protease inhibition
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Author(s):  
Priyanka Sivasubramanian ◽  
R. Gayatri Devi ◽  
J. Selvaraj ◽  
A. Jothi Priya

Introduction: Inflammation is said to be the response of the body to an injury. It is a body defence reaction to reduce or eliminate the spread of injurious agents. It is essential that steps should be taken to introduce new medicinal plants and to develop cheaper, effective and safe analgesic and anti-inflammatory drugs. The main aim of this study is to assess the potential anti-inflammatory activity of Tecoma stans, Acalypha indica and Abutilon indicum plant is being studied. Materials and Methods: Protease inhibition assay was done by Bovine serum albumin was added to plant samples with increase in concentrations as per the standard methods. In this study, Aspirin was used as a standard anti-inflammatory drug.The data were analyzed statistically by a one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test to see the statistical significance among the groups. The results with p<0.05 level were considered to be statistically significant. Results: In this study, it was observed that the plant leaf extract of Tecoma stans, Acalypha indica and Abutilon indicus contain anti-inflammatory activity. The protein denaturation inhibitory activity of leaf extract of Tecoma stans, Acalypha indica and Abutilon indicum, plant extract was represented graphically. Tecoma stans, was observed to contain the anti inflammatory activity. Conclusion: This study revealed that Tecoma stans, Acalypha indica and Abutilon indicum are important medicinal plants with diverse pharmacological spectrum and contain anti-inflammatory properties. Hence, this research has been taken to collect and compile the pharmacological uses of these plant extracts which will be useful to the society to venture into a field of alternative systems of medicine.


2021 ◽  
Vol 22 (22) ◽  
pp. 12210
Author(s):  
Yoshira M. Ayala-Marin ◽  
Alice H. Grant ◽  
Georgialina Rodriguez ◽  
Robert A. Kirken

Compared to other ethnicities, Hispanic children incur the highest rates of leukemia, and most cases are diagnosed as Acute Lymphoblastic Leukemia (ALL). Despite improved treatment and survival for ALL, disproportionate health outcomes in Hispanics persist. Thus, it is essential to identify oncogenic mutations within this demographic to aid in the development of new strategies to diagnose and treat ALL. Using whole-exome sequencing, five single nucleotide polymorphisms within mitogen-activated protein kinase 3 (MAP2K3) were identified in an ALL cancer patient library from the U.S./Mexico border. MAP2K3 R26T and P11T are located near the substrate-binding site, while R65L and R67W localized to the kinase domain. Truncated-MAP2K3 mutant Q73* was also identified. Transfection in HEK293 cells showed that the quadruple-MEK3 mutant (4M-MEK3) impacted protein stability, inducing degradation and reducing expression. The expression of 4M-MEK3 could be rescued by cysteine/serine protease inhibition, and proteasomal degradation of truncated-MEK3 occurred in a ubiquitin-independent manner. MEK3 mutants displayed reduced auto-phosphorylation and enzymatic activity, as seen by decreases in p38 phosphorylation. Furthermore, uncoupling of the MEK3/p38 signaling pathway resulted in less suppressive activity on HEK293 cell viability. Thus, disruption of MEK3 activation may promote proliferative signals in ALL. These findings suggest that MEK3 represents a potential therapeutic target for treating ALL.


Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 269-269
Author(s):  
Madhav R Seshadri ◽  
Lorena Fontan ◽  
Prasanna Sreevatsan ◽  
David Scott ◽  
John Hatcher ◽  
...  

