Self-propelled assembly of nanoparticles with self-catalytic regulation for tumour-specific imaging and therapy.

Targeted assembly of nanoparticles in biological systems holds great promise for disease-specific imaging and therapy. However, the current manipulation of nanoparticle dynamics is primarily limited to organic pericyclic reactions, which necessitate the introduction of synthetic functional groups as bioorthogonal handles on the nanoparticles, leading to complex and laborious design processes. Here, we report the synthesis of tyrosine (Tyr)-modified peptides-capped iodine (I) doped CuS nanoparticles (CuS-I@P1 NPs) as self-catalytic building blocks that undergo self-propelled assembly inside tumour cells via Tyr-Tyr condensation reactions catalyzed by the nanoparticles themselves. Upon cellular internalization, the CuS-I@P1 NPs undergo furin-guided condensation reactions, leading to the formation of CuS-I nanoparticle assemblies through dityrosine bond. The tumour-specific furin-instructed intracellular assembly of CuS-I NPs exhibits activatable dual-modal imaging capability and enhanced photothermal effect, enabling highly efficient imaging and therapy of tumours. The robust nanoparticle self-catalysis-regulated in situ assembly, facilitated by natural handles, offers the advantages of convenient fabrication, high reaction specificity, and biocompatibility, representing a generalizable strategy for target-specific activatable biomedical imaging and therapy.

Tibetan medicine Liuwei Muxiang pills (LWMX pills) effectively protects mice from chronic non-atrophic gastritis.

BACKGROUND: Chronic non-atrophic gastritis (CNG) is the most common type of chronic gastritis. If not actively treated, it may induce gastric cancer (GC). Western medicine is effective in CNG, but there are more adverse reactions after long-term medication, and it is easy to relapse after treatment, which affects patients' health and life. Tibetan medicine Liuwei Muxiang Pills (LWMX pills) is a traditional Tibetan medicine compound, which has a unique curative effect in the treatment of gastric inflammation, especially chronic non-atrophic gastritis. However, the mechanisms of LWMX pills for treatment CNG still remain poor known. PURPOSE: The aim of this study was to evaluate the therapeutic intervention potential of Tibetan medicine LWMX pills on CNG and explore its potential mechanisms in mice models. METHODS: The mice models was established to evaluate the therapeutic effect of LWMX pills on CNG. The main components of LWMX pills were analyzed by GC-MS. HE staining, immunohistochemistry, proteomics and Western Blot were used to analyze the potential mechanism of LWMX pills for CNG treatment. RESULTS: In the present study, LWMX pills containing costunolide, dehydrocostuslactone and antioxidants were found. IF results showed that the expression of ALDH1B1 in the control group was significantly lower than that in the model group in the gastric mucosa tissue, and the expression of ALDH1B1 was significantly lower in the 25 mg/ml LWMX Pills group (one month) and 25 mg/ml LWMX Pills group (two months) than in the model group. IHC revealed that model group samples expressed higher levels of Furin than 25 mg/ml LWMX Pills group samples, as evidenced by very strong staining of Furin in gastric mucosal cells. However, AMY2 staining in gastric mucosal cells did not differ significantly between the treated and control groups. the protein expression levels of these proteins were decreased in 25 mg/mL LWMX pills. Meanwhile, we found that the CAM1 protein expression in the in 25 mg/ml LWMX pills group (two mouths) was increased compared to the in 25 mg/ml LWMX pills group (one mouths).Western blotting showed that the protein expression levels of Furin, AMY2A, CPA3, ALDH1B1, Cam1, COXII, IL-6, IL-1ß were decreased in 25 mg/mL LWMX pills. Meanwhile, that the CAM1 protein expression in the in 25 mg/ml LWMX pills group (two mouths) was increased compared to the in 25 mg/ml LWMX pills group (one mouths). CONCLUSION: 25mg/ml LWMX pill treatment for one month had better therapeutic effect on mice CNG. Further proteomic results showed that LWMX pills maintain gastric function by inhibiting inflammation and oxidative stress, and we also found that LWMX pills regulate the expression of proteins associated with cancer development (Amy2, Furin).

