Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

10.26434/chemrxiv.12213125 ◽

2020 ◽

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

10.26434/chemrxiv.12213125.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ existing drugs and offers a potential treatment option for COVID-19, currently in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. We showed, analysing our model, that the synergy was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, we found that available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Exploiting the synergy may improve the deployability of drug combinations targeting host proteases required for SARS-CoV-2 entry.

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008461 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1008461

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra M. Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Limiting Carryover Events Resulting from Cell Surface Retention of VSV-G Pseudotyped HIV-Lentivector Particles on Hematopoietic Targets

Blood ◽

10.1182/blood.v110.11.3735.3735 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3735-3735

Author(s):

Lee O’Neill ◽

Yung-Wei Pan ◽

Amy M. Skinner ◽

Peter Kurre

Keyword(s):

Gene Transfer ◽

Cell Surface ◽

Ex Vivo ◽

Target Cells ◽

Vector Particle ◽

Dependent Manner ◽

Dose Dependent ◽

Vector Particles

Abstract Preclinical evidence and clinical trials speak to the therapeutic potential of retrovirus vectors for the heritable genetic modification of cells. Careful evaluation of the antecedent risks is critical to move these applications forward. Others previously demonstrated the persistence of intact vector particles on the surface of target cells. Inadvertent particle transfer after in vivo applications could lead to the transduction of bystander tissues, or provoke immunological responses. We recently demonstrated prolonged adherence of VSV-G pseudotyped, HIV-1 derived lentivirus particles after ex vivo transduction culture of murine hematopoietic target cells (1°) with subsequent transduction of secondary (2°) targets in vitro and in vivo. Extended particle adherence is independent of Env pseudotype and routine wash procedures (Pan et al., J Virol. Jan 2007). We hypothesized that unwanted carryover could be minimized by disrupting the vector particle attachment to 2° cells while maintaining uptake to 1° targets. Initial studies indicated that the transduction of 1° targets at 4°C (to prevent uptake) for up to 6 hours followed by serial PBS washes and subsequent direct co-culture with fibroblasts resulted in undiminished 2° gene transfer compared to transduction at 37°C. Conversely, post-transduction exposure to escalating concentrations of citric acid resulted in a systematic decrease in both 1° and 2° gene transfer rates. This is consistent with separable mechanisms for pH sensitive VSV-G mediated uptake of particles in 1° targets and the receptor independent attachment responsible for carryover and 2° transduction, respectively. Glycosaminoglycans, including heparin, quantitatively bind to pseudotyped vector particles. We found that exposure of particles to heparin effectively abrogated subsequent transduction of cells by disrupting attachment. Remarkably, serial heparin washes at the conclusion of transduction had only minimal effects on gene transfer to 1° targets, but resulted in a two-log reduction in 2° gene transfer. Increases in the concentration of protamine sulfate (a polycation) during transduction partly reversed the effect of heparin (a polyanion), demonstrating the residual impact of electrostatic interactions on attachment of retrovirus particles from the 1° cell. In further studies we showed that trypsin washes following vector exposure incompletely cleaved 1° cell surface bound particles while pronase effectively degraded cell surface bound particles in a dose dependent manner, abrogating carryover. Because pronase at high concentrations also compromised cell surface epitope integrity we studied the expression of chemokine receptor (CXCR) 4, both a critical mediator of progenitor cell homing to the bone marrow and a representative protease-sensitive surface molecule. These experiments revealed a dose dependent degradation of CXCR4 on the cell surface of 1° target cells and rapid regeneration within three hours, critical for applications involving the injection of ex vivo modified hematopoietic cells. In conclusion, our results demonstrate that select wash procedures can disrupt the ability of virus particles to bind secondary targets, degrade residual surface bound particles and reduce gene transfer to inadvertent 2° targets in vitro by up to 99%. These studies are important first steps in understanding and limiting inadvertent carryover in the context of gene therapy while maximizing target cell transduction.

Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans

Scientific Reports ◽

10.1038/s41598-021-92991-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Benjamin N. Nelson ◽

Savannah G. Beakley ◽

Sierra Posey ◽

Brittney Conn ◽

Emma Maritz ◽

...

Keyword(s):

Antifungal Activity ◽

Cryptococcus Neoformans ◽

Mammalian Cells ◽

Cysteine Proteases ◽

Dependent Manner ◽

Life Threatening ◽

Opportunistic Fungal Pathogen ◽

Dose Dependent ◽

Lysosomal Proteins

AbstractCryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

Anti-tumor activity of a novel proteasome inhibitor D395 against multiple myeloma and its lower cardiotoxicity compared with carfilzomib

Cell Death and Disease ◽

10.1038/s41419-021-03701-z ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Xuxing Shen ◽

Chao Wu ◽

Meng Lei ◽

Qing Yan ◽

Haoyang Zhang ◽

...

