Determining the Mechanism of Propofol-induced Neurotoxicity

Background and Hypothesis: Propofol is an IV anesthetic agent with many clinical applications, including general anesthesia, sedation, delirium, and palliative care. Despite its versatility and effectiveness, Propofol has been shown to have detrimental effects on the vascular interface between the brain and circulation. The Blood-Brain Barrier (BBB) acts as a selective interface that serves to protect the brain from its cerebrovascular network. BBB dysfunction can have lethal complications such as cerebral edema and stroke. Matrix Metalloproteinases (MMP’s), specifically MMP-2 and MMP-9 have been linked to BBB breakdown. Using human induced pluripotent stem cell (IPSC) derived brain microvascular endothelial cells (BMECs), we evaluated the impact of inhibiting MMP-2 on restoring BBB integrity following exposure to Propofol with the intent to reveal the mechanism by which Propofol disrupts the BBB. We hypothesized that inhibiting MMP-2 would lead to phenotype recovery after exposure to Propofol. Methods: IPSC-differentiated BMECs were treated with MMP inhibitors at varying time points and concentrations relative to exposure to Propofol for 3 hours. Trans-endothelial electrical resistance (TEER) and sodium fluorescein permeability was used to assess BBB structural integrity. A MTT assay was conducted to assess cell viability. Results: Inhibiting MMP-2 did not result in in recovery of BBB integrity following exposure to Propofol as no significant differences were observed in TEER and sodium fluorescein permeability between the Propofol control and Propofol + MMP-2 inhibitor groups. Conclusions & Future Directions: Propofol-induced disruption of BBB integrity does not appear to be through MMP-2 activity. High concentrations of MMP-inhibitor compounds result in increased disruption of BBB tightness and permeability. It is plausible to suspect that Propofol may act through other MMP’s to facilitate BBB break-down, thus future studies should investigate the effects of the other selective MMP inhibitors in their ability to achieve phenotype recovery following exposure to Propofol.

Towards Optimized Bioavailability of 99mTc-Labeled Barbiturates for Non-invasive Imaging of Matrix Metalloproteinase Activity

Introduction Dysregulated activity of matrix metalloproteinases (MMPs) drives a variety of pathophysiological conditions. Non-invasive imaging of MMP activity in vivo promises diagnostic and prognostic value. However, current targeting strategies by small molecules are typically limited with respect to the bioavailability of the labeled MMP binders in vivo. To this end, we here introduce and compare three chemical modifications of a recently developed barbiturate-based radiotracer with respect to bioavailability and potential to image MMP activity in vivo. Methods Barbiturate-based MMP inhibitors with an identical targeting unit but varying hydrophilicity were synthesized, labeled with technetium-99m, and evaluated in vitro and in vivo. Biodistribution and radiotracer elimination were determined in C57/BL6 mice by serial SPECT imaging. MMP activity was imaged in a MMP-positive subcutaneous xenograft model of human K1 papillary thyroid tumors. In vivo data were validated by scintillation counting, autoradiography, and MMP immunohistochemistry. Results We prepared three new 99mTc‐labeled MMP inhibitors, bearing either a glycine ([99mTc]MEA39), lysine ([99mTc]MEA61), or the ligand HYNIC with the ionic co-ligand TPPTS ([99mTc]MEA223) yielding gradually increasing hydrophilicity. [99mTc]MEA39 and [99mTc]MEA61 were rapidly eliminated via hepatobiliary pathways. In contrast, [99mTc]MEA223 showed delayed in vivo clearance and primary renal elimination. In a thyroid tumor xenograft model, only [99mTc]MEA223 exhibited a high tumor-to-blood ratio that could easily be delineated in SPECT images. Conclusion Introduction of HYNIC/TPPTS into the barbiturate lead structure ([99mTc]MEA223) results in delayed renal elimination and allows non-invasive MMP imaging with high signal-to-noise ratios in a papillary thyroid tumor xenograft model.