Baicalin inhibited both the Furin/TGFß1/Smad3/TSP-1 pathway in endothelial cells and the AKT/Ca2+/ROS pathway in platelets to ameliorate inflammatory coagulopathy.

Inflammatory coagulopathy is resulted from endothelial dysfunction and platelet hyperactivation in inflammatory diseases. In this study, the effects of baicalin, an active component of the traditional Chinese medicine Huangqin, on inflammatory coagulopathy were observed both in vivo and in vitro. In LPS-induced rats, baicalin ameliorated coagulation indexes, inhibited platelet hyperactivation and decreased the expression of thrombospondin-1 (TSP-1) in vessels. In cultured endothelial cells, baicalin decreased the expression of TSP-1 and collagen as well as the TNF-α-induced increase in the levels of TSP-1 and ICAM-1. Baicalin could significantly decrease the platelet adhesion on endothelial cells treated with TNF-α. Baicalin also could inhibit the increase of ROS level and the activation of the NLRP3/Caspase-1/GSDMD pathway in TNF-α-induced endothelial cells. Furin was found to be the direct target of baicalin in HUVECs. Knockdown of Furin using siRNA could ameliorate the effects of baicalin on the activation of TGFß1/Smad3 pathway, TSP-1 expression and the adhesion of platelets on TNF-α-treated endothelial cells. At the same time, baicalin inhibited platelet aggregation induced by collagen or combination of collagen and TSP-1 peptide. Collagen-induced Ca2+ mobilization, ROS level increase, AKT1 phosphorylation, platelet degranulation and TSP-1 release could be all inhibited by baicalin. In all, baicalin ameliorated endothelial dysfunction by inhibiting Furin/TGFß1/Smad3/TSP-1 pathway and also ameliorated platelet activation by inhibiting AKT-related pathway. Both the inhibiting effects of baicalin on endothelial dysfunction and platelet activation might contribute to its ameliorating effects on inflammatory coagulopathy.

Role of Vitamin D, ACE2 and the Proteases as TMPRSS2 and Furin on SARS-CoV-2 Pathogenesis and COVID-19 Severity.

BACKGROUND: COVID-19, the 21st century pandemic disease caused by SARS-CoV-2, has shown a wide clinical spectrum ranging from asymptomatic to deadly serious pneumonia. OBJECTIVE: In our study, the relationship between the pathogenesis and clinical severity of COVID-19 and vitamin D, ACE2, Furin and TMPRSS2 was investigated. METHODS: Serum 25(OH)D, 1,25(OH)2D and ACE2 protein were measured in 85 COVID-19 cases, divided into 5 groups, according to disease severity, from asymptomatic to severe and including a healthy control group. Expression levels of ACE2, VDR, TMPRSS2 and Furin mRNAs in PBMC were also measured. The relationship of the parameters within each group, the severity of the disease and the effect on the patients' fate were investigated. RESULTS: Statistically significant differences were found between the severity of COVID-19 and all study parameters, except for serum 25(OH)D. A strong negative correlation was found between serum ACE2 protein, 1,25(OH)2D, and ACE2 mRNA, and disease severity, length of hospital stay and death/survival rate. Vitamin D deficiency increased the death risk by 5.6-fold (95% CI 0.75-41.47), and the levels of 1,25(OH)2D lower than 1 ng/mL increased the risk of death by 3.8-fold (95% CI 1.07-13.30). CONCLUSION: This study suggests that vitamin D supplementation could be beneficial in the treatment and/or prevention of COVID-19.

Loss of hypothalamic Furin affects POMC to proACTH cleavage and feeding behavior in high-fat diet-fed mice.

OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.

Promising natural products against SARS-CoV-2: Structure, function, and clinical trials.

The corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus type 2 (SARS-COV-2) poses a severe threat to human health and still spreads globally. Due to the high mutation ratio and breakthrough infection rate of the virus, vaccines and anti-COVID-19 drugs require continual improvements. Drug screening research has shown that some natural active products can target the critical proteins of SARS-CoV-2, including 3CLpro, ACE2, FURIN, and RdRp, which could produce great inhibitory effects on SARS-COV-2. In addition, some natural products have displayed activities of immunomodulation, antiinflammatory, and antihepatic failure in COVID-19 clinical trials, which may relate to their non-monomeric structures. However, further evaluation and high-quality assessments, including safety verification tests, drug interaction tests, and clinical trials, are needed to substantiate natural products' multi-target and multi-pathway effects on COVID-19. Here, we review the literature on several promising active natural products that may act as vaccine immune enhancers or provide targeted anti-COVID-19 drugs. The structures, mechanisms of action, and research progress of these natural products are analyzed, to hopefully provide effective ideas for the development of targeted drugs that possess better structure, potency, and safety.

Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Tumour enzyme affinity mediated peptide molecular crowding for targeted disruption of hyperactivated glucose uptake.

An unconventional environment-responsive molecular crowding via specific binding between small molecule peptide inhibitor derivatives and an overexpressed tumour enzyme has been developed. Assemblies of such short peptides selectively localize on tumour surfaces and exhibited unique functions in disrupting hyperactivated glucose uptake, providing novel insights towards strategic tumour treatment.

Semysinthetic biflavonoid Morelloflavone-7,4',7″,3‴,4‴-penta-O-butanoyl is a more potent inhibitor of Proprotein Convertases Subtilisin/Kexin PC1/3 than Kex2 and Furin.

BACKGROUND: Garcinia brasiliensis is a species native to the Amazon forest. The white mucilaginous pulp is used in folk medicine as a wound healing agent and for peptic ulcer, urinary, and tumor disease treatments. The activity of the proprotein convertases (PCs) Subtilisin/Kex is associated with the development of viral, bacterial and fungal infections, osteoporosis, hyperglycemia, atherosclerosis, cardiovascular, neurodegenerative and neoplastic diseases. METHODS: Morelloflavone (BF1) and semisynthetic biflavonoid (BF2, 3 and 4) from Garcinia brasiliensis were tested as inhibitor of PCs Kex2, PC1/3 and Furin, and determined IC50, Ki, human proinflammatory cytokines secretion in Caco-2 cells, mechanism of inhibition, and performed molecular docking studies. RESULTS: Biflavonoids were more effective in the inhibition of neuroendocrine PC1/3 than mammalian Furin and fungal Kex2. BF1 presented a mixed inhibition mechanism for Kex2 and PC1, and competitive inhibition for Furin. BF4 has no good interaction with Kex2 and Furin since carboxypropyl groups results in steric hindrance to ligand-protein interactions. Carboxypropyl groups of BF4 promote steric hindrance with Kex2 and Furin, but effective in the affinity of PC1/3. BF4 was more efficient at inhibiting PCl/3 (IC50 = 1.13 µM and Ki = 0,59 µM, simple linear competitive mechanism of inhibition) than Kex2, Furin. Also, our results strongly suggested that BF4 also inhibits the endogenous cellular PC1/3 activity in Caco-2 cells, since PC1/3 inhibition by BF4 causes a large increase in IL-8 and IL-1ß secretion in Caco-2 cells. CONCLUSIONS: BF4 is a potent and selective inhibitor of PC1/3. GENERAL SIGNIFICANCE: BF4 is the best candidate for further clinical studies on inhibition of PC1/3.

Host Cell and SARS-CoV-2-Associated Molecular Structures and Factors as Potential Therapeutic Targets.

