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Matrix metalloproteinases (MMPs) serve as prognostic factors in several pathophysiological conditions, including chronic wounds. Therefore, they are considered important therapeutic targets in the intervention and treatment of these conditions. In this study, computational tools such as molecular docking and molecular dynamics simulations were used to gain insight into protein‒ligand interactions and determine the free binding energy between Ehretia species phytoconstituents and gelatinases (MMP2 and MMP9). A total of 74 phytoconstituents from Ehretia species were compiled from the literature, and 46 of these compounds were identified as potential inhibitors of at least one type of MMP. Molecular docking revealed that lithospermic acid B, rosmarinic acid, and danshensu had stronger binding affinities against the two enzymes than the reference ligands. Furthermore, (9S, 10E, 12Z, 15Z)-9-hydroxy-10,12,15-octadecatrienoic (∗-octadecatrienoic) had a higher binding energy for MMP2, whereas caffeic anhydride and caffeic acid established stronger binding energy with MMP9 than the reference ligand. These complexes also demonstrated relatively stable, favourable, and comparable conformational changes with those of unbound proteins at 500 ns. The free energy decomposition results further provide detailed insights into the contributions of active site residues and different types of interactions to the overall binding free energy. Finally, most of the hit phytoconstituents (rosmarinic acid, caffeic anhydride, caffeic acid, and danshensu) had good physicochemical, drug-likeness, and pharmacokinetic properties. Collectively, our findings showed that phytoconstituents from Ehretia species could be beneficial in the search for novel MMP inhibitors as therapeutic agents for the treatment of chronic wounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Angiogenesis, which results in the formation of new blood and lymph vessels, is required to serve metastatic cancer progression. Cancer medications may target these two interconnected pathways. Phytocompounds have emerged as promising options for treating cancer. In this study, we used a reverse docking strategy to find new candidate molecules for cancer treatment that target both pathways. Following a literature study, the important cancer-causing proteins vascular endothelial growth factor D (VEGF-D) and basic fibroblast growth factor (bFGF) for angiogenesis and matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) for the metastatic pathway were targeted. Protein Data Bank was used to retrieve the structures of chosen proteins. 22 significant plant metabolites were identified as having anticancer activity. To determine the important protein binding residues, active site prediction was used. Using Lenvatinib and Withaferin A as reference ligands, the binding affinity of certain proteins for plant metabolites was determined by docking analysis. Homoharringtonine and viniferin, both have higher binding affinities when compared to reference ligands, with docking scores of -180.96 and -180.36 against the protein MMP-9, respectively. Moreover, Viniferin showed the highest binding affinity with both MMP-9 and MMP-2 proteins, which were then subjected to a 100-ns molecular dynamic simulation. where they were found to be significantly stable. In pharmacoinformatics investigations, the majority of our compounds were found to be non-toxic for the host. In this study, we suggested natural substances as cutting-edge anticancer treatments that target both angiogenesis and metastasis, which may aid in accelerating drug development and identifying viable therapeutic candidates.Communicated by Ramaswamy H. Sarma.

MMP-2 overexpression is strongly related to several diseases including cancer. However, none of the MMP-2 inhibitors have been marketed as drug candidates due to various adverse effects. Here, a set of sulphonyl pyrrolidines was subjected to validation of molecular modelling followed by binding mode analysis to explore the crucial structural features required for the discovery of promising MMP-2 inhibitors. This study revealed the importance of hydroxamate as a potential zinc-binding group compared to the esters. Importantly, hydrophobic and sterical substituents were found favourable at the terminal aryl moiety attached to the sulphonyl group. The binding interaction study revealed that the S1' pocket of MMP-2 similar to ' a basketball passing through a hoop ' allows the aryl moiety for proper fitting and interaction at the active site to execute potential MMP-2 inhibition. Again, the sulphonyl pyrrolidine moiety can be a good fragment necessary for MMP-2 inhibition. Moreover, some novel MMP-2 inhibitors were also reported. They showed the significance of the 3 rd position substitution of the pyrrolidine ring to produce interaction inside S2' pocket. The current study can assist in the design and development of potential MMP-2 inhibitors as effective drug candidates for the management of several diseases including cancers in the future.

Dental caries is a chronic and destructive disease and matrix metalloproteinase-2 (MMP-2) plays a major role in caries. The inhibitory mechanisms of theaflavins [theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3,3'-digallate (TF3)] on MMP-2 were investigated using techniques such as enzyme inhibition kinetics, multi-spectral methods, molecular docking, and molecular dynamics simulations. The results showed that TF1, TF2A, TF2B, and TF3 all competitively and reversibly inhibited MMP-2 activity. Fluorescence spectra and molecular docking indicated that four theaflavins spontaneously bind to MMP-2 through noncovalent interactions, driven by hydrogen bonds and hydrophobic interactions, constituting a static quenching mechanism and resulting in an altered tryptophan residue environment around MMP-2. Molecular dynamic simulations demonstrated that four theaflavins can form stable, compact complexes with MMP-2. In addition, the order of theaflavins' ability to inhibit MMP-2 was found to be TF1 > TF2B > TF2A > TF3. Interestingly, the order of binding capacity between MMP-2 and TF1, TF2A, TF2B, and TF3 was consistent with the order of inhibitory capacity, and was opposite to the order of steric hindrance of theaflavins. This may be due to the narrow space of the active pocket of MMP-2, and the smaller the steric hindrance of theaflavins, the easier it is to enter the active pocket and bind to MMP-2. This study provided novel insights into theaflavins as functional components in the exploration of natural MMP-2 inhibitors.

Background: Inflammation has an important role in the pathogenesis of schizophrenia. The aim of this study was to investigate the levels of tumor necrosis factor (TNF) and matrix metalloproteinase-2 (MMP-2) in male patients with treatment-resistant schizophrenia (TRS) and chronic medicated schizophrenia (CMS), and the relationship with psychopathology., Methods: The study enrolled 31 TRS and 49 cm male patients, and 53 healthy controls. Serum MMP-2 and TNF-α levels were measured by the Luminex liquid suspension chip detection method. Positive and Negative Syndrome Scale (PANSS) scores were used to evaluate symptom severity and Repeatable Battery for the Assessment of Neuropsychological Status was used to assess cognitive function., Results: Serum TNF-α and MMP-2 levels differed significantly between TRS, CMS and healthy control patients (F = 4.289, P = 0.016; F = 4.682, P = 0.011, respectively). Bonferroni correction demonstrated that serum TNF-α levels were significantly elevated in CMS patients (P = 0.022) and MMP-2 levels were significantly higher in TRS patients (P = 0.014) compared to healthy controls. In TRS patients, TNF-α was negatively correlated with age (r=-0.435, P = 0.015) and age of onset (r=-0.409, P = 0.022). In CMS patients, MMP-2 and TNF-α were negatively correlated with PANSS negative and total scores, and TNF-α was negatively correlated with PANSS general psychopathology scores (all P < 0.05). MMP-2 levels were positively correlated with TNF-α levels (P < 0.05), but not with cognitive function (P > 0.05)., Conclusion: The results indicate the involvement of inflammation in the etiology of TRS and CMS. Further studies are warranted., (© 2024. The Author(s).)

