Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of <scp>MMP</scp>‐9 and <scp>MMP</scp>‐14

Shirian, Jason; Arkadash, Valeria; Cohen, Itay; Sapir, Tamila; Radisky, Evette S.; Papo, Niv; Shifman, Julia M.

doi:10.1002/1873-3468.13016

Cited by 33 publications

(23 citation statements)

References 62 publications

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“…Metalloproteinases (MMPs) is a family of proteolytic enzymes that can maintain the dynamic equilibrium of extracellular matrix by degrading and remodeling extracellular matrix [ 19 ]. Several studies have shown that MMPs are related to inflammation, atherosclerosis, liver cirrhosis, connective tissue disease and cancer [ 20 , 21 ]. As the active center of mitochondrial ribosomes, mitochondrial ribosomal proteins (MRPs) are the basis for the normal expression of mitochondrial DNA and responsible for translation of encoding proteins of mitochondrial DNA [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9

Yang

Yan

et al. 2020

Bioscience Reports

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Colorectal cancer (CRC) is a common malignant tumor in digestive tract with highly invasive and metastatic capacity. Drug sensitivity remains a significant obstacle to successful chemotherapy in CRC patients. This study aimed to explore genes related to cetuximab (CTX) sensitivity in CRC by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Celigo image cytometer was used to detect suitable cells and optimal dosage of CTX. Inhibition rate of CTX on Caco-2 cells was evaluated by CCK-8 method before and after transfection. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was performed to explore suitable concentration of puromycin and multiplicity of infection (MOI). CRISPR-Cas9, sequencing data quality analysis and cell viability test were used for the selection of genes related to CTX sensitivity in CRC cells. Finally, the selected genes associated with CTX sensitivity in CRC cells were further validated by colony formation and CCK-8 assays. In this study, Caco-2 cells had a better prolificacy, and CTX 100 ug/ml exhibited a good inhibition trend on the 7th and 14th day of infection. MTT assay indicated that the minimum lethal concentration of puromycin was 2.5 μg/mL. Forty-six candidate genes were preliminarily screened via sequencing data quality analysis. Subsequently, we found that knockout of any of the four genes (MMP15, MRPL48, CALN1 and HADHB) could enhance CTX sensitivity in Caco-2 cells, which was further confirmed by colony formation assay. In summary, MMP15, MRPL48, CALN1 and HADHB genes are related to the mediation of CTX sensitivity in CRC.

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Section: Discussionmentioning

confidence: 99%

Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9

Yang

Yan

et al. 2020

Bioscience Reports

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“…MMP-14 has been suggested as a novel target for therapy. Thus far, however, no efficient inhibitor for MMP-14 for clinical use exists [28]. Liu et al [29] studied the effect of a synthetic broad-spectrum MMP inhibitor (MMPI), prinomastat, in an orthotopic lung cancer model in which tumors express MMP-14, MMP-2, and tissue inhibitor of metalloproteinases-2 (TIMP-2).…”