Matrixmetalloproteinase Inhibitors: Promising Therapeutic Targets Against Cancer

Background: Cancer is a wide range cellular level disease that occurs when cells go through uncontrolled division and growth. The mechanisms by which the cells undergo metastasis are complex and involve many interactions between the tumor cells and their cellular environment. Matrix metalloproteinases (MMPs) have been found to over-express at various stages of tumor progression and their inhibition using MMP inhibitors has been a subject of potential therapy against cancer. Objective: This review discusses recent research in MMP inhibitors (MMPI) used for preventing tumor progression. Methods: In this review, we explored the role of MMPs in cancer progression and summarized the current developments in MMPIs, their role in cancer suppression in in vitro and in vivo studies and their evaluation in clinical trials from the current research data. Results: MMPIs have shown to be very successful in in vitro models, cell lines and in some in vivo studies. Unfortunately, their efficacy in clinical trials has been found to be hit and miss. Recent studies have shown that the novel delivery approaches of MMP inhibitors may enhance their therapeutic effects towards the prevention of cancer. Conclusion: In this review, we presented different MMP inhibitors, their performance at different stages of models - in vitro, in vivo, small animal models and eventually clinical trials. We provide newer methods of MMPI delivery that may be better targeted to suppress only specific MMPs and avoid toxic side-effects in healthy cells.

Identification of Broad-Spectrum MMP Inhibitors by Virtual Screening

Matrix metalloproteinases (MMPs) are the family of proteases that are mainly responsible for degrading extracellular matrix (ECM) components. In the skin, the overexpression of MMPs as a result of ultraviolet radiation triggers an imbalance in the ECM turnover in a process called photoaging, which ultimately results in skin wrinkling and premature skin ageing. Therefore, the inhibition of different enzymes of the MMP family at a topical level could have positive implications for photoaging. Considering that the MMP catalytic region is mostly conserved across different enzymes of the MMP family, in this study we aimed to design a virtual screening (VS) workflow to identify broad-spectrum MMP inhibitors that can be used to delay the development of photoaging. Our in silico approach was validated in vitro with 20 VS hits from the Specs library that were not only structurally different from one another but also from known MMP inhibitors. In this bioactivity assay, 18 of the 20 compounds inhibit at least one of the assayed MMPs at 100 μM (with 5 of them showing around 50% inhibition in all the tested MMPs at this concentration). Finally, this VS was used to identify natural products that have the potential to act as broad-spectrum MMP inhibitors and be used as a treatment for photoaging.

The Pharmacological Tails of Matrix Metalloproteinases and Their Inhibitors

Matrix metalloproteinases (MMPs) have been demonstrated to have both detrimental and protective functions in inflammatory diseases. Several MMP inhibitors, with the exception of Periostat®, have failed in Phase III clinical trials. As an alternative strategy, recent efforts have been focussed on the development of more selective inhibitors or targeting other domains than their active sites (e.g., exosites, ectosites) through specific small molecule inhibitors or monoclonal antibodies. Here, we present some examples that aim to better understand the mechanisms of conformational changes/allosteric control of MMPs functions. In addition to MMP inhibitors, we discuss unbiased global approaches such as proteomics and N-terminomics to identify new MMP substrates and achieve a better understanding of the roles of these proteases in diseases.

The Pharmacological TAILS of Matrix Metalloproteinases and Their Inhibitors

Matrix metalloproteinases (MMPs) have been demonstrated to have both detrimental and protective functions in inflammatory diseases. Several MMP inhibitors, with the exception of Periostat®, have failed in Phase III clinical trials. As an alternative strategy, recent efforts have been focussed on the development of more selective inhibitors or targeting other domains than their active sites through specific small molecule inhibitors or monoclonal antibodies. Here, we present some examples that aim to better understand the mechanisms of conformational changes/allosteric control of MMPs functions. In addition to MMP inhibitors, we discuss unbiased global approaches, such as proteomics and N-terminomics, to identify new MMP substrates. We present some examples of new MMP substrates and their implications in regulating biological functions. By characterizing the roles and substrates of individual MMP, MMP inhibitors could be utilized more effectively in the optimal disease context or in diseases never tested before where MMP activity is elevated and contributing to disease progression.