MMP-2 has been reported as the most validated target for cancer progression and deserves further investigation. However, due to the lack of methods for obtaining large amounts of highly purified and bioactive MMP-2, identifying specific substrates and developing specific inhibitors of MMP-2 remains extremely difficult. In this study, the DNA fragment coding for pro-MMP-2 was inserted into plasmid pET28a in an oriented manner, and the resulting recombinant protein was effectively expressed and led to accumulation as inclusion bodies in E. coli. This protein was easy to purify to near homogeneity by the combination of common inclusion bodies purification procedure and cold ethanol fractionation. Then, our results of gelatin zymography and fluorometric assay revealed that pro-MMP-2 at least partially restored its natural structure and enzymatic activity after renaturation. We obtained approximately 11 mg refolded pro-MMP-2 protein from 1 L LB broth, which was higher than other strategies previously reported. In conclusion, a simple and cost-effective procedure for obtaining high amounts of functional MMP-2 was developed, which would contribute to the progress of studies on the gamut of biological action of this important proteinase. Furthermore, our protocol should be appropriate for the expression, purification, and refolding of other bacterial toxic proteins., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Cancer continues to be the second leading cause of death worldwide, with colorectal cancer (CRC) being the third most common type. Despite significant advances in cancer therapies, the current treatment of CRC remains suboptimal. In addition, the effectiveness of available chemotherapeutic drugs such as 5-Fluorouracil (5-FU) is limited by CRC-acquired resistance., Methods: In this study, we provide innovative approaches employed in synthesizing four novel nucleobase analogs. Equally, we describe the effects of these compounds on proliferation, migration, aggregation, and adhesion of 5-FU-sensitive (HCT116) and -resistant (5-FU-R-HCT116) human CRC cells. In either cell type, our synthesized novel analogs significantly inhibited cell viability in a concentration- and time-dependent manner. This highlights the higher potency of these novel analogs. In addition, these compounds attenuated migration and adhesion of both cell types while they promoted homotypic cell-cell interaction., Results: These changes were reflected by the downregulation of matrix metalloproteases (MMP-2 and MMP-9). Furthermore, our analogs exhibited potent anti-angiogenic activity in vivo., Conclusion: These novel nucleobase analogs reduced the level of secreted vascular endothelial growth factor (VEGF) and nitric oxide (NO) production in both 5-FU-sensitive and -resistant CRC cells. Taken together, our data highlight the potential chemotherapeutic properties of our novel analogs against CRC, including the 5-FU-resistant form., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Rheumatoid arthritis (RA) is an ordinarily occurring autoimmune disease with systemic inflammatory. Targeted drug delivery systems have many successful applications in the treatment of rheumatoid arthritis. In order to develop nanoparticles for targeted delivery of Celastrol (Cel) to rheumatoid arthritis and specific drug release, the dextran sulfate (DS) was modified as the targeting molecular by binding to the scavenger receptor of macrophage. The dextran-sulfate-PVGLIG-celastrol (DS-PVGLIG-Cel), named DPC, amphiphilic polymeric prodrug was synthesized and characterized. The resulting DPC@Cel micelles had the average size of 189.9 nm. Moreover, the micelles had ultrahigh entrapment efficiency (about 44.04%) and zeta potential of -11.91 mV. In the in vitro release study, due to the excessive production of matrix metalloproteinase-2 (MMP-2) at the inflammatory joint, the MMP-2 reactive peptide was used to crack in the inflammatory microenvironment to accelerate the release of Cel. The results have shown that the nanoparticles can effectively deliver Cel to activated macrophages and significantly improve the bioavailability. In vivo experiments showed that DPC@Cel have better anti-rheumatoid arthritis effects and lower systemic toxicity than free Cel. This study provided a new therapeutic strategy for the treatment of RA.

The matrix metalloproteinase family of Zn 2+ -dependent metalloenzymes are a group of proteases that possess the ability to degrade the extracellular matrix (ECM) and thus are involved in different biological processes. Being one of the prime members of this family, the matrix metalloproteinase-2 (MMP-2) is associated with several pathophysiological conditions including cancer, tumor progression, invasion, and metastasis. Due to its contribution toward human pathophysiology, MMP-2 is in the limelight of anticancer drug development research for more than three decades. Our study deals with a combination of classification-based modeling approaches with fragment-based data mining techniques on a large and diverse set of MMP-2 inhibitors. This has enabled the identification of important structural fragments/features of MMP-2 inhibitors essential for their potency and efficacy. Significantly, molecular properties such as AlogP, MW, hydrogen bond features, and the molecular fractional polar surface area can be utilized to discriminate the potent MMP-2 inhibitors from the lesser active/inactive ones. From this extensive study, it can be concluded that the hydroxamate and carboxylic acid groups as suitable zinc-binding groups (ZBGs) for potent MMP-2 inhibition. This study also concludes the importance of solvent accessibility, hydrogen bond donor and acceptor groups as well as the bulky aromatic groups for better interactions inside the MMP-2 active site. Meanwhile, the similarities of this current study with our previous observations validate the outcomes of this current study. Hence, these findings may provide useful information toward the development of effective MMP-2 inhibitors in the future., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase (MMP) and soluble epoxide hydrolase (sEH) have completely unrelated biological functions; however, their dysregulation produce similar effects on biological systems. Based on the similarity in the reported structural requirements for their inhibition, the current study aimed to identify a simultaneous inhibitor for MMP and sEH. Six compounds were identified as potential simultaneous MMP/sEH inhibitors and tested for their capacity to inhibit MMP and sEH. Inhibition of MMP and sEH activity using their endogenous and exogenous substrates was measured by liquid chromatography/mass spectrometry, spectrophotometry, and zymography. Two compounds, CTK8G1143 and ONO-4817, were identified to inhibit both MMP and sEH activity. CTK8G1143 and ONO-4817 inhibited the recombinant human sEH activity by an average of 67.4% and 55.2%, respectively. The IC 50 values for CTK8G1143 and ONO-4817 to inhibit recombinant human sEH were 5.2 and 3.5 µM, respectively, whereas their maximal inhibition values were 71.4% and 42.8%, respectively. Also, MMP and sEH activity of human cardiomyocytes were simultaneously inhibited by CTK8G1143 and ONO-4817. Regarding other compounds, they showed either MMP or sEH inhibitory activity but not both. In conclusion, these two simultaneous inhibitors of MMP and sEH could provide a promising intervention for the prevention and control of several diseases, especially cardiovascular diseases., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Cissus rotundifolia has been reported to possess various biological activities such as anti-diabetic, anti-fertility, anti-hyperlipidemic, anti-malarial, anti-osteoporotic, and anti-parasitic activities. Therefore in the present study, eleven selected constituents of Cissus rotundifolia which includes aconitic acid, astragalin, acteoside, aliospiroside A, beta amyrin, bergenin, formononetin, gallic acid, isovitexin, isoorientin, and isoquercitrin were studied on the docking behavior of human neutrophil elastase (HNE), matrix metalloproteinases (MMP 2 and MMP 9), and tyrosinase by using PatchDock method. Furthermore, molecular physicochemical, bioactivity score/drug-likeness, ADME (absorption, distribution, metabolism, and excretion), and toxicity analyses were also carried out using Molinspiration, Swiss ADME, and ProTox-II methods, respectively. The molecular physicochemical investigation showed that three ligands such as acteoside, aliospiroside A, and isoorientin have three violations for Lipinski's rule of five. Similarly, ADME analysis one ligand (formononetin) predicated to have high blood-brain barrier (BBB) permeability effect. The docking studies showed that isovitexin exhibited the highest atomic contact energy (-341.61 kcal/mol) for human neutrophil elastase (HNE), more over alliospiroside A has shown maximum atomic contact energy for both matrix metalloproteinases (MMP 2 [-618.00 kcal/mol] and MMP 9 [-634.73 kcal/mol]). Furthermore, isoquercitrin has exhibited the highest atomic contact energy (-145.70 kcal/mol) for tyrosinase. Thus, the present investigation outcome provides new knowledge in understanding eleven Cissus rotundifolia constituents as possible novel inhibitors against HNE, MMP 2, MMP 9, and tyrosinase., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Rapid proliferation, high stemness potential, high invasiveness and apoptotic evasion are the distinctive hallmarks of glioma malignancy. The dysregulation of the Wnt/β-catenin pathway is the key factor regulating glioma malignancy. Wnt antagonist, secreted frizzled-related protein 4 (sFRP4), which has a prominent pro-apoptotic role in glioma stem cells, has two functional domains, the netrin-like domain (NLD), and cysteine-rich domain (CRD) both of which contribute to apoptotic properties of the whole protein. However, there are no reports elucidating the specific effects of individual domains of sFRP4 in inhibiting the invasive properties of glioma. This study explores the efficacy of the domains of sFRP4 in inhibiting the key hallmarks of glioblastoma such as invasion, metastasis, and stemness. We overexpressed sFRP4 and its domains in the glioblastoma cell line, U87MG cells and observed that both CRD and NLD domains played prominent roles in attenuating cancer stem cell properties. Significantly, we could demonstrate for the first time that both NLD and CRD domains negatively impacted the key driver of metastasis and migration, the matrix metalloproteinase-2 (MMP-2). Mechanistically, compared to CRD, NLD domain suppressed MMP-2 mediated invasion more effectively in glioma cells as observed in matrigel invasion assay and a function-blocking antibody assay. Fluorescent matrix degradation assay further revealed that NLD reduces matrix degradation. NLD also significantly disrupted fibronectin assembly and decreased cell adhesion in another glioma cell line LN229. In conclusion, the NLD peptide of sFRP4 could be a potent short peptide therapeutic candidate for targeting MMP-2-mediated invasion in the highly malignant glioblastoma multiforme., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Acute lung injury (ALI) is one of the fatal symptoms of sepsis. However, there were no effective clinical treatments. TF accumulation-induced fibrin deposit formations and coagulation abnormalities in pulmonary vessels contribute to the lethality of ALI. Suppressor of cytokine signaling 3 (SOCS3) acts as an endogenous negative regulator of the TLR4/TF pathway. We hypothesized that inducing SOCS3 expression using lidocaine to suppress the TLR4/TF pathway may alleviate ALI. Hematoxylin and eosin (H&E), B-mode ultrasound, and flow cytometry were used to measure the pathological damage of mice. Gelatin zymography was used to measure matrix metalloproteinase-2/9 (MMP-2/9) activities. Western blot was used to assay the expression of protein levels. Here, we show that lidocaine could increase the survival rate of ALI mice and ameliorate the lung injury of ALI mice including reducing the edema, neutrophil infiltration, and pulmonary thrombosis formation and increasing blood flow velocity. Moreover, in vitro and in vivo , lidocaine could increase the expression of p-AMPK and SOCS3 and subsequently decrease the expression of p-ASK1, p-p38, TF, and the activity of MMP-2/9. Taken together, our study demonstrated that lidocaine could inhibit the TLR4/ASK1/TF pathway to alleviate ALI via activating AMPK-SOCS3 axis., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Binbin Zheng et al.)