Coronavirus disease 19 (COVID-19) is caused by an enveloped, positive-sense, single-stranded RNA virus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which belongs to the realm Riboviria, order Nidovirales, family Coronaviridae, genus Betacoronavirus and the species Severe acute respiratory syndrome-related coronavirus. This viral disease is characterized by a myriad of varying symptoms, such as pyrexia, cough, hemoptysis, dyspnoea, diarrhea, muscle soreness, dysosmia, lymphopenia and dysgeusia amongst others. The virus mainly infects humans, various other mammals, avian species and some other companion livestock. SARS-CoV-2 cellular entry is primarily accomplished by molecular interaction between the virus's spike (S) protein and the host cell surface receptor, angiotensin-converting enzyme 2 (ACE2), although other host cell-associated receptors/factors, such as neuropilin 1 (NRP-1) and neuropilin 2 (NRP-2), C-type lectin receptors (CLRs), as well as proteases such as TMPRSS2 (transmembrane serine protease 2) and furin, might also play a crucial role in infection, tropism, pathogenesis and clinical outcome. Furthermore, several structural and non-structural proteins of the virus themselves are very critical in determining the clinical outcome following infection. Considering such critical role(s) of the abovementioned host cell receptors, associated proteases/factors and virus structural/non-structural proteins (NSPs), it may be quite prudent to therapeutically target them through a multipronged clinical regimen to combat the disease.

Furin Enzyme and pH Synergistically Triggered Aggregation of Gold Nanoparticles for Activated Photoacoustic Imaging and Photothermal Therapy of Tumors.

Specific and effective accumulation of nanoparticles within tumors is highly crucial for precise cancer diagnosis and treatment. Therefore, spatiotemporally manipulating the aggregation of small gold nanoparticles (AuNPs) in a tumor microenvironment is of great significance for enhancing the diagnostic and therapeutic efficacy of tumors. Herein, we reported a novel furin enzyme/acidic pH synergistically triggered small AuNP aggregation strategy for activating the photoacoustic (PA) imaging and photothermal (PTT) functions of AuNPs in vivo. Smart gold nanoparticles decorated with furin-cleavable RVRR (Arg-Val-Arg-Arg) peptides (Au-RRVR) were rationally designed and fabricated. Both in vitro and in vivo experiments demonstrated that such Au-RRVR nanoparticles could be simultaneously induced by furin and acidic pH to form large aggregates within tumorous tissue resulting in improved tumor accumulation and retention, which can further activate the PA and PTT effect of AuNPs for sensitive imaging and efficient therapy of tumors. Thus, we believe that this dual-stimuli-responsive aggregation system may offer a universal platform for effective cancer diagnosis and treatment.

Screening for inhibitory effects of crude drugs on furin-like enzymatic activities.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contains a cleavage motif R-X-X-R for furin-like enzymes at the boundary of the S1/S2 subunits. The cleavage of the site by cellular proteases is essential for S protein activation and virus entry. We screened the inhibitory effects of crude drugs on in vitro furin-like enzymatic activities using a fluorogenic substrate with whole-cell lysates. Of the 124 crude drugs listed in the Japanese Pharmacopeia, aqueous ethanolic extract of Cnidii Monnieris Fructus, which is the dried fruit of Cnidium monnieri Cussion, significantly inhibited the furin-like enzymatic activities. We further fractionated the plant extract and isolated the two active compounds with the inhibitory activity, namely, imperatorin and osthole, whose IC50 values were 1.45 mM and 9.45 µM, respectively. Our results indicated that Cnidii Monnieris Fructus might exert inhibitory effects on furin-like enzymatic activities, and that imperatorin and osthole of the crude drug could be potential inhibitors of the motif cleavage.

L-Carnitine Tartrate Downregulates the ACE2 Receptor and Limits SARS-CoV-2 Infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for one of the worst pandemics in modern history. Several prevention and treatment strategies have been designed and evaluated in recent months either through the repurposing of existing treatments or the development of new drugs and vaccines. In this study, we show that L-carnitine tartrate supplementation in humans and rodents led to significant decreases of key host dependency factors, notably angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and Furin, which are responsible for viral attachment, viral spike S-protein cleavage, and priming for viral fusion and entry. Interestingly, pre-treatment of Calu-3, human lung epithelial cells, with L-carnitine tartrate led to a significant and dose-dependent inhibition of the infection by SARS-CoV-2. Infection inhibition coincided with a significant decrease in ACE2 mRNA expression levels. These data suggest that L-carnitine tartrate should be tested with appropriate trials in humans for the possibility to limit SARS-CoV-2 infection.