The Pharmacological Tails of Matrix Metalloproteinases and Their Inhibitors

Matrix metalloproteinases (MMPs) have been demonstrated to have both detrimental and protective functions in inflammatory diseases. Several MMP inhibitors, with the exception of Periostat®, have failed in Phase III clinical trials. As an alternative strategy, recent efforts have been focussed on the development of more selective inhibitors or targeting other domains than their active sites (e.g., exosites, ectosites) through specific small molecule inhibitors or monoclonal antibodies. Here, we present some examples that aim to better understand the mechanisms of conformational changes/allosteric control of MMPs functions. In addition to MMP inhibitors, we discuss unbiased global approaches such as proteomics and N-terminomics to identify new MMP substrates and achieve a better understanding of the roles of these proteases in diseases.

713 Antibody-based approach of MT1-MMP metalloprotease inhibition results in decreased invasive properties of pancreatic cancer cells

BackgroundPancreatic cancer (PC) is one of the most aggressive types of malignant tumors due to the fact, that early stages of the disease are asymptomatic and difficult to diagnose. Matrix metalloproteinases (MMP) play a key role in progression of early PC stages through proteolysis of collagen and a range of regulators that promotes tumor invasion and angiogenesis. MMPs are considered as promising therapeutic targets, but MMP inhibitors exhibit significant efficacy exclusively in a narrow time window, that makes it difficult to use them for prevention of local relapses. That is why MMP inhibitors and blocking antibodies demonstrated moderate results in clinical trials – progressive tumor stages could not be effectively treated with these agents, while their use in the early stages still looks very promising. The aim of the present work was to study the effects of long­term and preventive suppression of the activity of MT1­MMP (a key initiator of tumor proliferation and invasion) in pancreatic cancer using the approach of active immunization.MethodsWe performed active immunization of SPF C57BL/6 mice using different variants of the vaccine: peptide fragments of MT1-MMP protein or DNA expression vectors coding these peptide fragments. 2-fold vaccine administration and serums collection were performed according to the previously published protocol.1 The serums were collected on the 21 day of the experiment. We applied ELISA to estimate the levels of anti-MT1-MMP antibodies. The functional activity of the serums was tested using enzymatic assay and in vitro metastatic assay according to previously described protocols.2ResultsWe selected the serum containing high titers of anti-MT1-MMP antibodies which effectively suppressed MT1-­MMP activity in the absence of the effect on MMP­9. This feature of the serum is fundamental due to the fact, that MMP­9 is currently regarded as an undesirable target of anticancer therapy. The functional activity of selected serum and its ability to inhibit pancreatic cancer cell invasion was shown in an in vitro metastatic assay using the PC mouse cell line. In addition, we demonstrated the ability of this serum to inhibit the activity of MMP2 and TGF­β in conditioned mediums and lysates of PC cells, that suggests its additional anti­cancer properties associated with the suppression of the ability of MT1-­MMP to proteolytic activation of a number of tumor modulators.ConclusionsThe selected mode could be used for effective immunization against MT1-MMP.AcknowledgementsThis project is supported by grant 19-74-00096 from Russian Science Foundation.ReferencesPavshintsev VV, Mitkin NA, Frolova OY, Kushnir EA, Averina OA, Lovat ML. Individual roles of brain and serum alcohol dehydrogenase isoforms in regulation of alcohol consumption in SPF Wistar rats. Physiology & behavior 2017;179:458–466.Korneev KV, Sviriaeva EN, Mitkin NA, Gorbacheva AM, Uvarova AN, Ustiugova AS, Polanovsky OL, Kulakovskiy IV, Afanasyeva MA, Schwartz AM, Kuprash DV. Minor C allele of the SNP rs7873784 associated with rheumatoid arthritis and type-2 diabetes mellitus binds PU. 1 and enhances TLR4 expression. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease 2020;1866(3):165626.