Matrix metalloproteinases (MMPs) are an important factor in cancer progression and metastasis, especially gelatinases MMP-2 and MMP-9. A simple methodology for their detection and monitoring is highly desirable. Molecular probes have been very widely and successfully applied to study the activity of MMPs in cellular processes in vitro. We thus synthesized a small compound library of MMP-2 and MMP-9 binding probes based on drug molecules and endowed with free amine groups for the functionalization of transducer surfaces. In this study, we combined experimental results obtained by a kinetic fluorogenic peptide substrate cleavage assay with molecular modeling studies in order to assess the ability of the probe to bind to their target enzymes. The synthesized biphenyl substituted lysine derivatives showed IC 50 -values in the low nanomolar concentration range against MMP-2 (ligands 3a-d: 3 nM to 8 µM, ligands 4a-d: 45 nM to 350 µM) and low micromolar range against MMP-9 (ligands 3a-d: 350 nM to 60 µM, ligands 4a-d: 5 µM to 600 µM), with a selectivity up to more than 160-fold for MMP-2. The experimental results correlated well with molecular modelling with FleXAID and X-score functions. We showed that in our compound series, the side chain remained far away from the S1' cavity and the ligand for all the docked minima. Ligands 4a-d with their free amine group on the side chain may thus be bound to transducer surfaces for the fabrication of sensors, while retaining their activity against their target enzymes., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Uterine leiomyosarcoma is a rare malignant smooth muscle tumor originating in the uterine wall that generally responds poorly to chemotherapy and radiation., Aim: We investigated the in vitro effects of a novel nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract on the human leiomyosarcoma cell line SK-UT-1 by measuring cell proliferation, invasiveness, apoptosis, and expression of matrix metalloproteinases (MMP). We also tested the effects of nutrient mixture in vivo using nude mice., Materials and Methods: Human leiomyosarcoma SK-UT-1 cells were treated with different concentrations of nutrient mixture. Cell proliferation was determined by MTT assay; MMP expression by gelatinase zymography; invasion by Matrigel assay; migration by scratch test; apoptosis using Live Green caspase kit. In vivo studies were conducted on 5-6 weeks old female nude mice inoculated subcutaneously with 3 • 10 6  SK-UT-1 cells. The mice were fed a regular diet or a diet supplemented with 0.5% nutrient mixture. After four weeks, the mice were sacrificed and the tumors were weighed and processed for histology., Results: In vitro, nutrient mixture treatment was not toxic to SK-UT-1 cells at 250 µg/ml but exhibited 20% and 40% cytotoxicity at 500 and 1000 µg/ml respectively. Zymography did not show bands for either MMP-2 or MMP-9 in SK-UT-1 cells. However, treatment with phorbol myristate acetate stimulated the expression of MMP-9, both active and inactive forms in equal proportion. Nutrient mixture inhibited the secretion of both active and inactive forms in a dose dependent manner. Invasion through Matrigel and migration by scratch test were inhibited in a dose dependent fashion, with both invasion and migration inhibited at 250 µg/ml. Live Green Caspase apoptosis assay demonstrated slight apoptosis at 100 µg/ml and significant apoptosis at 250 to 1000 µg/ml. The results of in vitro studies were further confirmed in vivo by showing 50% decrease in tumor weight, 40% reduction in tumor burden compared to the tumors from mice fed regular diet., Conclusion: The results suggest a therapeutic potential for nutrient mixture in uterine leiomyosarcoma treatment.

