and blood clearance as well as tracer stability were sufficient. The CD44 targeting tracer showed initial potential warranting further development, as the tracer uptake was associated with myocardial inflammation. MMP-2/9 targeted imaging showed significant limitations due to tracer instability and slow clearance. In conclusion, imaging of αvβ3 integrin expression is a potential tool for the purpose of evaluating myocardial repair after MI.Sydänkudoksen infarktinjälkeisen paranemisen molekyylikuvantaminen : uusien 68Ga-leimattujen merkkiaineiden prekliininen arviointi Sydämen vajaatoiminta kehittyy pian akuutin sydäninfarktin jälkeen lähes 25 prosentille eloonjääneistä. Nykyaikaiset sydämen kuvantamismenetelmät ovat hyödyllisiä diagnostiikassa ja mahdollisesti sydänlihaksen muovautumiseen liittyvien molekyylimekanismien havaitsemisessa. Solupinnan glykoproteiini CD44 osallistuu erilaisiin soluvälitteisiin toimintoihin, kuten proliferaatioon, adheesioon, migraatioon ja lymfosyyttien aktivaatioon. Integriinit ovat transmembraaniproteiineja, jotka osallistuvat erilaisiin signalointireitteihin liittyen tulehdukseen, angiogeneesiin ja fibroosiin. Proteolyyttisten matriksin metalloproteinaasi 2:n ja 9:n (MMP-2/9) ilmentyminen liittyy niin ikään solunulkoisen matriksin uudelleenmuovautumiseen. Tämän väitöskirjan tarkoituksena on arvioida uusia Gallium-68-leimattuja koettimia positroniemissiotomografiaa (PET) varten. Tutkitut koettimet kohdistuvat joko αvβ3-integriiniin, MMP-2/9:ään tai CD44:ään. Tutkimus toteutettiin infarktinjälkeisen sydämen vajaatoiminnan kirurgisessa rottamallissa. MMP-2/9-koetinta arvioitiin myös ateroskleroottisten muutosten kuvantamiseen hyperkolesterolemisessa hiirimallissa. αvβ3-integriiniin kohdentuvan merkkiaineen kertymä näkyi selkeästi in vivo, ja veren puhdistuma sekä merkkiaineen stabiilisuus olivat riittävät. CD44 kuvantamiskohteena osoitti alkuvaiheen potentiaalia, joka mahdollistaa jatkokehityksen, sillä merkkiaineen kertymä assosioitui infarktinjälkeiseen tulehdusreaktioon. MMP-2/9- kohdennetulle kuvantamiselle puolestaan ilmeni merkittäviä rajoituksia merkkiaineiden epävakauden ja hitaan veripuhdistuman vuoksi Yhteenvetona voidaan todeta, että αvβ3-integriinifragmentin kuvantaminen on potentiaalinen työkalu sydänlihaksen paranemisprosessien arvioimiseksi akuutin sydäninfarktin jälkeen

A Novel Cell Based Assay for MMP Inhibitor Screening

Up to date, a plethora of protein based materials are used as implants to stimulate tissue regeneration or fillers to alleviate tissue or organ impairment. This includes glottal insufficiency, urinary bladder incontinence and especially in cosmetic industrial to improve facial contour. Once in vivo, protein-based materials are decomposed by cell secreted matrix metalloproteinases (MMP) and lose their volume within months. By introducing MMP inhibitor (MMPI), the extent of material degradation over time may be reduced. In this dissertation, the development of cell-based assay capable of identifying MMPI candidates for protein-based implant lifetime prolongation is described. To visualize the degradation, DQ-gelatin, (heavily fluorescence labeled gelatin that emits fluorescence signal proportional to its degradation) was used to represent the implant material. This gelatin was co-cultured with NIH-3T3 in a 96-well plate supplemented with growth media under standard tissue culture condition (5% CO2, 95% humidity at 37°C). Number of seeded cells, DQ-gelatin concentration and experiment run time were varied to optimize signal-to-noise ratio whilst taking into account more than 80% of seeded cells must remain viable. With optimized parameters, 0.8 million cells cultured on cell adhesion support scaffold in presence of 50 µg/mL DQ-gelatin for 5 days, the efficacy of BB-94 and TIMP-1 as synthetic and natural MMPI candidate were investigated. Both BB-94 and TIMP-1 were tested at different concentrations according to their IC50 and the approximated amount of MMPs in tissue fluid, 20-1000 nM and 0.1-2 µg/mL respectively, to determine their most efficient dosage. BB-94 and TIMP-1 demonstrated maximum potential at 72.59±4.75 % and 60.00±27.41 % at the concentration of 1000 nM and 2 µg/mL respectively. Statistical Analysis could not detect the significant difference from varying MMP inhibitor concentration, therefore, their concluded most efficient dosage in our experiment is the lowest concentration used for testing. Because our assay generated reliable statistically distinct signals and are capable of detecting quantitative inhibitory efficacy of MMP inhibitors, we believe our novel cell-based assay is a feasible method for MMP inhibitor screening that could better represent the complex degradation process of protein-based implants in biological systems than the current conventional enzyme-based methods

