A role of ROS-dependent defects in mitochondrial dynamic and autophagy in carbon black nanoparticle-mediated myocardial cell damage.

Carbon black nanoparticles (CBNPs) are widely distributed in the environment and are increasingly recognized as a contributor in the development of cardiovascular disease. A variety of cardiac injuries and diseases result from structural and functional damage to cardiomyocytes. This study explored the mechanisms of CBNPs-mediated myocardial toxicity. CBNPs were given to mice through intra-tracheal instillation and it was demonstrated that the particles can be taken up into the cardiac tissue. Exposure to CBNPs induced cardiomyocyte inflammation and apoptosis. In combination with in vitro experiments, we showed that CBNPs increased the ROS and induced mitochondria fragmentation. Functionally, CBNPs-exposed cardiomyocyte exhibited depolarization of the mitochondrial membrane potential, release of cytochrome c, and activation of pro-apoptotic BAX, thereby initiating programmed cell death. On the other hand, CBNPs impaired autophagy, leading to the inadequate removal of dysfunctional mitochondria. The excess accumulation of damaged mitochondria further stimulated NF-κB activation and triggered the NLRP3 inflammasome pathway. Both the antioxidant N-acetylcysteine and the autophagy activator rapamycin were effective to attenuate the damage of CBNPs on cardiomyocytes. Taken together, this study elucidated the potential mechanism underlying CBNPs-induced myocardial injury and provided a scientific reference for the evaluation and prevention of the CBNPs-related heart risk.

Identification and quantification of microplastics in salts by complementary approaches using pyrolysis-gas chromatography/quadrupole-time of flight mass spectrometry (Py-GC/QTOFMS) and laser direct infrared (LDIR) chemical imaging analysis.

Microplastics (MPs) and nanoplastics (NPs) have been suspected as contaminants in various foodstuffs, including salts, all over the world. Regarding the different sizes and polymer types, the mass concentrations of actual plastic particles in salt are not very clear. The purpose of this study is to develop a scalable method for qualitative and quantitative analysis of MPs and NPs by using Pyrolysis Gas Chromatography Quadrupole-Time of Flight mass spectrometry (Py-GC/QTOFMS) to detect their mass concentrations in salt samples. The targeted and suspected lists of polymers in salts were compiled based on the combined results of the high-resolution mass spectrometry (HRMS) full scanning with auxiliary MS dataset and the laser direct infrared (LDIR) chemical imaging analysis. The seven targeted MPs with polymer standards, i.e., polyvinyl chloride (PVC), polymethyl methacrylate (PMMA), polypropylene (PP), polystyrene (PS), polyethylene (PE), polyethylene terephthalate (PET), and polycarbonate (PC), were first subjected to a full MS scanning mode of the Py-GC/QTOFMS analysis. Subsequently, the parental masses of their pyrolysis compounds were used as the seeds to generate the related daughter masses. This process established both retention time and mass-pairs matching for the target MS/MS mode for enabling the identification and quantification of the particles. The suspected MPs with a matching degree >0.65 in the LDIR list were explored either by the full scan MS. Only PVC was identified, and PET was suspected. The Py-GC/QTOFMS result is complementary and comparable to the LDIR detection with the matching degree >0.85. We identified that PVC and PET (suspected) can be measured in both commercial and bulk sea salts, and their concentrations in sea salts are much higher than in rock salts, implying heavy contamination of the seawater.

Activation of prefrontal cortex and striatal regions in rats after shifting between rules in a T-maze.

Prefrontal cortical and striatal areas have been identified by inactivation or lesion studies to be required for behavioral flexibility, including selecting and processing of different types of information. In order to identify these networks activated selectively during the acquisition of new reward contingency rules, rats were trained to discriminate orientations of bars presented in pseudorandom sequence on two video monitors positioned behind the goal sites on a T-maze with return arms. A second group already trained in the visual discrimination task learned to alternate left and right goal arm visits in the same maze while ignoring the visual cues still being presented. In each experimental group, once the rats reached criterion performance, the brains were prepared after a 90-min delay for later processing for c-fos immunohistochemistry. While both groups extinguished a prior strategy and acquired a new rule, they differed by the identity of the strategies and previous learning experience. Among the 28 forebrain areas examined, there were significant increases in the relative density of c-fos immunoreactive cell bodies after learning the second rule in the prefrontal cortex cingulate, the prelimbic and infralimbic areas, the dorsomedial striatum and the core of the nucleus accumbens, the ventral subiculum, and the central nucleus of the amygdala. These largely correspond to structures previously identified in inactivation studies, and their neurons fire synchronously during learning and strategy shifts. The data suggest that this dynamic network may underlie reward-based selection for action-a type of cognitive flexibility.