Gelatinases [gelatinase A - matrix metalloproteinase-2 (MMP-2), gelatinase B - matrix metalloproteinase-9 (MMP-9)] play key roles in many disease conditions including cancer. Despite some research work on gelatinases inhibitors both jointly and individually had been reported, challenges still exist in achieving potency as well as selectivity. Here in part I of a series of work, we have reported the structural requirement of some arylsulfonamides. In particular, regression-based 2D-QSARs, topomer CoMFA (comparative molecular field analysis) and Bayesian classification models were constructed to refine structural features for attaining better gelatinase inhibitory activity. The 2D-QSAR models exhibited good statistical significance. The descriptors nsssN, SHBint6, SHBint7, PubchemFP629 were directly correlated with the MMP-2 binding affinities whereas nsssN, SHBint10 and AATS2i were directly proportional to MMP-9 binding affinities. The topomer CoMFA results indicated that the steric and electrostatic fields play key roles in gelatinase inhibition. The established Naïve Bayes prediction models were evaluated by fivefold cross validation and an external test set. Furthermore, important molecular descriptors related to MMP-2 and MMP-9 binding affinities and some active/inactive fragments were identified. Thus, these observations may be helpful for further work of aryl sulphonamide based gelatinase inhibitors in future.

Stroke is one of the leading causes of death and disability worldwide. However, treatment options for ischemic stroke remain limited. Matrix-metalloproteinases (MMPs) contribute to brain damage during ischemic strokes by disrupting the blood-brain barrier (BBB) and causing brain edemas. Carnosine, an endogenous dipeptide, was found by us and others to be protective against ischemic brain injury. In this study, we investigated whether carnosine influences MMP activity. Brain MMP levels and activity were measured by gelatin zymography after permanent occlusion of the middle cerebral artery (pMCAO) in rats and in vitro enzyme assays. Carnosine significantly reduced infarct volume and edema. Gelatin zymography and in vitro enzyme assays showed that carnosine inhibited brain MMPs. We showed that carnosine inhibited both MMP-2 and MMP-9 activity by chelating zinc. Carnosine also reduced the ischemia-mediated degradation of the tight junction proteins that comprise the BBB. In summary, our findings show that carnosine inhibits MMP activity by chelating zinc, an essential MMP co-factor, resulting in the reduction of edema and brain injury. We believe that our findings shed new light on the neuroprotective mechanism of carnosine against ischemic brain damage.

Human α 2 -macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The present study evaluated the anticancer potential of celastrol through down-regulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. HeLa cells were incubated with different concentrations of celastrol (1, 10 and 100 µM) for 48h. Doxorubicin was used as a reference drug. Cancer cell migration, apoptosis, cell viability and mitochondrial fragmentation were evaluated following celastrol treatment. In addition, the expression level of MMP-2, MMP-9 and caspase-3 was evaluated following celastrol treatment. HeLa cell viability was 94.1 ± 7, 53.4 ± 4 and 36.3 ± 2% at 1-100 µM of celastrol, respectively. Apoptotic cell numbers were increased, and inhibition of larger wounds in cancer cells was observed following celastrol treatment. Celastrol-treated cells showed condensed nuclei and clumped mitochondria. Reduced expression of MMP-2 and MMP-9 and increased expression of caspase-3 were observed following celastrol treatment. Based on the experimental results, we are concluding that the celastrol was effective against HeLa cervical cancer cells., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: We developed a preclinical protocol for the screening of candidate drugs able to control myopia and prevent its progression. The protocol uses zebrafish, C57BL/6 mice, and golden Syrian hamster models of myopia., Methods: A morpholino (MO) targeting the zebrafish lumican gene (zlum) was injected into single-cell zebrafish embryos, causing excessive expansion of the sclera. A library of 640 compounds with 2 matrix metalloproteinase (MMP) inhibitors (marimastat and batimastat), which have the potential to modulate scleral remodelling, was screened to identify candidates for mitigating scleral diameter expansion in zlum-MO-injected embryos. The myopia-prevention ability of compounds discovered to have superior potency to inhibit scleral expansion was validated over 4 weeks in 4-week-old C57BL/6 mice and 3-week-old golden Syrian hamsters with form-deprivation myopia (FDM). Changes in the refractive error and axial length were investigated. Scleral thickness, morphology of collagen fibrils in the posterior sclera, messenger RNA (mRNA) expressions, and protein levels of transforming growth factor-β2 (TGF-β2), tissue inhibitor of metalloproteinase-2 (TIMP-2), MMP-2, MMP-7, MMP-9, and collagen, type I, alpha 1 (collagen Iα1) were investigated in C57BL/6 mice, and MMP-2, MMP-9, and MMP activity assays were conducted in these mice., Findings: In the zebrafish experiment, atropine, marimastat, batimastat, doxycycline, and minocycline were the drugs that most effectively reduced expansion of scleral equatorial diameter. After 28-day treatment in diffuser-wearing mice and 21-day treatment in lid-sutured hamsters, myopic shift and axial elongation were significantly mitigated by eye drops containing 1% atropine, 50 µM marimastat, 5 µM batimastat, or 200 µM doxycycline. MMP-2 mRNA expression in mouse sclera was lower after treatment with atropine, marimastat, batimastat, or doxycycline. The protein levels and activity of MMP-2 and MMP-7 were significantly reduced after treatment with atropine, marimastat, batimastat, doxycycline, and minocycline. Furthermore, scleral thickness and collagen fibril diameter were not lower after treatment with atropine, marimastat, batimastat, or doxycycline than those of occluded eyes., Interpretation: Stepwise drug screening in a range of models from zlum-MO-injected zebrafish to rodent FDM models identified effective compounds for preclinical myopia control or prevention. On the basis of the 640 compounds that were screened, MMP inhibitors may offer alternatives for clinical trials., Funding: This research was supported by grants from Taiwan's Ministry of Science and Technology and Ministry of Health and Welfare., Competing Interests: Conflict of Interest All authors declare that they have no financial or commercial conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of "selectome"; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family-MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events., Competing Interests: The authors have declared that no competing interests exist.

This report describes the development of a non-genetic cell-surface modification method, in which a self-assembling small molecule is combined with Halo-tag proteins. Cell-surface functionalization with cancer-linked extracellular proteins led to enhanced cell motility, angiogenesis, and immune shielding of the cells, paving the way for translational opportunities for cell therapy.

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.

Introduction of α-cyano α,β-unsaturated carbonyl moiety into natural cyclic compounds markedly improves their bioactivities, including inhibitory potential against tumor growth and metastasis. Previously, we showed that cyano enone-bearing derivatives of 18βH-glycyrrhetinic (GA) and deoxycholic acids displayed marked cytotoxicity in different tumor cell lines. Moreover, GA derivative soloxolone methyl (SM) was found to induce ER stress and apoptosis in tumor cells in vitro and inhibit growth of carcinoma Krebs-2 in vivo. In this work, we studied the effects of these compounds used in non-toxic dosage on the processes associated with metastatic potential of tumor cells. Performed screening revealed SM as a hit compound, which inhibits motility of murine melanoma B16 and human lung adenocarcinoma A549 cells and significantly suppresses colony formation of A549 cells. Further study showed that SM effectively blocked transforming growth factor β (TGF-β)-induced epithelial-mesenchymal transition (EMT) of A549 cells: namely, inhibited TGF-β-stimulated motility and invasion of tumor cells as well as loss of their epithelial characteristics, such as, an acquisition of spindle-like phenotype, up- and down-regulation of mesenchymal (vimentin, fibronectin) and epithelial (E-cadherin, zona occludens-1 (ZO-1)) markers, respectively. Network pharmacology analysis with subsequent verification by molecular modeling revealed that matrix metalloproteinases MMP-2/-9 and c-Jun N-terminal protein kinase 1 (JNK1) can be considered as hypothetical primary targets of SM, mediating its marked anti-EMT activity. The inhibitory effect of SM on EMT revealed in vitro was further confirmed in a metastatic model of murine B16 melanoma: SM was found to effectively block metastatic dissemination of melanoma B16 cells in vivo, increase expression of E-cadherin and suppress expression of MMP-9 in lung metastatic foci. Altogether, our data provided valuable information for a better understanding of the antitumor activity of cyano enone-bearing semisynthetic compounds and revealed SM as a promising anti-metastatic drug candidate.