Development of Targeted Therapeutics for the Treatment of Glioblastoma

Glioblastoma is the most aggressive cancer of the brain. Despite recent advances in cancer biology and multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome of patients with high grade glioma remains fatal. The major drawback of current glioma chemotherapeutics is their inability to cross the blood brain barrier, lack of tumour specificity agents and their consequent side effects. Matrix metalloprotease (MMP) activity is central to cancer development, angiogenesis and invasion. They are highly active in the tumour environment and absent or inactive in normal tissues, therefore they represent viable targets for cancer drug discovery. A better understanding of the role of MMPs in human gliomas could potentially have diagnostic, prognostic and therapeutic implications. This study aims to assess the expression of specific MMPs in preclinical human glioma models and clinical glioma samples; evaluate in silico docking to rationalise substrate binding preferences of homologous MMPs; rationally design MMP-subtype-selective tumour activated prodrugs; and determine the feasibility of targeting MMP-selective anticancer prodrugs conjugated to graphene oxide as a local drug delivery approach for glioblastoma. This study found significant overexpression of MMP-10 in glioma relative to histologically normal brain tissues. Strong correlation was observed between MMP-10 protein and gene expression of glioma cell lines relative to low expression in a normal brain cell line. MMP-10 activity, as measured by fluorogenic substrate cleavage assay, also demonstrated a strong correlation between MMP-10 activity and gene expression levels. Following demonstration of selective overexpression of MMP-10 in glioma, a reiterative in silico proteolytic docking coupled in vitro biochemical assessment was utilised to rationalise functional similarity and differentiate substrate binding selectivity of homologous MMPs. The binding modes of MMP-substrates within the active site of closely related MMPs were able to accurately predict the cleavage subsites by specific MMPs, as confirmed by in vitro cleavage assay. The success of computational and experimental methodology provided a robust tool for identifying MMP-subtype differences and subsequent development of MMP-10 selective peptide prodrugs. MMP-subtype selective and MMP-10 selective prodrugs were designed by rational exploitation of MMP-docked complexes of substrates. Peptide residues were modified to achieve selectivity for MMP-2 and MMP-10 (over MMP-3 and MMP-9) demonstrating predicted cleavage at distinct subsites. This selectivity was further exploited to attain MMP-10 selectivity, over MMP-2, MMP-3 and MMP-9. The rationally designed peptide prodrugs were synthesised and were shown to be preferentially cleaved by MMPs at predicted subsites and demonstrated no activation by engineered-out MMPs, as predicted. Compared to MJ02 (MMP-2 and MMP-10 selective doxorubicin prodrug), MJ04 (MMP-10 selective doxorubicin prodrug) demonstrated selective metabolism by glioma cell lines to release chemotherapeutic agents. This therapeutic approach against glioma cell lines depended upon the involvement of MMPs, confirmed using pharmacological inhibition. MJ04 demonstrated negligible activity in the presence of an MMP-10 selective inhibitor, suggesting MMP-10 selective activation of the prodrug in glioma cells relative to normal glial cells. Following successful development of MMP-10 selective prodrugs, the feasibility of targeting glioma tumour with local delivery of chemotherapeutics from functionalised graphene-oxide tethered prodrug implants, was assessed as a therapeutic strategy to circumvent the blood brain barrier. Graphene oxide conjugated prodrug was synthesised which is shown to be preferentially cleaved in MMP expressing glioma cell lines relative to normal glial cells. This study demonstrates that MMP-10 is overexpressed in glioblastoma and can be used to metabolise anticancer prodrugs that can be activated selectively by local tumour environment

Molecular docking and inhibition of matrix metalloproteinase-2 by novel difluorinatedbenzylidene curcumin analog

We recently described the synthesis and characterization of a novel difluorinatedbenzylidene analog of curcumin, commonly referred as CDF, which demonstrated significantly enhanced bioavailability and in vivo anticancer activity. CDF targets many factors similar to curcumin, albeit with more potency, as reported previously. To further highlight this differential behavior of CDF, we chose matrix metalloproteinase protein MMP-2 which is involved in the processes of invasion and metastasis of human tumors. Both curcumin and CDF were characterized for their binding characteristics using in silico docking studies; they were also evaluated via biological assays involving gelatin zymography, miRNA analysis, invasion assays and ELISA. CDF was found to inhibit MMP-2 expression and activity in A549 and H1299 NSCLC cells much more effectively than curcumin, validating molecular modeling results. miR-874, an MMP-2-targeting miRNA, was up-regulated by CDF. Thus, it appears that CDF can inhibit MMP-2 through multiple mechanisms. Our results are suggestive of a more potent inhibition of invasion and metastasis by CDF, compared to curcumin, thus warranting its further evaluation as an effective anticancer agent

Proteinase activity regulation by glycosaminoglycans

There are few reports concerning the biological role and the mechanisms of interaction between proteinases and carbohydrates other than those involved in clotting. It has been shown that the interplay of enzymes and glycosaminoglycans is able to modulate the activity of different proteases and also to affect their structures. From the large number of proteases belonging to the well-known protease families and also the variety of carbohydrates described as widely distributed, only few events have been analyzed more deeply. The term family is used to describe a group of proteases in which every member shows an evolutionary relationship to at least one other protease. This relationship may be evident throughout the entire sequence, or at least in that part of the sequence responsible for catalytic activity. The majority of proteases belong to the serine, cysteine, aspartic or metalloprotease families. By considering the existing limited proteolysis process, in addition to the initial idea that the proteinases participate only in digestive processes, it is possible to conclude that the function of the enzymes is strictly limited to the cleavage of intended substrates since the destruction of functional proteins would result in normal tissue damage. In addition, the location as well as the eventual regulation of protease activity promoted by glycosaminoglycans can play an essential role in the development of several physiopathological conditions.Universidade de Mogi das Cruzes Centro Interdisciplinar de Investigação BioquímicaUniversidade Federal de São Paulo (UNIFESP) Escola Paulista de Medicina Departamento de BiofísicaUNIFESP, EPM, Depto. de BiofísicaSciEL