Abstract Introduction ABC-DLBCL is characterized by chronic activation of NFκB, which is dependent on the CARD11-BCL10-MALT1 (CBM) complex. MALT1 activates NFκB transcription factors via distinct protease and scaffold functions. Clinical allosteric MALT1 protease inhibitors are in development, however these agents partially preserve NFκB activation via MALT1 scaffold function. In addition, MALT1 protease is critical for regulatory T cell (Treg) function, and its inactivation is associated with increased inflammatory cytokine expression by T-effector cells. However, overt inflammation is not seen with MALT1 knockout. It is therefore possible that MALT1 protease inhibition with preserved scaffold function will lead to autoimmune toxicities. As an alternative strategy, we developed MALT1-directed proteolysis targeting chimera (PROTAC) compounds which consist of a MALT1-binding domain linked to an IMiD-based cereblon (CRBN) binding domain, which induce ubiquitination and proteasomal degradation of MALT1. Here we present preliminary studies of a lead compound, PS-II-115, to assess feasibility of this approach for ABC-DLBCL. Results To assess MALT1 protein degradation, OCI-Ly3 cells were treated with DMSO or PS-II-115 10 µM for 24 hours and immunoblots of cell lysates were performed. PS-II-115 induced degradation of MALT1 (degradation ±SEM 73.94 ±9.8% vs DMSO, N = 3). No significant degradation of CRBN neosubstrates IKZF1, IKZF3, or GSPT1 was seen. To assess effects downstream of MALT1, we treated OCI-Ly3 cells with PS-II-115 for 23.5 hours followed by stimulation with PMA/IO for 30 minutes, and immunoblots were performed. We found increased IκB, an inhibitor of NFκB which is degraded following MALT1 scaffold mediated IKK activation, in PS-II-115 treated cells (IkB 206% compared to DMSO), suggesting that MALT1 degradation inhibits MALT1 scaffold-mediated NFκB activation pathways. To measure MALT1 protease inhibition, we used the GloSensor split luciferase method, in which a chimeric protein is cleaved by MALT1 resulting in functional luciferase. Raji cells expressing this construct were treated with DMSO or PS-II-115 for 23.5 hours followed by stimulation with PMA/IO for 30 minutes, and GloSensor assay was performed. PS-II-115 induced dose-dependent inhibition of MALT1 protease activity compared to DMSO (IC 50 1.203 µM, 95% CI 0.374-4.230, N=4). To assess growth inhibition, ABC-DLBCL and GCB-DLBCL cell lines were treated with DMSO or PS-II-115 for 96 hours, and CellTiter Glo assay was performed. GCB-DLBCLs are not dependent on chronic NFκB activation and were expected to be resistant to PS-II-115. PS-II-115 induced growth inhibition of ABC-DLBCL cell lines (OCI-Ly3 IC 50 2.54 µM, OCI-Ly10 IC 50 9.65 µM, TMD8 IC 50 1.87 µM, N=4) more potently than GCB-DLBCL cell lines (OCI-Ly1 IC 50 34.6 µM, OCI-Ly7 IC 50 &gt;50 µM, N=4), suggesting the effect of PS-II-115 is based on MALT1 rather than off-target effects. We then assessed the impact of PS-II-115 on T cell phenotypes. Human T cells were isolated from healthy donors' peripheral blood. Cells were stimulated with CD3/CD28 beads and treated with DMSO, PS-II-115, or an allosteric MALT1 inhibitor for 48 or 96 hours and flow cytometry was performed. We found a significant decrease in CD4+ FOXP3+ CD25+ CD127- Tregs among cells treated with PS-II-115 1 µM or allosteric inhibitor compared to DMSO at 48 hours (PS-II-115 3.89%, allo inh 3.85%, DMSO 9.72% Tregs among CD4+ T cells, P&lt;0.005). We also found decreased pro-inflammatory CD4+ CD45RA+ CCR7- terminal effector T cells expressing RA (TEMRA) among cells treated with PS-II-115 20 µM for 96 hours compared to DMSO (35.67% vs 56.97% TEMRA among CD4+ T cells, p&lt;0.05). The allosteric inhibitor did not affect TEMRA cells. Conclusion We describe a MALT1-directed PROTAC which induces (1) degradation of MALT1 with preservation of other IMiD-associated CRBN neosubstrates, (2) blockade of MALT1 scaffold mediated NFκB activation pathways, (3) MALT1 protease inhibition, (4) selective growth inhibition of ABC-DLBCL cell lines, (5) decrease in Tregs, and (6) decrease in CD4+ TEMRA cells. These results provide proof of principle that MALT1 degradation using a PROTAC compound is a feasible strategy against ABC-DLBCL, and may have distinct effects on T-effector phenotypes compared to MALT1 protease inhibition. Further studies are needed to elucidate immunologic effects of MALT1 degradation and to validate findings in vivo. Disclosures Fontan: Janssen Pharmaceuticals: Current Employment. Gray: Gatekeeper: Consultancy, Current holder of individual stocks in a privately-held company; Syros: Consultancy, Current holder of individual stocks in a privately-held company; Petra: Consultancy, Current holder of individual stocks in a privately-held company; C4: Consultancy, Current holder of individual stocks in a privately-held company; Allorion: Consultancy, Current holder of individual stocks in a privately-held company; Jengu: Consultancy, Current holder of individual stocks in a privately-held company; B2S: Consultancy, Current holder of individual stocks in a privately-held company; Inception: Consultancy, Current holder of individual stocks in a privately-held company; EcoCys: Consultancy, Current holder of individual stocks in a privately-held company; Soltego: Consultancy, Current holder of individual stocks in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Takeda: Research Funding; Astellas: Research Funding; Taiho: Research Funding; Janssen: Research Funding; Kinogen: Research Funding; Her2IIc: Research Funding; Deerfield: Research Funding; Sanofi: Research Funding. Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees.