The C-terminal domain (CTD) of MMP-2, which includes a hemopexin-like domain, has been increasingly studied as an alternative target in developing selective intervention strategies towards MMP-2. Moreover, The CTD itself has been implicated in a growing number of biological events, either MMP-dependent or -independent. The production of CTD, however, has been mostly based on the uncontrolled lysis of the latent ProMMP-2 or fusion protein expression that leaves a fusion tag. In this work we present a facile production of the untagged CTD in E. coli. The target protein was expressed as inclusion bodies, and we established an efficient wash and refolding strategy that allows us to obtain the target protein in extremely high purity. The yield was established at ~6 mg/L of the culture medium, which would greatly facilitate the production and hence the biological study of CTD. The method described herein might also prove useful for related (domain) proteins in MMP family and beyond., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 IC 50 of 3.7 nM versus MMP-2 and IC 50 of 46 nM versus MMP-9., (© 2020 Wiley-VCH GmbH.)

Matrix metalloproteinases (MMPs) are evolutionarily conserved extracellular matrix proteinases. Genetic analysis of the Drosophila MMPs, Mmp1 and Mmp2, in vivo reveal that they play vital roles in tissue remodeling. Although the catalytic domain (CD) undertakes most MMP functions, few studies have sought to demonstrate the biochemical properties of the CDs of fly MMPs. Here, we identified the overexpression, purification, and refolding of the CDs of Drosophila Mmp1 and Mmp2 for biochemical studies. Zymography assays and substrate degradation analysis showed that both Mmp1-CD and Mmp2-CD were able to digest casein, gelatin, fibronectin, collagen (types I, IV, and V), while Mmp2-CD showed much higher degradation activity compared with Mmp1-CD. Moreover, human collagen III could be degraded by Mmp1-CD but not Mmp2-CD, and rat collagen I and laminin could be degraded by Mmp2-CD but not Mmp1-CD, suggesting that Drosophila Mmp1 and Mmp2 might have overlapping yet distinct substrate specificity. Using synthetic fluorescent substrates, we further demonstrated that the enzymatic activity of Mmp1-CD and Mmp2-CD could be inhibited by human tissue inhibitors of metalloproteinases (TIMPs). These results reveal the context of the cooperative yet distinct roles of Mmp1 and Mmp2 in tissue remodeling., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Inspired by the excellent catalytic activity of Au@Pt bimetallic nanorods, we construct a H 2 O 2 -free electrochemical peptide biosensor based on Au@Pt bimetallic nanorods for highly efficient and sensitive sensing of MMP-2 for the first time, not only simplifying the traditional testing steps but also avoiding the potential damage caused by H 2 O 2 to peptides and proteins.

Corneal neovascularization (CNV), a pathological process of angiogenesis, can lead to serious consequences in the cornea. CNV is generally proved to associate with inflammation in the cornea closely, which is mainly elicited by the disruption of equilibrium between angiogenic and antiangiogenic factors. Angiogenic factors including vascular endothelial growth factors (VEGFs), basic fibroblast growth factors (bFGFs), and matrix metalloproteinases (MMPs) are vital factors in the formation of CNV. Especially VEGFs are convinced to be the core angiogenic factors in CNV, and MMPs are proved to exert dual effects on the process. Strikingly, matrix metalloproteinase 2 (MMP-2) and matrix metalloproteinase 9 (MMP-9) are determined to play key roles in the formation of CNV, while the mechanism is still vague. In this review, the latest researches are reviewed to discuss the role of MMP-2 and MMP-9 in CNV, respectively, and some inhibitors of them are presented. We hope to provide a new direction of drug research for CNV., (© 2020 John Wiley & Sons A/S.)

Self-assembly of peptide-based building units into supramolecular nanostructures creates an important class of biomaterials with robust mechanical properties and improved resistance to premature degradation. Yet, upon aggregation, substrate-enzyme interactions are often compromised because of the limited access of macromolecular proteins to the peptide substrate, leading to either a reduction or loss of responsiveness to biomolecular cues. Reported here is the supramolecular design of unsymmetric reverse bolaamphiphiles (RBA) capable of exposing a matrix metalloproteinase (MMP) substrate on the surface of their filamentous assemblies. Upon addition of MMP-2, these filaments rapidly break into fragments prior to reassembling into spherical micelles. Using 3D cell culture, it is shown that drug release is commensurate with cell density, revealing more effective cell killing when more cancer cells are present. This design platform could serve as a cell-responsive therapeutic depot for local chemotherapy., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Multidrug resistance (MDR) is one of the major obstacles for tumor therapy. Intake by receptor-mediated endocytosis enables molecules to bypass ABC transporter efflux, which is the primary mechanism of MDR. Here, we developed a novel pH/enzyme dual-responsive polypeptide prodrug to reverse multidrug resistance. This drug is composed of pH/MMP2-sensitive nanoparticles (MSNPs) self-assembled from mPEG-peptide-DOX. MSNPs can overcome sequential physiological barriers of multidrug resistance by prolonging the circulation time through PEGylation, enhancing tumor accumulation through passive targeting, increasing tumor penetration by enzyme-sensitive PEG deshielding, bypassing ABC transporter efflux by undergoing receptor-mediated endocytosis, and inducing sufficient DOX release from nanoparticles triggered by lysosomal pH. The reversal of MDR by MSNPs was evaluated in MCF-7/ADR cells and nude mice bearing tumors consisting of MCF-7/ADR cells. Both in vitro and in vivo studies showed that the MSNPs can effectively reverse MDR. Thus, MSNPs may constitute a potentially promising strategy for overcoming MDR in clinical applications.

