Analysis of the toxicological and pharmacokinetic profile of Kaempferol-3-O-β-D-(6”-E-p-coumaryl) glucopyranoside - Tiliroside: in silico, in vitro and ex vivo assay

Tiliroside is a glycosidic flavonoid present in many plants species including Helicteres velutina K. Schum (Malvaceae sensu lato), commonly known in Brazil as “pitó”. This molecule has been shown to have many biological activities, however no study has been carried out to investigate the toxicity of this substance. The present work aimed to evaluate the possible cellular toxicity in silico, in vitro and ex-vivo of the kaempferol-3-O-β-D-(6”-E-p-coumaroyl) glucopyranoside (tiliroside), through chemical structure analysis, toxicity assessment and predictive bioactive properties, using human samples for in vitro and ex-vivo tests. The in silico analysis suggests that tiliroside exhibited great absorption index when penetrating biological membranes. In addition, it also displayed considerable potential for cellular protection against free radicals, and anticarcinogenic, antioxidant, antineoplastic, anti-inflammatory, anti-hemorrhagic and antithrombotic activities. The assessment of the hemolytic and genotoxic effects of tiliroside showed low hemolysis rates in red blood cells and absence of cellular toxicity in the oral mucosa cells. The data obtained indicate that this molecule could be a promising therapeutic approach as a possible new drug with biotechnological potential.

Stimulation of Angiotensin Converting Enzyme 2: A Novel Treatment Strategy for Diabetic Nephropathy

Despite current therapies for diabetic nephropathy, many patients continue to progress to end-stage renal disease requiring renal replacement therapy. While the precise mechanisms underlying diabetic nephropathy remain to be determined, it is well established that chronic activation of the renin angiotensin aldosterone system (RAAS) plays a substantial role in the pathogenesis of diabetic nephropathy. Angiotensin converting enzyme 2 (ACE2), the enzyme responsible for activating the reno-protective arm of the RAAS converts angiotensin (Ang) II into Ang 1-7 which exerts reno-protective effects. Chronic RAAS activation leads to kidney inflammation and fibrosis, and ultimately lead to end-stage kidney disease. Currently, angiotensin converting enzyme inhibitors and Ang II receptor blockers are approved for renal fibrosis and inflammation. Targeting the reno-protective arm of the RAAS should therefore, provide further treatment options for kidney fibrosis and inflammation. In this review, we examine how targeting the reno-protective arm of the RAAS can ameliorate kidney inflammation and fibrosis and rescue kidney function in diabetic nephropathy. We argue tissue ACE2 stimulation provides a unique and promising therapeutic approach for diabetic nephropathy.

NRF2 Participates in the Suppressive Tumor Immune Microenvironment of KRAS/KEAP1 Co-Mutant Non-Small Cell Lung Cancer by Inhibiting the STING Pathway

Background: KRAS/KEAP1 (KK) co-mutant lung adenocarcinoma (LUAD) exhibited poor response to immune checkpoint inhibitors (ICI) via shaping a suppressive tumor immune microenvironment, the mechanism remains to be elucidated. Methods: The mRNA and protein expression of target molecules were analyzed by qRT-PCR and Western blot, respectively. The subcellular location of NRF2 was observed by immunofluorescence staining, and nuclear and cytoplasm isolation. After exogenous over-expression and knockdown of NRF2 and the addition of a STING pathway inhibitor in tumor cells, the effects on the CD8+ T cell recruitment was detected using chemotaxis assay, and the secretion of chemokines CCL5 and CXCL10 was analyzed by ELISA. The potential NRF2 target BRCA1 was identified using bioinformatic approaches and verified by a dual luciferase reporter assay. Results: NRF2, the target of KEAP1, was overexpressed and activated in KK type cells. NRF2 effected as a negative regulator of CD8+ T cells recruitment by decreasing CCL5 and CXCL10 chemokines in KK type LUAD. Mechanistically, NRF2 promoted the transcription and expression of BRCA1 to repair DNA damage, resulting in STING pathway inactivation. Conclusion: The combination of NRF2 inhibitor or STING agonist with ICI may be a promising therapeutic approach for patients with KK type LUAD.