Terapias naturales para la reducción de complicaciones en pacientes con diabetes tipo 2 y COVID-19 / Natural therapies for the reduction of complications in patients with type 2 diabetes and COVID-19

Background: In December 2019, a series of cases occurred in Wuhan (China), caused by a new coronavirus. On March 11, 2020, the WHO declared the COVID-19 disease, caused by SARS-CoV-2, as a pandemic. In Peru, the first person infected with SARS-CoV-2 was confirmed on March 6, 2020. The number of infected has been constantly increasing to this day. Purpose: It is relevant to the current pandemic, understanding the mechanism of infection of SARS-CoV-2 in diabetic patients and in this way to be able to provide natural alternatives to reduce the complications that these patients can carry out until death. Methodology: An information search was carried out between April 6 and August 25 of 2020 in databases and indexed journals, whose articles have been published between 2011 and 2020. Results: It was found regarding inhibitors of dipeptidyl peptidase (DPP-4) evaluated in in vitro tests, that the Berberis aristata species has a metabolite called "berberine", which has the highest inhibitory capacity among the mentioned species, and, concerning furine inhibition, among the in vitro tests, catechin has a significant inhibitory capacity; it also has DPP-4 inhibitory activity. Conclusion: There is a great variety of medicinal plants with inhibitory properties for DPP-4 and some for furin. These properties are beneficial in patients with type 2 diabetes, since they reduce the activity of these proteases and, consequently, the complications in SARS-CoV-2 infection

Antecedentes: En diciembre de 2019, se registraron una serie de casos producidos por un nuevo coronavirus en Wuhan (China). El 11 de marzo de 2020, la Organización Mundial de la Salud declaró a la COVID-19, provocada por el coronavirus 2 causante del síndrome respiratorio agudo grave (SARS-CoV-2), como una pandemia. En el Perú, la primera persona infectada por SARS-CoV-2 fue confirmada el 6 de marzo de 2020; desde entonces, la cifra de infectados ha ido en constante aumento hasta el día de hoy. Propósito: Es relevante, ante la actual pandemia, entender el mecanismo de infección del SARS-CoV-2 en pacientes diabéticos, para, de esta manera, poder dar alternativas naturales para disminuir las complicaciones que pueden llevar a estos pacientes hasta la muerte. Metodología: se realizó una búsqueda de información entre el 6 de abril y el 25 de agosto de 2020, en bases de datos y revistas indexadas, cuyos artículos han sido publicados entre 2011 y 2020. Resultados: Se encontró, en cuanto a los inhibidores de la dipeptidilpeptidasa 4 (DPP-4) evaluados en ensayos in vitro, que la especie Berberis aristata posee un metabolito denominado "berberina", el cual presentó la mayor capacidad inhibitoria entre las especies analizadas. Con respecto a la inhibición de la furina, en los ensayos in vitro, la catequina posee una capacidad inhibitoria significativa; además de actividad inhibitoria para la DPP-4. Conclusión: Existe una gran variedad de plantas medicinales con propiedades inhibitorias para la DPP-4, y algunas de ellas para la furina. Estas propiedades son beneficiosas en pacientes con diabetes tipo 2, dado que reducen la actividad de estas proteasas y, por consiguiente, las complicaciones causadas por la infección por SARS-CoV-2

Phytopharmaceuticals mediated Furin and TMPRSS2 receptor blocking: can it be a potential therapeutic option for Covid-19?