2021 ◽  
Vol 8 (Supplement_1) ◽  
pp. S354-S354
Author(s):  
Khalid Eljaaly ◽  
Hani Asfour ◽  
Tarek Ibrahim ◽  
Osama Ahmed ◽  
Nabil Alhakamy ◽  
...  

Abstract Background The outbreak of COVID-19 pandemic in China regarded as a major health/economic hazard. The importance of coming up with mechanisms for preventing or treating COVID-19 has been felt across the world. This work aimed at examining the efficiency of Sitagliptin (SIT) and human immunodeficiency virus type 1 (HIV-1) trans-activator transcription peptide (TAT) against SARS-CoV-2. Methods SIT-TAT nano-conjugates were prepared according to a full three-factor bi-level (23) factorial design. SIT concentration (mM, X1), TAT concentration (mM, X2), and pH (X3) were selected as the factors. Particle size (nm, Y1) and zeta potential (mV, Y2) were assessed as responses. Characterization of the optimized formula for Fourier-transformed infrared (FTIR) and Transmission electron microscope was carried out. In addition, IC50 in Vero E6 cells, In vitro 3CL-protease inhibition and docking tests were investigated. Results The prepared complex’s formula was as follows 1: 1 SIT: TAT molar ratio, whereas zeta potential and particle size values were at 34.17 mV and 97.19 nm, respectively. This combination did exhibit its antiviral potentiality against SARS-CoV-2 via IC50 values of 9.083 5.415, and 16.14 µM for TAT, SIT-TAT, and SIT, respectively. In addition, the complex SIT-TAT showed a significant (P &lt; 0.001) viral-3CL-protease inhibitory effect (IC50 = 3.959 µM ± 0.011) in comparison to isolated components (IC50 = 10.93 µM ± 0.25) and TAT (IC50 = 8.128 µM ± 0.42). This was further confirmed via in silico study. Molecular docking investigation has shown promising binding affinity of the formula components towards SARS-CoV-2 main protease (3-CL). Conclusion While offering significant binding interactions with protein’s key pocket residues, an optimized formulation of SIT-TAT could guarantee both the enhanced delivery to the target cells and the improved cellular uptake. The presented findings would guarantee further investigations regarding formula optimization against SARS-CoV-2. Disclosures All Authors: No reported disclosures


2021 ◽  
Author(s):  
Stephan Brinkmann ◽  
Sandra Semmler ◽  
Christian Kersten ◽  
Maria A. Patras ◽  
Micheal Kurz ◽  
...  

Protease inhibitors represent a promising therapeutic option for the treatment of parasitic diseases such as malaria and human African trypanosomiasis. Falcitidin was the first member of a new class of inhibitors of falcipain 2, a cysteine protease of the malaria parasite Plasmodium falciparum. Using a metabolomics dataset of 25 Chitinophaga strains for molecular networking enabled identification of over 30 natural analogs of falcitidin. Based on MS/MS spectra, they vary in their amino acid chain length, sequence, acyl residue, and C terminal functionalization; therefore, they were grouped into the four falcitidin peptide families A-D. The isolation, characterization and absolute structure elucidation of two falcitidin-related pentapeptide aldehyde analogs by extensive MS/MS spectrometry and NMR spectroscopy in combination with advanced Marfey's analysis was in agreement with the in silico analysis of the corresponding biosynthetic gene cluster. Total synthesis of chosen pentapeptide analogs followed by in vitro testing against a panel of proteases revealed selective parasitic cysteine protease inhibition and additionally low-micromolar inhibition of α-chymotrypsin. The pentapeptides investigated here showed superior inhibitory activity compared to falcitidin.


2021 ◽  
Vol 55 (5) ◽  
pp. 605-617

Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.


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