A critical challenge to the development of tissue engineering small-diameter vascular grafts is to achieve rapid endothelialization and long-term anticoagulation. It is necessary to graft both adhesion and antithrombus factors onto the surface of polycaprolactone without burst release to promote endothelial cell affinity and antithrombogenicity. A bionic structure with a nanocoating that allows a biologically responsive, long-term release was employed in this work to enable the grafting of various bioactive molecules such as gelatin, polylysine, and heparin. This approach involved orienting the biomimetic vascular structures; the self-assembly grafting of gelatin, polylysine, and heparin nanoparticles; and genipin crosslinking to form a multiphase crosslinked nanocoating. In this biologically inspired design, vascular endothelialization and long-term anticoagulation were successfully induced through a matrix metallopeptidase 2 regulative mechanism by delivering both adhesion and antithrombus factors with a responsive, long-term release without burst release. The method provided a simple and effective approach for delivering dual factors for tissue engineering small-diameter vascular grafts., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Matrix metalloproteinases (MMPs) play critical roles in a multiple number of autoimmunity diseases progression and metastasis of solid tumor. Gelatinases including MMP-2 and MMP-9 are extremely overexpressed in multiple pathological processes. MMP-9 and MMP-2 breakdown the extracellular matrix component gelatin very efficaciously. Therefore, designing and expansion of MMPs inhibitors can be an engrossing plan for therapeutic intermediacy. Anyway, a wide range of MMPs inhibitors face failure in several clinical trials. Due to sequence and structural conservation across the various MMPs, achieving specific and selective inhibitors is very demanding. In the current study, a phage-displayed peptide library was screened using active human recombinant MMP-9 protein and evaluated by enzyme-linked immunosorbent assay. Here, we isolate novel peptide sequence from phage display peptide libraries that can be a specific gelatinase inhibitor. Interestingly, in silico molecular docking showed strong interactions between the peptide three-dimensional models and some important residues of the MMP-9 and MMP-2 proteins at the fibronectin domain. A consensus peptide sequence was then synthesized (named as RSH-12) to evaluate its inhibitory potency by in vitro assays. Zymography assay was employed to evaluate the effect of RSH-12 on gelatinolysis activity of MMP-2 and MMP-9 secretion from the HT1080 cells using different concentrations of RSH-12 and inhibiting MMP-9- and MMP-2-driven gelatin proteolysis, measured by fluorescein isothiocyanate-gelatin degradation assay and HT1080 cell invasion assay on Matrigel (gelatinous protein mixture). The negative control peptide (CP) with the irrelevant sequence and no MMP inhibition properties and the positive control compound (GM6001) as a potent inhibitor of MMPs were used to assess the selectivity and specificity of gelatinases inhibition by RSH-12. Therefore, RSH-12 decreased the gelatin degradation by specifically preventing gelatin binding to MMP-9 and MMP-2. Selective gelatinase inhibitors may prove the usefulness of the new peptide discovered in tumor targeting and anticancer and anti-inflammation therapies., (© 2019 Wiley Periodicals, Inc.)

The 72-kDa type IV collagenase or gelatinase A is the second member of the matrix metalloproteinase family, MMP-2. Since the discovery of its first two substrates within components of the extracellular matrix, denatured interstitial type I collagen and native type IV collagen, the roles and various levels of regulation of MMP-2 have been intensively studied, mainly in vitro. Its (over)expression in most if not all tumors was considered a hallmark of cancer aggressiveness and boosted investigations aiming at its inhibition. Unfortunately, the enthusiasm subsided like a soufflé after clinical trial failures, mostly because of insufficient knowledge of in vivo MMP-2 activities and detrimental side effects of broad-spectrum MMP inhibition. Nowadays, MMP-2 remains a major topic of interest in research, the second in the MMP family after MMP-9. This review presents a broad overview of the major features of this protease. This knowledge is crucial to identify diagnostic or therapeutic strategies focusing on MMP-2. In this sense, recent publications and clinical trials underline the potential value of measuring circulating or tissular MMP-2 levels as diagnostic or prognostic tools, or as a useful secondary outcome for therapies against other primary targets. Direct MMP-2 inhibition has benefited from substantial progress in the design of more specific inhibitors but their in vivo application remains challenging but certainly worth the efforts it receives., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The role of metalloproteinases (MMPs) on the migration and invasion of cancer cells has been correlated with tumor aggressiveness, namely with the up-regulation of MMP-2 and 9. Herein, two pyridine-containing macrocyclic compounds, [15]pyN 5 and [16]pyN 5 , were synthesized, chemically characterized and evaluated as potential MMP inhibitors for breast cancer therapy using 3D and 2D cellular models. [15]pyN 5 and [16]pyN 5 (5-20 µM) showed a marked inhibition of MMPs activity (100% at concentrations ≥ 7.5 μM) when compared to ARP-100, a known MMP inhibitor. The inhibitory activity of [15]pyN 5 and [16]pyN 5 was further supported through in silico docking studies using Goldscore and ChemPLP scoring functions. Moreover, although no significant differences were observed in the invasion studies in the presence of all MMPs inhibitors, cell migration was significantly inhibited by both pyridine-containing macrocycles at concentrations above 5 μM in 2D cells ( p < 0.05). In spheroids, the same effect was observed, but only with [16]pyN 5 at 20 μM and ARP-100 at 40 μM. Overall, [15]pyN 5 and [16]pyN 5 led to impaired breast cancer cell migration and revealed to be potential inhibitors of MMPs 2 and 9.

This work explored the application of matrix metalloproteinase 2-targeted superparamagnetic nanoprobes for magnetic resonance imaging (MRI), near infrared (NIR) fluorescence imaging and photodynamic therapy of tumors. PEG, PAMAM (G5) and matrix metalloproteinase 2 (MMP2) were attached to the surface of carboxylated Fe3O4 nanoparticles (NPs) using a chemical coupling method and then finally loaded with the photosensitizer chlorin e6 (Ce6). In vitro and in vivo experiments demonstrated that the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes exhibited excellent stability, precise tumor targeting and biocompatibility. Furthermore, the fluorescence properties of Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes were analogous to Ce6 and could be employed for fluorescence imaging. Meanwhile, the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes have also been shown to be effective as contrast agents for T2-weighted MRI. The target molecule MMP2 enhanced the tumor targeting ability of Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes. Additionally, the Fe3O4-PEG-G5-MMP2@Ce6 nanoprobes significantly inhibited tumor growth compared with PBS and free Ce6. This work will inspire greater enthusiasm for the construction of multifunctional magnetic nanoplatforms for biomedical applications.

Increased activity of matrix metalloproteinases (MMPs) is associated with worse prognosis in different cancer types. The wild-type protective antigen (PA-WT) of the binary anthrax lethal toxin was modified to form a pore in cell membranes only when cleaved by MMPs (to form PA-L1). Anthrax lethal factor (LF) is then able to translocate through these pores. Here, we used a 111 In-radiolabeled form of LF with the PA/LF system for noninvasive in vivo imaging of MMP activity in tumor tissue by SPECT. Methods: MMP-mediated activation of PA-L1 was correlated to anthrax receptor expression and MMP activity in a panel of cancer cells (HT1080, MDA-MB-231, B8484, and MCF7). Uptake of 111 In-radiolabeled PA-L1, 111 In-PA-WT K563C , or 111 In-LF E687A (a catalytically inactive LF mutant) in tumor and normal tissues was measured using SPECT/CT imaging in vivo. Results: Activation of PA-L1 in vitro correlated with anthrax receptor expression and MMP activity (HT1080 > MDA-MB-231 > B8484 > MCF7). PA-L1-mediated delivery of 111 In-LF E687A was demonstrated and was corroborated using confocal microscopy with fluorescently labeled LF E687A Uptake was blocked by the broad-spectrum MMP inhibitor GM6001. In vivo imaging showed selective accumulation of 111 In-PA-L1 in MDA-MB-231 tumor xenografts (5.7 ± 0.9 percentage injected dose [%ID]/g) at 3 h after intravenous administration. 111 In-LF E687A was selectively delivered to MMP-positive MDA-MB-231 tumor tissue by MMP-activatable PA-L1 (5.98 ± 0.62 %ID/g) but not by furin-cleavable PA-WT (1.05 ± 0.21 %ID/g) or a noncleavable PA variant control, PA-U7 (2.74 ± 0.24 %ID/g). Conclusion: Taken together, our results indicate that radiolabeled forms of mutated anthrax lethal toxin hold promise for noninvasive imaging of MMP activity in tumor tissue., (© 2019 by the Society of Nuclear Medicine and Molecular Imaging.)