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitor ◽

Osteoclast Differentiation ◽

Dependent Manner ◽

Serum Enzyme ◽

Herg Channels ◽

Anti Tumor Activity ◽

AbstractCarfilzomib, a second-generation proteasome inhibitor, has significantly improved the survival rate of multiple myeloma (MM) patients, but its clinical application is still restricted by drug resistance and cardiotoxicity. Here, we identified a novel proteasome inhibitor, D395, and assessed its efficacy in treating MM as well as its cardiotoxicity at the preclinical level. The activities of purified and intracellular proteasomes were measured to determine the effect of D395 on the proteasome. CCK-8 and flow cytometry experiments were designed to evaluate the effects of D395 on cell growth and apoptosis. The effects of D395 and carfilzomib on serum enzyme activity, echocardiography features, cardiomyocyte morphology, and hERG channels were also compared. In our study, D395 was highly cytotoxic to MM cell lines and primary MM cells but not normal cells, and it was well tolerated in vivo. Similar to carfilzomib, D395 inhibited osteoclast differentiation in a dose-dependent manner. In particular, D395 exhibited lower cardiotoxicity than carfilzomib in all experiments. In conclusion, D395 is a novel irreversible proteasome inhibitor that has remarkable anti-MM activity and mild cardiotoxicity in vitro and in vivo.

Effect of Synchronous Versus Sequential Regimens on the Pharmacokinetics and Biodistribution of Regorafenib with Irradiation

Pharmaceutics ◽

10.3390/pharmaceutics13030386 ◽

2021 ◽

Vol 13 (3) ◽

pp. 386

Author(s):

Tung-Hu Tsai ◽

Yu-Jen Chen ◽

Li-Ying Wang ◽

Chen-Hsi Hsieh

Keyword(s):

Stereotactic Body Radiation Therapy ◽

In Vivo Studies ◽

Control Group ◽

High Dose ◽

Dependent Manner ◽

Hep G2 ◽

Time Curve ◽

This study was performed to evaluate the interaction between conventional or high-dose radiotherapy (RT) and the pharmacokinetics (PK) of regorafenib in concurrent or sequential regimens for the treatment of hepatocellular carcinoma. Concurrent and sequential in vitro and in vivo studies of irradiation and regorafenib were designed. The interactions of RT and regorafenib in vitro were examined in the human hepatoma Huh-7, HA22T and Hep G2 cell lines. The RT–PK phenomenon and biodistribution of regorafenib under RT were confirmed in a free-moving rat model. Regorafenib inhibited the viability of Huh-7 cells in a dose-dependent manner. Apoptosis in Huh-7 cells was enhanced by RT followed by regorafenib treatment. In the concurrent regimen, RT decreased the area under the concentration versus time curve (AUC)regorafenib by 74% (p = 0.001) in the RT2 Gy × 3 fraction (f’x) group and by 69% (p = 0.001) in the RT9 Gy × 3 f’x group. The AUCregorafenib was increased by 182.8% (p = 0.011) in the sequential RT2Gy × 1 f’x group and by 213.2% (p = 0.016) in the sequential RT9Gy × 1 f’x group. Both concurrent regimens, RT2Gy × 3 f’x and RT9Gy × 3 f’x, clearly decreased the biodistribution of regorafenib in the heart, liver, lung, spleen and kidneys, compared to the control (regorafenib × 3 d) group. The concurrent regimens, both RT2Gy × 3 f’x and RT9Gy × 3 f’x, significantly decreased the biodistribution of regorafenib, compared with the control group. The PK of regorafenib can be modulated both by off-target irradiation and stereotactic body radiation therapy (SBRT).

Papaya shoot tip associated endophytic bacteria isolated from in vitro cultures and host–endophyte interaction in vitro and in vivo

Canadian Journal of Microbiology ◽

10.1139/w06-141 ◽

2007 ◽

Vol 53 (3) ◽

pp. 380-390 ◽

Cited By ~ 36

Author(s):

Pious Thomas ◽

Sima Kumari ◽

Ganiga K. Swarna ◽

T.K.S. Gowda

Keyword(s):