Retinitis pigmentosa 2 pathogenic mutants degrade through BAG6/HUWE1 complex.

Retinitis pigmentosa (RP) is the most common inherited retinal degenerative disease which is the major cause of vision loss. X-linked RP patients account for 5%-15% of all inherited RP cases and mutations in RP2 (Retinitis pigmentosa 2) were responsible for about 20% X-linked RP families. A majority of RP2 pathogenic mutations displayed a vulnerable protein stability and degraded rapidly through ubiquitin-proteasome system (UPS). Though the RP2 protein could be readily recovered by proteasome inhibitors, e.g., MG132, their applications for RP2-related RP therapy were limited by their nonspecific characterization. In the present study, we aimed to identify UPS-related factors, such as E3 ligases, which are specifically involved in degradation of RP2 pathogenic mutants. We identified several E3 ligases, such as HUWE1, and the co-chaperon BAG6 specifically interacting with RP2 pathogenic mutants. Knockdown of HUWE1 and BAG6 could partially rescue the reduced protein levels of RP2 mutants. BAG6 is required for recruitment of HUWE1 to ubiquitinate RP2 mutants at the K268 site. The HUWE1 inhibitor BI8622 could restore the levels of RP2 mutant and then the binding to its partner ARL3 in retina cell lines. This study revealed the details of UPS-related degradation of RP2 mutants and possibly provided a potential treatment for RP2-related RP.

Upregulation of wild-type p53 by small molecule-induced elevation of NQO1 in non-small cell lung cancer cells.

The tumor suppressor p53 is usually inactivated by somatic mutations in malignant neoplasms, and its reactivation represents an attractive therapeutic strategy for cancers. Here, we reported that a new quinolone compound RYL-687 significantly inhibited non-small cell lung cancer (NSCLC) cells which express wild type (wt) p53, in contract to its much weaker cytotoxicity on cells with mutant p53. RYL-687 upregulated p53 in cells with wt but not mutant p53, and ectopic expression of wt p53 significantly enhanced the anti-NSCLC activity of this compound. RYL-687 induced production of reactive oxygen species (ROS) and upregulation of Nrf2, leading to an elevation of the NAD(P)H:quinoneoxidoreductase-1 (NQO1) that can protect p53 by inhibiting its degradation by 20S proteasome. RYL-687 bound NQO1, facilitating the physical interaction between NQO1 and p53. NQO1 was required for RYL-687-induced p53 accumulation, because silencing of NQO1 by specific siRNA or an NQO1 inhibitor uridine, drastically suppressed RYL-687-induced p53 upregulation. Moreover, a RYL-687-related prodrug significantly inhibited tumor growth in NOD-SCID mice inoculated with NSCLC cells and in a wt p53-NSCLC patient-derived xenograft mouse model. These data indicate that targeting NQO1 is a rational strategy to reactivate p53, and RYL-687 as a p53 stabilizer bears therapeutic potentials in NSCLCs with wt p53.

Novel quinolone derivatives targeting human dihydroorotate dehydrogenase suppress Ebola virus infection in vitro.

Ebola virus (EBOV) has emerged as a significant public health concern since the 2013-2016 outbreak in West Africa. Currently, no effective antiviral treatments have been approved for clinical use. Compound 1 RYL-634 is a quinolone-derived compound that can inhibit dihydroorotate dehydrogenase, a rate-limiting enzyme in the de novo pyrimidine synthesis pathway and it exhibited antiviral activity against multiple RNA virus infection. In this study, we evaluated the efficacy of a panel of newly developed compounds based on RYL-634 against EBOV infection. Our data showed that RYL-634 as well as its derivatives are effective against EBOV transcription- and replication-competent virus-like particle (trVLP) infection and authentic EBOV infection in vitro at low nanomolar IC50 values and relatively high CC50. Of note, the new derivative RYL-687 had the lowest IC50 at approximately 7 nM and was almost 6 times more potent than remdesivir (GS-5734). Exogenous addition of different metabolites in the pyrimidine de novo synthesis pathway confirmed DHODH as the target of RYL-687. These data provide evidence that such quinolone-derived compounds are promising therapeutic candidates against EBOV infection.

Discovery of a first-in-class CDK2 selective degrader for AML differentiation therapy.

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.

Design, Synthesis, and Evaluation of Highly Potent FAK-Targeting PROTACs.

Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase, exerts kinase-dependent enzymatic functions and kinase-independent scaffolding functions, both of which are crucial in cancer development, early embryonic development, and reproduction. However, previous efforts for FAK blocking mainly focus on kinase inhibitors. Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that allow direct post-translational knockdown of proteins via ubiquitination of a target protein by E3 ubiquitin ligase and subsequent proteasomal degradation. Here, we designed and synthesized a FAK PROTAC library with FAK inhibitor (PF562271 or VS6063) and CRBN E3 ligand. A novel FAK-targeting PROTAC, FC-11, showed a rapid and reversible FAK degradation with a picomolar of DC50 in various cell lines in vitro, which imply that FAK-PROTACs could be useful as expand tools for studying functions of FAK in biological system and as potential therapeutic agents.

Global PROTAC Toolbox for Degrading BCR-ABL Overcomes Drug-Resistant Mutants and Adverse Effects.

The BCR-ABL fusion oncoprotein causes chronic myeloid leukemia or acute lymphoblastic leukemia in Ph+ patients because the ABL kinase is constitutively activated. However, current clinical treatment with ABL inhibitors is seriously limited by drug resistance and adverse effects. Although the emerging proteolysis-targeting chimeras (PROTACs) have been introduced to degrade BCR-ABL, most of them showed limited activity and could not overcome the common drug-resistant mutants, especially for T315I mutant. Herein, we systematically designed a set of unique PROTACs by globally targeting all the three binding sites of BCR-ABL, including dasatinib-, ponatinib-, and asciminib-based PROTACs. Our ponatinib-based PROTACs showed practical activity as dasatinib-based PROTACs, while no reported ponatinib-based PROTACs could degrade BCR-ABL before. As a proof of concept, some additional dasatinib-based PROTACs were then designed to degrade T315I mutant too. We provided a global PROTAC toolbox for degrading both wild-type and T315I-mutated BCR-ABL from each binding site. More importantly, these PROTACs showed better selectivity and less adverse effects than the inhibitors, indicating that PROTACs had great potential for overcoming clinical drug resistance and safety issues.

Degradation versus Inhibition: Development of Proteolysis-Targeting Chimeras for Overcoming Statin-Induced Compensatory Upregulation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is an eight-pass transmembrane protein in the endoplasmic reticulum (ER) and a classical drug target to treat dyslipidemia. Statins including the well-known atorvastatin (Lipitor; Pfizer) have been widely used for the prevention and treatment of cardiovascular disease for decades. However, statins can elicit a compensatory upregulation of HMGCR protein and cause adverse effects including skeletal muscle damage. They are ineffective for patients with statin intolerance. Inspired by the recently emerging proteolysis-targeting chimeras (PROTACs), we set out to eliminate HMGCR protein using PROTAC-mediated degradation. One PROTAC designated as P22A was found to reduce HMGCR protein level and block cholesterol biosynthesis potently with less compensatory upregulation of HMGCR. To the best of our knowledge, HMGCR is the first ER-localized, polytopic transmembrane protein successfully degraded by the PROTAC technique. This finding may provide a new strategy to lower cholesterol levels and treat the associated diseases.

PROTACs: great opportunities for academia and industry.

Although many kinds of therapies are applied in the clinic, drug-resistance is a major and unavoidable problem. Another disturbing statistic is the limited number of drug targets, which are presently only 20-25% of all protein targets that are currently being studied. Moreover, the focus of current explorations of targets are their enzymatic functions, which ignores the functions from their scaffold moiety. As a promising and appealing technology, PROteolysis TArgeting Chimeras (PROTACs) have attracted great attention both from academia and industry for finding available approaches to solve the above problems. PROTACs regulate protein function by degrading target proteins instead of inhibiting them, providing more sensitivity to drug-resistant targets and a greater chance to affect the nonenzymatic functions. PROTACs have been proven to show better selectivity compared to classic inhibitors. PROTACs can be described as a chemical knockdown approach with rapidity and reversibility, which presents new and different biology compared to other gene editing tools by avoiding misinterpretations that arise from potential genetic compensation and/or spontaneous mutations. PRTOACs have been widely explored throughout the world and have outperformed not only in cancer diseases, but also in immune disorders, viral infections and neurodegenerative diseases. Although PROTACs present a very promising and powerful approach for crossing the hurdles of present drug discovery and tool development in biology, more efforts are needed to gain to get deeper insight into the efficacy and safety of PROTACs in the clinic. More target binders and more E3 ligases applicable for developing PROTACs are waiting for exploration.

Potent and Preferential Degradation of CDK6 via Proteolysis Targeting Chimera Degraders.