Section: Discussionmentioning

confidence: 99%

High serum MMP-14 predicts worse survival in gastric cancer

et al. 2018

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Matrix metalloproteinases (MMPs), endopeptidases with diverse biochemical functions, can promote cancer cell invasion and metastasis by degrading the extracellular matrix. A high matrix metalloproteinase-14 (MMP-14) expression in gastric cancer tissue has been associated with metastasis and poor prognosis. To further understand this association, we investigated serum MMP-14 as a biomarker in gastric cancer patients. The patient cohort consisted of 240 gastric adenocarcinoma patients who underwent surgery at Helsinki University Hospital, Finland, between 2000 and 2009. We determined the soluble MMP-14 serum levels using an enzyme-linked immunosorbent assay. We then calculated the associations between serum levels and clinicopathologic variables using the Mann-Whitney U-test or the Kruskal-Wallis test. We constructed survival curves using the Kaplan-Meier method and calculating the hazard ratios using the Cox proportional hazard model. We revealed a positive association between a high serum MMP-14 level and stages III–IV (p = 0.029), and between a high serum MMP-14 and distant metastasis (p = 0.022). Patients with a low serum MMP-14 had a 5-year disease-specific survival of 49.2% (95% confidence interval [CI] 45.5–52.9), whereas patients with a high serum MMP-14 had a 5-year survival of 22.1% (95% CI 15.2–29.0; p = 0.001). High serum MMP-14 was a statistically significant prognostic factor among patients with an intestinal type of cancer (hazard ratio [HR] 3.54; 95% CI 1.51–8.33; p = 0.004), but not among patients with a diffuse type. The serum MMP-14 level remained an independent prognostic factor in our multivariate survival analysis (HR 1.55; 95% CI 1.02–2.35; p = 0.040). This study indicates for the first time that high serum soluble MMP-14 levels in gastric cancer serves as a marker for a poor prognosis, possibly indicating the presence of distant metastases.

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“…This study developed a novel and complementary approach able to switch the inhibition selectivity among closely related protease family members. More specifically, such selectivity change was achieved by performing FACS selection and counter-selection simultaneously, a similar method has been recently applied for engineering MMP-specific TIMPs [ 49 ]. As proof of concept, we converted an MMP-14 inhibitor to a panel of MMP-9 inhibitors with 690–4,500-fold selectivity shift.…”

Section: Discussionmentioning

confidence: 99%

Selectivity Conversion of Protease Inhibitory Antibodies

Lopez

Ramirez

Benitez

et al. 2018

Antibody Therapeutics

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Background: Proteases are one of the largest pharmaceutical targets for drug developments. Their dysregulations result in a wide variety of diseases. Because proteolytic networks usually consist of protease family members that share high structural and catalytic homology, distinguishing them using small molecule inhibitors is often challenging. To achieve specific inhibition, this study described a novel approach for the generation of protease inhibitory antibodies. As a proof of concept, we aimed to convert a matrix metalloproteinase (MMP)-14 specific inhibitor to MMP-9 specific inhibitory antibodies with high selectivity. Methods: An error-prone single-chain Fv (scFv) library of an MMP-14 inhibitor 3A2 was generated for yeast surface display. A dual-color competitive FACS was developed for selection on MMP-9 catalytic domain (cdMMP-9) and counter-selection on cdMMP-14 simultaneously, which were fused/conjugated with different fluorophores. Isolated MMP-9 inhibitory scFvs were biochemically characterized by inhibition assays on MMP-2/-9/-12/-14, proteolytic stability tests, inhibition mode determination, competitive ELISA with TIMP-2 (a native inhibitor of MMPs), and paratope mutagenesis assays. Results: We converted an MMP-14 specific inhibitor 3A2 into a panel of MMP-9 specific inhibitory antibodies with dramatic selectivity shifts of 690–4,500 folds. Isolated scFvs inhibited cdMMP-9 at nM potency with high selectivity over MMP-2/-12/-14 and exhibited decent proteolytic stability. Biochemical characterizations revealed that these scFvs were competitive inhibitors binding to cdMMP-9 near its reaction cleft via their CDR-H3s. Conclusions: This study developed a novel approach able to convert the selectivity of inhibitory antibodies among closely related protease family members. This methodology can be directly applied for mAbs inhibiting many proteases of biomedical importance. Statement of SignificanceTo achieve high selectivity required for therapies, we developed a novel approach for the generation of protease inhibitory antibodies with nM potency and decent proteolytic stability. The methodology demonstrated here can be readily applied to many proteases of biomedical importance.

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Converting a broad matrix metalloproteinase family inhibitor into a specific inhibitor of MMP‐9 and MMP‐14

Cited by 33 publications

References 62 publications

Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9

Detection of genes responsible for cetuximab sensitization in colorectal cancer cells using CRISPR-Cas9

High serum MMP-14 predicts worse survival in gastric cancer

Selectivity Conversion of Protease Inhibitory Antibodies

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