Culture Medium ◽

Carica Papaya ◽

In Vitro Cultures ◽

Dependent Manner ◽

16S Rdna Sequence ◽

16S Rdna Sequence Analysis ◽

Single Organism ◽

Fourteen distinct bacterial clones were isolated from surface-sterilized shoot tips (~1 cm) of papaya (Carica papaya L. ‘Surya’) planted on Murashige and Skoog (MS)-based papaya culture medium (23/50 nos.) during the 2–4 week period following in vitro culturing. These isolates were ascribed to six Gram-negative genera, namely Pantoea ( P. ananatis ), Enterobacter ( E. cloacae ), Brevundimonas ( B. aurantiaca ), Sphingomonas , Methylobacterium ( M. rhodesianum ), and Agrobacterium ( A. tumefaciens ) or two Gram-positive genera, Microbacterium ( M. esteraromaticum ) and Bacillus ( B. benzoevorans ) based on 16S rDNA sequence analysis. Pantoea ananatis was the most frequently isolated organism (70% of the cultures) followed by B. benzoevorans (13%), while others were isolated from single stocks. Bacteria-harboring in vitro cultures often showed a single organism. Pantoea, Enterobacter, and Agrobacterium spp. grew actively on MS-based normal papaya medium, while Microbacterium, Brevundimonas, Bacillus, Sphingomonas, and Methylobacterium spp. failed to grow in the absence of host tissue. Supplying MS medium with tissue extract enhanced the growth of all the organisms in a dose-dependent manner, indicating reliance of the endophyte on its host. Inoculation of papaya seeds with the endophytes (20 h at OD550 = 0.5) led to delayed germination or slow seedling growth initially. However, the inhibition was overcome by 3 months and the seedlings inoculated with Pantoea, Microbacterium, or Sphingomonas spp. displayed significantly better root and shoot growths.

Triphenyltin chloride induces spindle microtubule depolymerisation and inhibits meiotic maturation in mouse oocytes

Reproduction Fertility and Development ◽

10.1071/rd12332 ◽

2014 ◽

Vol 26 (8) ◽

pp. 1084 ◽

Cited By ~ 2

Author(s):

Yu-Ting Shen ◽

Yue-Qiang Song ◽

Xiao-Qin He ◽

Fei Zhang ◽

Xin Huang ◽

...

Keyword(s):

Polar Body ◽

Meiotic Maturation ◽

Dependent Manner ◽

Germinal Vesicle Breakdown ◽

Mouse Oocytes ◽

First Polar Body ◽

Triphenyltin Chloride ◽

Meiosis produces haploid gametes for sexual reproduction. Triphenyltin chloride (TPTCL) is a highly bioaccumulated and toxic environmental oestrogen; however, its effect on oocyte meiosis remains unknown. We examined the effect of TPTCL on mouse oocyte meiotic maturation in vitro and in vivo. In vitro, TPTCL inhibited germinal vesicle breakdown (GVBD) and first polar body extrusion (PBE) in a dose-dependent manner. The spindle microtubules completely disassembled and the chromosomes condensed after oocytes were exposed to 5 or 10 μg mL–1 TPTCL. γ-Tubulin protein was abnormally localised near chromosomes rather than on the spindle poles. In vivo, mice received TPTCL by oral gavage for 10 days. The general condition of the mice deteriorated and the ovary coefficient was reduced (P < 0.05). The number of secondary and mature ovarian follicles was significantly reduced by 10 mg kg–1 TPTCL (P < 0.05). GVBD decreased in a non-significant, dose-dependent manner (P > 0.05). PBE was inhibited with 10 mg kg–1 TPTCL (P < 0.05). The spindles of in vitro and in vivo metaphase II oocytes were disassembled with 10 mg kg–1 TPTCL. These results suggest that TPTCL seriously affects meiotic maturation by disturbing cell-cycle progression, disturbing the microtubule cytoskeleton and inhibiting follicle development in mouse oocytes.

The Antiinfective Effects of Velvet Antler of Formosan Sambar Deer (Cervus unicolor swinhoei) onStaphylococcus aureus-Infected Mice

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2011/534069 ◽

2011 ◽

Vol 2011 ◽

pp. 1-9 ◽

Cited By ~ 16

Author(s):

Ting-Yeu Dai ◽

Chih-Hua Wang ◽

Kun-Nan Chen ◽

I-Nung Huang ◽

Wei-Sheng Hong ◽

...

Keyword(s):

Lipoteichoic Acid ◽

Lavage Fluid ◽

Dependent Manner ◽

Protective Mechanisms ◽

Velvet Antler ◽

Sambar Deer ◽

Dose Dependent ◽

Cervus Unicolor

We assayed the effects of velvet antler (VA) of Formosan sambar deer (Cervus unicolor swinhoei) and its extracts on the anti-infective activity against pathogenicStaphylococcus aureus in vitroandin vivoin this study.In vitrodata indicated that the VA extracts stimulated the proliferation of resting splenocytes and macrophages in a dose-dependent manner up to the highest concentration used (150 μg mL−1). The production of proinflammatory cytokines (TNF-α, IL-6, IL-12) by lipoteichoic acid was significantly suppressed after being cocultured with the VA extracts in a dose-dependent manner. Animal test inS. aureus-infected mice demonstrated that the numbers of bacteria determined in the kidneys and peritoneal lavage fluid ofS. aureus-infected mice were significantly higher than those found in the same organs of mice pretreated with the VA samples. Moreover, the highly enhanced phagocytic activity of macrophages was further verified afterin vitrotreatment with the VA samples. The protective mechanisms of the VA samples might include an immune enhancer and an inflammatory cytokine suppressor.