Proteases in the cancer microenvironment have been studied for some time, with a general conclusion that such proteases facilitate the spread of cancer, although there is some controversy regarding that conclusion in later-stage cancer development. More recently, a very large collection of data regarding mutant amino acids in the potential substrates of cancer microenvironment proteases have become available. To better understand the potential impact of these mutant amino acids on protease function and cancer progression, we established a bioinformatics approach to assessing the impact of melanoma mutants, among a previously defined set of extracellular matrix (ECM) structural proteins, on the sensitivity of matrix metalloproteinase-2 (MMP2), extensively associated with melanoma. The results indicated that tumor samples with mutant amino acids adjacent to the ECM structural protein, MMP2 sites also represented a better survival rate and a larger proportion of mutant peptides with high HLA class I-binding affinities, particularly in comparison with melanoma samples with a reduced or absent T-cell infiltrate. Furthermore, even better HLA class I binders were identified among the samples representing the ECM structural protein mutants resistant to MMP2. Samples representing only MMP2-resistant mutants also represented a worse overall survival. Overall, this analysis suggested that MMP2 has the capacity of freeing mutant peptides that could facilitate an anti-tumor response and a better survival rate, and this analysis has the potential of resolving some of the controversy surrounding the role of cancer proteases in cancer progression.

Although macrophage migration inhibitory factor (MIF) is known to have antioxidant property, the role of MIF in cardiac fibrosis has not been well understood. We found that MIF was markedly increased in angiotension II (Ang-II)-infused mouse myocardium. Myocardial function was impaired and cardiac fibrosis was aggravated in Mif-knockout (Mif-KO) mice. Functionally, overexpression of MIF and MIF protein could inhibit the expression of fibrosis-associated collagen (Col) 1a1, COL3A1 and α-SMA, and Smad3 activation in mouse cardiac fibroblasts (CFs). Consistently, MIF deficiency could exacerbate the expression of COL1A1, COL3A1 and α-SMA, and Smad3 activation in Ang-II-treated CFs. Interestingly, microRNA-29b-3p (miR-29b-3p) and microRNA-29c-3p (miR-29c-3p) were down-regulated in the myocardium of Ang-II-infused Mif-KO mice but upregulated in CFs with MIF overexpression or by treatment with MIF protein. MiR-29b-3p and miR-29c-3p could suppress the expression of COL1A1, COL3A1 and α-SMA in CFs through targeting the pro-fibrosis genes of transforming growth factor beta-2 (Tgfb2) and matrix metallopeptidase 2 (Mmp2). We further demonstrated that Mif inhibited reactive oxygen species (ROS) generation and Smad3 activation, and rescued the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Smad3 inhibitors, SIS3 and Naringenin, and Smad3 siRNA could reverse the decrease of miR-29b-3p and miR-29c-3p in Ang-II-treated CFs. Taken together, our data demonstrated that the Smad3-miR-29b/miR-29c axis mediates the inhibitory effect of macrophage migration inhibitory factor on cardiac fibrosis., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Matrix metaloproteinase-2 (MMP-2) is an extracellular Zn 2+ protease specific to type I and IV collagens. Its expression is associated with several inflammatory, degenerative, and malignant diseases. Conformational properties, domain movements, and interactions between MMP-2 and its associated metal ions were characterized using a 1.0 µs molecular dynamics simulation. Dihedral principle component analysis revealed ten families of conformations with the greatest degree of variability occurring in the link region connecting the catalytic and hemopexin domains. Dynamic cross-correlation analysis indicated domain movements corresponding to the opening and closing of the hemopexin domain in relation to the fibronectin and catalytic domains facilitated by the link region. Interaction energies were calculated using the molecular mechanics Poisson Boltzman surface area-interaction entropy (MMPBSA-IE) analysis method and revealed strong binding energies for the catalytic Zn 2+ ion 1, Ca 2+ ion 1, and Ca 2+ ion 3 with significant conformational stability at the binding sites of Zn 2+ ion 1 and Ca 2+ ion 1. Ca 2+ ion 2 diffuses freely away from its crystallographically defined binding site. Zn 2+ ion 2 plays a minor role in conformational stability of the catalytic domain while Ca 2+ ion 3 is strongly attracted to the highly electronegative sidechains of the Asp residues around the central β-sheet core of the hemopexin domain; however, the interacting residue sidechain carboxyl groups are outside of Ca 2+ ion 3's coordination sphere.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases at the intersection of health and disease due to their involvement in processes such as tissue repair and immunity as well as cancer and inflammation. Because of the high structural conservation in the catalytic domains and shallow substrate binding sites, selective, small-molecule inhibitors of MMPs have remained elusive. In a tour-de-force peptide engineering approach combining phage-display selections, rational design of enhanced zinc chelation, and d-amino acid screening, we succeeded in developing a first synthetic MMP-2 inhibitor that combines high potency (K i =1.9±0.5 nm), high target selectivity, and proteolytic stability, and thus fulfills all the required qualities for in cell culture and in vivo application. Our work suggests that selective MMP inhibition is achievable with peptide macrocycles and paves the way for developing specific inhibitors for application as chemical probes and potentially therapeutics., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Protein-polymer conjugation is a clinically validated approach to enhanced pharmacokinetic properties. However, the permanent attachment of polymers often leads to irreversibly reduced protein bioactivity and poor tissue penetration. As such, the use of protein-polymer conjugates for solid tumors remains elusive. Herein, we report a simple strategy using enzyme-activatable and size-shrinkable protein-polypeptide conjugates to overcome this clinical challenge. Briefly, a matrix metalloproteinase (MMP)-responsive peptide sequence is introduced between a therapeutic protein interferon (IFN) and a synthetic polypeptide P(EG 3 Glu) 20 . The resulting site-specific MMP-responsive conjugate, denoted as PEP 20 -M-IFN, can, therefore, release the attached P(EG 3 Glu) 20 to achieve both protein activation and deep penetration into the tumor microenvironment (TME). Compared to a similarly produced nonresponsive analogue conjugate PEP 20 -IFN, our results find PEP 20 -M-IFN to show higher bioactivity in vitro, improved tumor retention, and deeper penetration in a MMP2-dependent manner. Moreover, systemic administration of PEP 20 -M-IFN shows outstanding antitumor efficacy in both OVCAR3 and SKOV3 ovarian tumor models in mice. This work highlights the releasable PEPylation strategy for protein drug potentiation at the TME and opens up new opportunities in clinics for the treatment of malignant solid tumors.

Stem cell-derived exosomes have been recognized as a potential therapy for cardiovascular disease. However, the low retention rate of exosomes after transplantation in vivo remains a major challenge in clinical applications. The aim of this study is to investigate whether human umbilical cord mesenchymal stem cell derived exosomes (UMSC-Exo) encapsulated in functional peptide hydrogels could increase the retention and stability of exosomes and improve heart function in a rat myocardial infarction model. Our results demonstrated that the PA-GHRPS peptide protected H9C2 cells from H2O2-induced oxidative stress. The gelatinization ability of PA-GHRPS can be enhanced by peptide NapFF. Therefore, these two peptides were mixed to form the PGN hydrogel, which was used to encapsulate exosomes. Our data showed that the PGN hydrogel was able to encapsulate exosomes effectively and ensured a stable and sustained release of exosomes. The exosome/PGN hydrogel mixture was injected into the infarcted border zone of rat hearts. Compared to the exosome treatment alone, the mixture improved the myocardial function by reducing inflammation, fibrosis and apoptosis, and by promoting angiogenesis. The strategy used in this study provided a practical and effective method to harness exosomes for myocardial regeneration.