BACKGROUND: Currently, novel coronavirus disease (Covid-19) outbreak creates global panic across the continents, as people from almost all countries and territories have been affected by this highly contagious viral disease. The scenario is deteriorating due to lack of proper & specific target-oriented pharmacologically safe prophylactic agents or drugs, and or any effective vaccine. drug development is urgently required to back in the normalcy in the community and to combat this pandemic. PURPOSE: Thus, we have proposed two novel drug targets, Furin and TMPRSS2, as Covid-19 treatment strategy. We have highlighted this target-oriented novel drug delivery strategy, based on their pathophysiological implication on SARS-CoV-2 infection, as evident from earlier SARS-CoV-1, MERS, and influenza virus infection via host cell entry, priming, fusion, and endocytosis. STUDY DESIGN &  METHODS: An earlier study suggested that Furin and TMPRSS2 knockout mice had reduced level of viral load and a lower degree of organ damage such as the lung. The present study thus highlights the promise of some selected novel and potential anti-viral Phytopharmaceutical that bind to Furin and TMPRSS2 as target. RESULT: Few of them had shown promising anti-viral response in both preclinical and clinical study with acceptable therapeutic safety-index. CONCLUSION: Hence, this strategy may limit life-threatening Covid-19 infection and its mortality rate through nano-suspension based intra-nasal or oral nebulizer spray, to treat mild to moderate SARS-COV-2 infection when Furin and TMPRSS2 receptor may initiate to express and activate for processing the virus to cause cellular infection by replication within the host cell and blocking of host-viral interaction.

Aframomum melegueta secondary metabolites exhibit polypharmacology against SARS-CoV-2 drug targets: in vitro validation of furin inhibition.

COVID-19 pandemic is currently decimating the world's most advanced technologies and largest economies and making its way to the continent of Africa. Weak medical infrastructure and over-reliance on medical aids may eventually predict worse outcomes in Africa. To reverse this trend, Africa must re-evaluate the only area with strategic advantage; phytotherapy. One of the many plants with previous antiviral potency is against RNA viruses is Aframomum melegueta. In this study, one hundred (100) A. melegueta secondary metabolites have been mined and computational evaluated for inhibition of host furin, and SARS-COV-2 targets including 3C-like proteinase (Mpro /3CLpro ), 2'-O-ribose methyltransferase (nsp16) and surface glycoprotein/ACE2 receptor interface. Silica-gel column partitioning of A. melegueta fruit/seed resulted in 6 fractions tested against furin activity. Diarylheptanoid (Letestuianin A), phenylpropanoid (4-Cinnamoyl-3-hydroxy-spiro[furan-5,2'-(1'H)-indene]-1',2,3'(2'H,5H)-trione), flavonoids (Quercetin, Apigenin and Tectochrysin) have been identified as high-binding compounds to SARS-COV-2 targets in a polypharmacology manner. Di-ethyl-ether (IC50 = 0.03 mg/L), acetone (IC50 = 1.564 mg/L), ethyl-acetate (IC50 = 0.382 mg/L) and methanol (IC50 = 0.438 mg/L) fractions demonstrated the best inhibition in kinetic assay while DEF, ASF and MEF completely inhibited furin-recognition sequence containing Ebola virus-pre-glycoprotein. In conclusion, A. melegueta and its secondary metabolites have potential for addressing the therapeutic needs of African population during the COVID-19 pandemic.

Enzyme-instructed self-aggregation of Fe3O4 nanoparticles for enhanced MRI T2 imaging and photothermal therapy of tumors.

The aggregation of superparamagnetic iron oxide (SPIO) nanoparticles (NPs) can greatly enhance magnetic resonance imaging (MRI) T2-weighted imaging and near-infrared (NIR) absorption in experiments. In this study, an Ac-Arg-Val-Arg-Arg-Cys(StBu)-Lys-CBT probe was designed and coupled with monodispersed carboxyl-decorated SPIO NPs to form SPIO@1NPs, which use it for intracellular self-aggregation. In vitro experiments showed that the self-aggregation of SPIO@1NPs was induced by a condensation reaction mediated by the enzyme furin in furin-overexpressing tumor cells. Moreover, the NPs in the aggregated state showed significantly higher MR r2 values and photothermal conversion efficiency than the NPs in the monodisperse state. Then, the in vivo SPIO@1NP self-aggregation in tumors can facilitate accurate MRI T2 imaging-guided photothermal therapy for effectively killing cancer cells. We believe that this basic technique, based on tumor-specific enzyme-instructed intracellular self-aggregation of NPs, could be useful for the rational synthesis of other inorganic NPs for use in the fields of tumor diagnosis and treatment.