A focused PROTAC library hijacking cancer therapeutic target CDK6 was developed. A design principle as "match/mismatch" was proposed for understanding the degradation profile differences in these PROTACs. Notably, potent PROTACs with specific and remarkable CDK6 degradation potential were generated by linking CDK6 inhibitor palbociclib and E3 ligase CRBN recruiter pomalidomide. The PROTAC strongly inhibited proliferation of hematopoietic cancer cells including multiple myeloma and robustly degraded copy-amplified/mutated forms of CDK6, indicating future potential clinical applications.

Gram-scale total synthesis of teixobactin promoting binding mode study and discovery of more potent antibiotics.

Teixobactin represents a new class of antibiotics with novel structure and excellent activity against Gram-positive pathogens and Mycobacterium tuberculosis. Herein, we report a one-pot reaction to conveniently construct the key building block L-allo-Enduracidine in 30-gram scale in just one hour and a convergent strategy (3 + 2 + 6) to accomplish a gram-scale total synthesis of teixobactin. Several analogs are described, with 20 and 26 identified as the most efficacious analogs with 3~8-fold and 2~4-fold greater potency against vancomycin resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus respectively in comparison with teixobactin. In addition, they show high efficiency in Streptococcus pneumoniae septicemia mouse model and neutropenic mouse thigh infection model using methicillin-resistant Staphylococcus aureus. We also propose that the antiparallel ß-sheet of teixobactin is important for its bioactivity and an antiparallel dimer of teixobactin is the minimal binding unit for lipid II via key amino acids variations and molecular docking.

In Vitro-In Vivo Extrapolation of OATP1B-Mediated Drug-Drug Interactions in Cynomolgus Monkey.

Hepatic organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are clinically relevant transporters associated with significant drug-drug interactions (DDIs) and safety concerns. Given that OATP1Bs in cynomolgus monkey share >90% degree of gene and amino acid sequence homology with human orthologs, we evaluated the in vitro-in vivo translation of OATP1B-mediated DDI risk using this preclinical model. In vitro studies using plated cynomolgus monkey hepatocytes showed active uptake Km values of 2.0 and 3.9 µM for OATP1B probe substrates, pitavastatin and rosuvastatin, respectively. Rifampicin inhibited pitavastatin and rosuvastatin active uptake in monkey hepatocytes with IC50 values of 3.0 and 0.54 µM, respectively, following preincubation with the inhibitor. Intravenous pharmacokinetics of 2H4-pitavastatin and 2H6-rosuvastatin (0.2 mg/kg) and the oral pharmacokinetics of cold probes (2 mg/kg) were studied in cynomolgus monkeys (n = 4) without or with coadministration of single oral ascending doses of rifampicin (1, 3, 10, and 30 mg/kg). A rifampicin dose-dependent reduction in i.v. clearance of statins was observed. Additionally, oral pitavastatin and rosuvastatin plasma exposure increased up to 19- and 15-fold at the highest dose of rifampicin, respectively. Use of in vitro IC50 obtained following 1 hour preincubation with rifampicin (0.54 µM) predicted correctly the change in mean i.v. clearance and oral exposure of statins as a function of mean unbound maximum plasma concentration of rifampicin. This study demonstrates quantitative translation of in vitro OATP1B IC50 to predict DDIs using cynomolgus monkey as a preclinical model and provides further confidence in application of in vitro hepatocyte data for the prediction of clinical OATP1B-mediated DDIs.

Synthesis and evaluation of osimertinib derivatives as potent EGFR inhibitors.

Osimertinib has been identified as a promising therapeutic drug targeting for EGFR T790M mutant non-small cell lung cancer (NSCLC). A new series of N-oxidized and fluorinated osimertinib derivatives were designed and synthesized. The cellular anti-proliferative activity, kinase inhibitory activity and the activation of EGFR signaling pathways of 1-6 in vitro were determined against L858R/T790M and wild-type EGFR, the antitumor efficacy in NCI-H1975 xenografts in vivo were further studied. Compound 2, the newly synthesized N-oxide metabolite in N,N,N'-trimethylethylenediamine side chain of osimertinib, showed a comparable kinase selectivity in vitro and a slightly better antitumor efficacy in vivo to osimertinib, making it valuable and suitable for the potential lung cancer therapy.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides.