Scorpion Venom-Functionalized Quercetin Phytosomes for Breast Cancer Management: In Vitro Response Surface Optimization and Anticancer Activity against MCF-7 Cells

Breast cancer is a dangerous type of cancer in women. Quercetin (QRT), a naturally occurring flavonoid, has wide biological effects including antioxidant, anticarcinogenic, anti-inflammatory, antiallergic, and antiviral activities. The anticancer activity is considered the most valuable effect of QRT against several types of cancer, including prostate, liver, lung, colon, and breast cancer. Scorpion venom peptides (SV) has been found to induce apoptosis and aggravate cancer cells, making it a promising anticancer agent. QRT, SV, and Phospholipon® 90H (PL) were incorporated in a nano-based delivery platform to assess QRT’s cellular uptake and antiproliferative efficacy against a lung cancer cell line derived from human breast cancer cells MCF-7. Several nanovesicles were prepared and optimized, using four-factor Box–Behnken, in an experimental design. The optimized phytosomes showed vesicle size and zeta potential values of 116.9 nm and 31.5 mV, respectively. The IC50 values revealed that MCF-7 cells were significantly more sensitive to the optimized QRT formula than the plain formula and raw QRT. Cell cycle analysis revealed that optimized QRT formula treatment resulted in significant cell cycle arrest at the S phase. The results also indicated that treatment with QRT formula significantly increased caspase-9, Bax, Bcl-2, and p53 mRNA expression, compared with the plain formula and QRT. In terms of the inflammatory markers, the QRT formula significantly reduced the activity of TNF-α and NF-κB, in comparison with the plain formula and QRT only. Overall, the findings from the study proved that a QRT formulation could be a promising therapeutic approach for the treatment of breast cancer.

Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer

The switch/sucrose non-fermentable (SWI/SNF) complex has a crucial role in chromatin remodelling1 and is altered in over 20% of cancers2,3. Here we developed a proteolysis-targeting chimera (PROTAC) degrader of the SWI/SNF ATPase subunits, SMARCA2 and SMARCA4, called AU-15330. Androgen receptor (AR)+ forkhead box A1 (FOXA1)+ prostate cancer cells are exquisitely sensitive to dual SMARCA2 and SMARCA4 degradation relative to normal and other cancer cell lines. SWI/SNF ATPase degradation rapidly compacts cis-regulatory elements bound by transcription factors that drive prostate cancer cell proliferation, namely AR, FOXA1, ERG and MYC, which dislodges them from chromatin, disables their core enhancer circuitry, and abolishes the downstream oncogenic gene programs. SWI/SNF ATPase degradation also disrupts super-enhancer and promoter looping interactions that wire supra-physiologic expression of the AR, FOXA1 and MYC oncogenes themselves. AU-15330 induces potent inhibition of tumour growth in xenograft models of prostate cancer and synergizes with the AR antagonist enzalutamide, even inducing disease remission in castration-resistant prostate cancer (CRPC) models without toxicity. Thus, impeding SWI/SNF-mediated enhancer accessibility represents a promising therapeutic approach for enhancer-addicted cancers.

Structure-Based De Novo Design and Docking Studies of 5(S)-Methyl-L-Proline Containing Peptidomimetic Compounds as Dipeptidyl Peptidase-4 Inhibitors

Background: Diabetes affects millions of people worldwide, with predicted numbers of about 700 million adults affected by 2045. Among the several anti-diabetic drug therapies available in the market, Dipeptidyl Peptidase-4 (DPP-4) inhibitors have emerged as a promising therapeutic approach with scope for exploration in the segment of peptidomimetics. Objective: Series of proline-containing peptidomimetic compounds were designed and investigated for their drug-likeness through Lipinski’s rule of five, lead-likeness through the rule of three, predictive pharmacokinetic studies (absorption, distribution, metabolism, and excretion), and toxicity properties through in-silico approaches. The designed compounds were evaluated for their interactions with binding sites of the enzyme DPP-4 using an extra precision docking approach. Methods: Proline-containing peptidomimetic compounds were designed rationally. Drug-likeness and lead-likeness properties were calculated using Schrödinger Maestro v11.2 software. ADME and toxicity properties were predicted using PreADMET version 2.0. Docking study was performed using Schrödinger Maestro v11.2 software, and ligands for the study were designed using MarvinSketch software. Results: 5(S)-methyl-L-proline containing 17 ligands were designed. All of them were found to obey Lipinski’s rule of five. Compounds were found to have good ADME profile and low toxicity predictions. Conclusion: Four compounds were found to have good interactions with DPP-4 binding sites and hence created the scope to develop a DPP-4 inhibitors containing 5(S)-methyl-L-proline moiety.

Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas

Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent HBV covalently closed circular DNA (cccDNA) and integrated DNA under antiviral therapy form the major barrier to eradication of HBV infection. CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, for HBV cure. However, the cleavage of integrated HBV DNA by CRISPR-Cas9 will cause double-strand break (DSB) of host genome, raising a serious safety concern about genome instability and carcinogenesis. The newly developed CRISPR-derived base editors (BEs), which fuse a catalytically disabled nuclease with a nucleobase deaminase enzyme, can be used to permanently inactivate HBV genome by introducing irreversible point mutations for generation of premature stop codons without DSBs of host genome. Although promising, CRISPR-mediated base editing still faces daunting challenges before its clinical application, including the base-editing efficacy, the off-target effect, the difficulty in finding conserved target HBV sequences, and in vivo delivery efficiency. Several strategies have been adopted to optimize the efficiency and specificity of CRISPR-BEs and to improve in vivo delivery efficacy through novel viral and non-viral delivery approaches. Particularly, the non-viral delivery of Cas9 mRNA and ribonucleoprotein by lipid nanoparticles exhibits attractive potential for liver-targeted delivery in clinical. Along with all progress above, the CRISPR-mediated gene therapy will ultimately achieve HBV cure.

Klotho and Mesenchymal Stem Cells: A Review on Cell and Gene Therapy for Chronic Kidney Disease and Acute Kidney Disease

Chronic kidney disease (CKD) and acute kidney injury (AKI) are public health problems, and their prevalence rates have increased with the aging of the population. They are associated with the presence of comorbidities, in particular diabetes mellitus and hypertension, resulting in a high financial burden for the health system. Studies have indicated Klotho as a promising therapeutic approach for these conditions. Klotho reduces inflammation, oxidative stress and fibrosis and counter-regulates the renin-angiotensin-aldosterone system. In CKD and AKI, Klotho expression is downregulated from early stages and correlates with disease progression. Therefore, the restoration of its levels, through exogenous or endogenous pathways, has renoprotective effects. An important strategy for administering Klotho is through mesenchymal stem cells (MSCs). In summary, this review comprises in vitro and in vivo studies on the therapeutic potential of Klotho for the treatment of CKD and AKI through the administration of MSCs.

A three-dimensional immune-oncology model for studying in vitro human NK cell cytotoxic activity v1

Immunotherapy has emerged as a promising therapeutic approach for treating several forms of cancer. Adoptive cell transfer of immune cells, such as natural killer (NK) cells, provides a powerful therapeutic potential against tumor cells. In the past decades, two-dimensional (2D) tumor models have been used to investigate the effectiveness of immune cell killing. However, the 2D tumor models exhibit less structural complexity and cannot recapitulate the physiological condition of the tumor microenvironment. Thus, the effectiveness of immune cells against tumor cells using these models cannot fully be translated to clinical studies. In order to gain a deeper insight into immune cell-tumor interaction, more physiologically relevant in vivo-like three-dimensional (3D) tumor models have been developed. These 3D tumor models can mimic the dynamic cellular activities, making them much closer to the in vivo tumor profiles. Here, we describe a simple and effective protocol to study the cytotoxic activity of primary human NK cells toward the 3D tumor spheroids. Our protocol includes isolation and expansion of human NK cells, labeling and formation of tumor spheroids, co-culture of NK cells and tumor spheroids, and evaluation of cytotoxic activity using a confocal microscope. This protocol is also applicable to other types of tumors and immune cells.