FURIN Inhibition Reduces Vascular Remodeling and Atherosclerotic Lesion Progression in Mice.

Objective- Atherosclerotic coronary artery disease is the leading cause of death worldwide, and current treatment options are insufficient. Using systems-level network cluster analyses on a large coronary artery disease case-control cohort, we previously identified PCSK3 (proprotein convertase subtilisin/kexin family member 3; FURIN) as a member of several coronary artery disease-associated pathways. Thus, our objective is to determine the role of FURIN in atherosclerosis. Approach and Results- In vitro, FURIN inhibitor treatment resulted in reduced monocyte migration and reduced macrophage and vascular endothelial cell inflammatory and cytokine gene expression. In vivo, administration of an irreversible inhibitor of FURIN, α-1-PDX (α1-antitrypsin Portland), to hyperlipidemic Ldlr-/- mice resulted in lower atherosclerotic lesion area and a specific reduction in severe lesions. Significantly lower lesional macrophage and collagen area, as well as systemic inflammatory markers, were observed. MMP2 (matrix metallopeptidase 2), an effector of endothelial function and atherosclerotic lesion progression, and a FURIN substrate was significantly reduced in the aorta of inhibitor-treated mice. To determine FURIN's role in vascular endothelial function, we administered α-1-PDX to Apoe-/- mice harboring a wire injury in the common carotid artery. We observed significantly decreased carotid intimal thickness and lower plaque cellularity, smooth muscle cell, macrophage, and inflammatory marker content, suggesting protection against vascular remodeling. Overexpression of FURIN in this model resulted in a significant 67% increase in intimal plaque thickness, confirming that FURIN levels directly correlate with atherosclerosis. Conclusions- We show that systemic inhibition of FURIN in mice decreases vascular remodeling and atherosclerosis. FURIN-mediated modulation of MMP2 activity may contribute to the atheroprotection observed in these mice.

Luteolin escape mutants of dengue virus map to prM and NS2B and reveal viral plasticity during maturation.

We previously showed that luteolin, a well-known plant-derived component found in the "heat clearing" class of Traditional Chinese Medicine (TCM) herbs, is an uncompetitive inhibitor (Ki 58.6 µM) of the host proprotein convertase furin, an endoprotease that is required for maturation of flaviviruses in the trans-Golgi compartment. Luteolin also weakly inhibited recombinant dengue virus NS2B/NS3 protease (Ki 140.36 µM) non-competitively. In order to further explore the mechanism of inhibition we isolated resistant mutants by continuous passaging of DENV2 in the presence of increasing concentrations of luteolin. Nucleotide sequence analysis of the luteolin-resistant escape mutants revealed nucleotide changes that lead to amino acid substitutions in the prM (T79R) and NS2B (I114M) genes. These mutations were introduced into a DENV2 infectious clone and tested for replication in Huh-7 cells. Interestingly we found that the replication kinetics of prM T19R-NS2B I114M double-mutant (DM) was similar to wild-type virus (WT). On the other hand the prM T79R single mutant (SM1) was attenuated and the NS2B I114M single mutant (SM2) showed enhanced replication. Time of drug addition assay with luteolin showed that the mutant viruses were able to produce more mature virions than WT in the order DM > SM2>SM1>WT. Exogenous addition of furin to purified immature WT or mutant viruses revealed that luteolin blocked the prM cleavage of WT and SM2 at a similar level. On the other hand the SM1 immature virus showed some cleavage while the DM immature virus revealed efficient furin cleavage of prM even in the presence of 50 µM luteolin. Our findings suggest that luteolin inhibition of furin may occur at host/pathogen interface that permits the virus to escape the suppression by mutating key residue that may lead to an altered interface.