In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free Cmax values (0.06, 0.66, 2.57, and 7.79 µM, respectively) spanning the reported in vitro IC50 values for OATP1B1 and OATP1B3 (≤1.7 µM). As expected, the area under the plasma concentration-time curve (AUC) of an OATP probe drug (i.v. 2H4-pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of the plasma BA pool, a robust RIF dose response (maximal ≥50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)]. In vitro, RIF (≤100 µM) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocyte-mediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited (≥70%) by RIF (5 µM); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF (≤21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey OATPs and that exploration of their utility as circulating human OATP biomarkers is warranted.

Constituents from Zhuyeqing Liquor with hepatoprotective effect on alcohol-induced HepaG 2 toxicity.

An unprecedented new skeleton compound (1R, 10R, 11S)-10,11-dimethyl-4-formyl-2,9-dioxa-bicyclo [5.4.0] undeca-4,6-dien-3-one (1), monoterpenoids and monoterpene glycoside picrocrocinic ester (2), epijasminoside B (3) and 6'-O-(3-methoxyl-4-hydroxyl-coumaroyl)-epijasminoside B (4), along with 26 known compounds, were obtained from Zhuyeqing Liquor. These compounds were identified mainly by analyzing their NMR, HR-ESI-MS and CD data. The isolated compounds were screened against alcohol induced HepaG 2 toxicity for hepatoprotective assay. Compounds 10, 19, 21 and 26 displayed the highest potency against alcohol induced HepaG 2 toxicity with the cell viability ratio 41.21, 56.91, 67.69 and 70.32% respectively.

[Liver and heart iron deposition status in patients with ß thalassemia major: a multicenter study].

OBJECTIVE: To observe the status of iron deposition in patient with ß thalassemia major, and to formulate appropriate treatment strategies. METHOD: The data of status of transfusion and chelation in 135 patients aged from 6 years and 4 months to 17 years and 11 months with ß thalassemia major were collected and analyzed. Serum ferritin levels were determined and cardiac and hepatic iron deposition was determined using MRI T2(*) technology. RESULT: Of the 135 cases studied, 66 were male, and 69 were female, their average age was 12.1 years. Serum ferritin (SF) was determined for 111 cases, it varied from 1 086.8 µg/L to 15 011.5 µg/L. Among them, 16 cases had SF level <2 000 µg/L (14.5%) , in 41 cases SF were between 2 000 and 4 000 µg/L (36.0%) ;in 54 cases SF >4 000 µg/L (48.7%) . Liver MRI T2(*) results showed that in only 8 cases (5.9%) iron content in the liver was in normal range, 19 cases (14.9%) showed mild liver iron deposition;34 (25.2%) moderate and 74 (54.8%, the youngest one was only 6 years and 4 months of age) had severe iron deposition respectively. Cardiac MRI T2(*) showed that in 89 cases (65.9%) iron content in the heart was in normal range;19 cases (14.1%) had mild cardiac iron deposition and 27 (20.0%) presented severe iron deposition (the youngest one was only 9 years and 3 months of age) . SF level was obviously related to liver and cardiac iron deposition (MRI T2(*)) r and P value were -0.284, 0.003 and -0.374, 0.000 respectively. In 108 cases regular transfusion and chelation were delayed due to financial problem. The late and insufficient dosage administered and irregular chelation caused the higher SF level and the severe iron deposition. CONCLUSION: The survival status of ß thalassemia major in China is worrisome. Majority of them had not received regular transfusion and chelation. Liver and cardiac iron deposition occur early and had a high incidence.

Protective effect of Zhuyeqing liquor, a Chinese traditional health liquor, on acute alcohol-induced liver injury in mice.

The study first evaluated the hepatoprotective effect of Zhuyeqing Liquor (ZYQL) against acute alcohol-induced liver injury in mice. Animals were administered orally with 50% alcohol 12 ml/kg at 4 h after the doses of ZYQL everyday for fourteen consecutive days except mice in normal group. The protective effect was evaluated by biochemical parameters including serum aspartate transaminase (AST), alanine transferase (ALT), total-bilirubin (TBIL) and reduced glutathione (GSH), malondialdehyde (MDA), superoxide dismutase (SOD) in liver tissue. The result were confirmed histopathologically and the expression of TNF-α in mice liver was determined by immunohistochemistry analysis. HPLC-PDA was used for phytochemical analysis of ZYQL, and the plant source of each compound was claritied by UPLC-TOF-MS. The result showed that pretreatment with ZYQL exhibited a significant protective effect by reversing the biochemical parameters and histopathological changes in a dose depended manner. HPLC analysis indicated that ZYQL contained flavonoids, iridoids, terpenoids and phenolic acids, which might be the active chemicals. This study demonstrated the hepatoprotective activity of ZYQL, thus scientifically supported the function of its health care.