Dysregulation of matrix metalloproteinases (MMPs) activity is known in many pathological conditions with which most of the conditions are related to elevate MMPs activities. Ficus deltoidea (FD) is a plant known for its therapeutic properties. In order to evaluate the therapeutic potential of FD leaf extract, we study the enzymatic inhibition properties of FD leaf extract and its major bioactive compounds (vitexin and isovitexin) on a panel of MMPs (MMP-2, MMP-8 and MMP-9) using experimental and computational approaches. FD leaf extract and its major bioactive compounds showed pronounced inhibition activity towards the MMPs tested. Computational docking analysis revealed that vitexin and isovitexin bind to the active site of the three tested MMPs. We also evaluated the cytotoxicity and cell migration inhibition activity of FD leaf extract in the endothelial EA.hy 926 cell line. Conclusively, this study provided additional information on the potential of FD leaf extract for therapeutical application.

Sorsby fundus dystrophy (SFD) is a macular degeneration caused by mutations in TIMP3, the majority of which introduce a novel cysteine. However, the exact molecular mechanisms underlying SFD remain unknown. We aimed to provide novel insights into the functional consequences of a distinct N-terminal mutation. Haplotype reconstruction in three SFD families revealed that the identified c.113C>G, p.(Ser38Cys) mutation is a founder in Belgian and northern French families with a late-onset SFD phenotype. Functional consequences of the p.(Ser38Cys) mutation were investigated by high-resolution Western blot analysis of wild type and mutant TIMP3 using patient fibroblasts and in vitro generated proteins, and by molecular modeling of TIMP3 and its interaction partners. We could not confirm a previous hypothesis on dimerization of mutant TIMP3 proteins. However, we identified aberrant intramolecular disulfide bonding. Our data provide evidence for disruption of the established Cys36-Cys143 disulfide bond and formation of a novel Cys36-Cys38 bond, possibly associated with increased glycosylation of the protein. In conclusion, we propose a novel pathogenetic mechanism underlying the p.(Ser38Cys) TIMP3 founder mutation involving intramolecular disulfide bonding. These results provide new insights into the pathogenesis of SFD and other retinopathies linked to mutations in TIMP3, such as age-related macular degeneration., (© 2019 The Authors. Human Mutation published by Wiley Periodicals, Inc.)

MMP2 and MMP9, also called gelatinases, play a primary role in the angiogenic switch, as a fundamental step of tumor progression, and show high degree of structural similarity. Clinically successful gelatinase inhibitors need to be highly selective as opposite effects have been found for the two enzymes, and the S1' subsite is the major driver to attain selective and potent inhibitors. The synthesis of d-proline-derived hydroxamic acids containing diverse appendages at the amino group, varying in length and decoration allowed to give insight on the MMP2/MMP9 selectivity around the S1' subsite, resulting in the identification of sub-nanomolar compounds with high selectivity up to 730. Molecular docking studies revealed the existence of an additional hydrophobic channel at the bottom of S1' subsite for MMP2 enzyme useful to drive selectivity towards such gelatinase., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cell-based therapy is a promising approach to restoring lost functions to compromised organs. However, the issue of inefficient cell engraftment remains to be resolved. Herein, we take a chemical approach to facilitate cell engraftment by using self-assembling molecules which modify two cellular traits: cell survival and invasiveness. In this system, the self-assembling molecule induces syndecan-4 clusters on the cellular surface, leading to enhanced cell viability. Further integration with Halo-tag technology provided this self-assembly structure with matrix metalloproteinase-2 to functionalize cells with cell-invasion activity. In vivo experiments showed that the pretreated cells were able to survive injection and then penetrate and engraft into the host tissue, demonstrating that the system enhances cell engraftment. Therefore, cell-surface modification via an alliance between self-assembling molecules and ligation technologies may prove to be a promising method for cell engraftment.

In the war against cancer, nanotechnology-based drug delivery systems may play a significant role by enhancing the efficacy of conventional therapies. Here, we tried to address some major limitations plaguing anticancer drugs, namely, poor water solubility and off-target toxicity. The systems we propose are cross-linked polyelectrolyte nanocapsules based on an oil-core and a matrix metalloproteinase-2 (MMP-2)-cleavable shell. They can load hydrophobic drugs, prevent their systemic leakage, and release their payload upon an endogenous stimulus. Both the stability enhancement and the stimuli-responsive drug release mechanisms were achieved by cross-linking the nanocapsule shell with an MMP-2-sensitive peptide. On the basis of this strategy, the system maintained its stability in PBS up to one month. Further, when tested on 3D tumor and healthy spheroid models, the nanocapsules were able to disrupt their integrity preferentially in the tumor-like microenvironment. The high level of MMP-2 enzymes expressed by tumor spheroids, indeed, catalyzed the disassembly of the nanocapsules, which ultimately leads to drug release. Therefore, this device holds great potential as a smart system that allows for the safe transport of hydrophobic drugs and for a spatially controlled release upon an endogenous stimulus coming from the very nature of the tumor itself. STATEMENT OF SIGNIFICANCE: The performance of nanoparticle-based therapeutic approaches is often hindered by some intrinsic limitations typically including laborious drug loading methods, synthetic routes of preparation and stability issues. In this work, we implemented for the first time a smart drug delivery strategy into oil-core multilayer nanocapsules, which represent an ideal family of nanocarriers. To this aim, we developed a robust method enabling the use of soft matters like oil-core nanocapsules combined with a microenvironmentally triggered release mechanism. The efficacy of nanocapsules was tested on tumor and healthy spheroids. Results clearly demonstrate their selective drug release, triggered by a stimulus intrinsically present in tumor microenvironment. We believe this study is of particular interest for cancer research and paves the way for the use of these robust stimuli-responsive nanocapsules in vivo., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Hypoxic injury of cardiovascular system is one of the most frequent complications following ischaemia. Heart injury arises from increased degradation of contractile proteins, such as myosin light chains (MLCs) and troponin I by matrix metalloproteinase 2 (MMP-2). The aim of the current research was to study the effects of 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate (MMP-2-inhibitor-NO-donor hybrid) on hearts subjected to ischaemia/reperfusion (I/R) injury. Primary human cardiac myocytes and Wistar rat hearts perfused using Langendorff method have been used. Human cardiomyocytes or rat hearts were subjected to I/R in the presence or absence of tested hybrid. Haemodynamic parameters of heart function, markers of I/R injury, gene and protein expression of MMP-2, MMP-9, inducible form of NOS (iNOS), asymmetric dimethylarginine (ADMA), as well as MMP-2 activity were measured. Mechanical heart function, coronary flow (CF) and heart rate (HR) were decreased in hearts subjected to I/R Treatment of hearts with the hybrid (1-10 µmol/L) resulted in a concentration-dependent recovery of mechanical function, improved CF and HR. This improvement was associated with decreased tissue injury and reduction of synthesis and activity of MMP-2. Decreased activity of intracellular MMP-2 led to reduced degradation of MLC and improved myocyte contractility in a concentration-dependent manner. An infusion of a MMP-2-inhibitor-NO-donor hybrid into I/R hearts decreased the expression of iNOS and reduced the levels of ADMA. Thus, 5-phenyloxyphenyl-5-aminoalkyl nitrate barbiturate protects heart from I/R injury., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)