Fragment-Based Screen of SARS-CoV-2 Papain-like Protease (PLpro).

Coronaviruses have been responsible for numerous viral outbreaks in the past two decades due to the high transmission rate of this family of viruses. The deadliest outbreak is the recent Covid-19 pandemic, which resulted in over 7 million deaths worldwide. SARS-CoV-2 papain-like protease (PLPro) plays a key role in both viral replication and host immune suppression and is highly conserved across the coronavirus family, making it an ideal drug target. Herein we describe a fragment-based screen against PLPro using protein-observed NMR experiments, identifying 77 hit fragments. Analyses of NMR perturbation patterns and X-ray cocrystallized structures reveal fragments bind to two distinct regions of the protein. Importantly none of the fragments identified belong to the same chemical class as the few reported inhibitors, allowing for the discovery of a novel class of PLPro inhibitors.

Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization.

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

Bone marrow mesenchymal stem cell-derived exosomes inhibit chondrocyte apoptosis and the expression of MMPs by regulating Drp1-mediated mitophagy.

Osteoarthritis (OA) is a joint degenerative disease commonly seen in the elderly. Bone marrow mesenchymal stem cell-exosomes (BMSC-exosomes) are closely associated with the progression of OA. Here, we investigated whether BMSC-exosomes can affect OA development by regulating mitophagy. Primary rat chondrocytes were treated with advanced glycation end products (AGEs) to induce cell damage. The results of flow cytometry showed that AGEs treatment significantly promoted apoptosis of chondrocytes. AGEs treatment also enhanced the expression of matrix metalloproteinases (MMPs), MMP-3 and MMP-13, and dynamin-related protein 1 (Drp1) in chondrocytes. To investigate the impact of BMSC-exosomes on chondrocytes, chondrocytes were treated with BMSC-exosomes. AGEs-mediated increase of apoptosis and up-regulation of MMP-3, MMP-13, and Drp1 in chondrocytes were abrogated by BMSC-exosomes. Western blot analysis of autophagy-related proteins and Mito-Keima assay revealed that BMSC-exosome treatment elevated the expression of autophagy-related proteins, LC3-II/LC3-I and Beclin-1, and promoted mitophagy in the AGEs-treated chondrocytes. Moreover, Drp1 overexpression repressed the expression of LC3-II/LC3-I and Beclin-1, and enhanced apoptosis and the expression of MMP-3 and MMP-13 in AGEs-treated chondrocytes. BMSC-exosomes reversed the impact of Drp1 overexpression on AGEs-treated chondrocytes. In conclusion, this work demonstrates that BMSC-exosomes inhibit chondrocyte apoptosis and the expression of MMPs, which attributes to regulate Drp1-mediated mitophagy. Thus, BMSC-exosomes may be a potential treatment for OA.

Suture repair of patellar inferior pole fracture: Transosseous tunnel suture compared with anchor suture.

Patellar inferior pole fracture is difficult to treat due to the inherent weakness of small comminuted distal fragments. However, suture fixation was recently introduced and reported. The aim of the present study was to evaluate and compare the clinical outcomes of two suture techniques, transosseous tunnel suture (TTS) and anchor suture (AS), for the fixation of patellar inferior pole fracture. A total of 35 patients with patellar inferior pole fracture treated at the Second Affiliated Hospital of Nanchang University (Nanchang, China) between June 2014 and April 2018 were retrospectively reviewed. Of these, 14 were treated with the TTS technique and 21 using AS fixation. The operation time, incision length and total cost were determined and compared. Functional outcomes were analyzed with the visual analog scale (VAS), Bostman and Lysholm scores and knee joint ranges of motion (ROMs). Postoperative complications were also observed and recorded. The mean follow-up was 22.6±9.7 and 18.7±5.9 months for TTS and AS, respectively. The groups were similar regarding age, sex, operative side and time to surgery. A smaller incision length and shorter operation time but higher hospital costs were observed in the AS group (P<0.01). For functional evaluation, there was no significant difference in VAS, Bostman and Lysholm scores or ROM between the 2 groups (P>0.05). No postoperative complications were observed in the TTS group. Only one patient in the AS group experienced a superficial minor wound infection. The TTS and AS techniques provided similarly satisfactory clinical outcomes for treating patellar inferior pole fracture. TTS had the advantage of cost-effectiveness due to saving anchors, while AS had a shorter operation time and a smaller incision length.

Design, synthesis and biological evaluation of novel chroman derivatives as non-selective acetyl-CoA carboxylase inhibitors.

Acetyl-CoA carboxylases (ACCs) are the rate-limiting enzymes in the de no lipogenesis, which play an important role in the synthesis and oxidation of fatty acid. Recent research reveals that ACCs are tightly relevant to many kinds of metabolic diseases and cancers. In this study, we synthesized a series of chroman derivatives and evaluated their ACCs inhibitory activities, obtaining compound 4s with IC50 value of 98.06 nM and 29.43 nM of binding activities in ACC1 and ACC2, respectively. Compound 4s exhibited the most potent anti-proliferation activity against A549, H1975, HCT116 and H7901 cell lines (values of IC50: 0.578 µΜ, 1.005 µΜ, 0.680 µΜ and 1.406 µΜ, respectively). Docking studies were performed to explain the structure-activity relationships. These results indicate that compound 4s is a promising ACC1/2 inhibitor for the potent treatment of cancer.

Activation of Sigma-1 Receptor Enhanced Pericyte Survival via the Interplay Between Apoptosis and Autophagy: Implications for Blood-Brain Barrier Integrity in Stroke.

Stroke is a cerebrovascular disorder that affects many people worldwide. Pericytes play an important role in stroke progression and recovery. The sigma-1 receptor (σ-1R) signaling pathway has been suggested as having promising neuroprotective potential in treating stroke; however, whether σ-1R activation regulates pericyte function remains unknown. The aim of this study was to elucidate the role of σ-1R and a novel σ-1R agonist in pericytes following ischemic stroke. An ischemic stroke animal model was induced by photothrombotic middle cerebral artery occlusion (pMCAO) in σ-1R knockout (KO) and wild-type (WT) mice. After pMCAO, there was significant pericyte loss and coverage in σ-1R KO mice compared with WT mice as determined using transmission electron microscopy, immunofluorescence staining, and western blot. Interestingly, a novel σ-1R agonist decreased infarct volume and blood-brain barrier damage with a concomitant amelioration of pericyte loss, as determined by western blot. Further studies indicated that cell apoptosis and autophagy were induced in an in vivo pMCAO ischemic stroke animal model and an in vitro oxygen glucose deprivation-treatment group. Inhibition of autophagy using a pharmacological approach significantly mitigated pericyte apoptosis, suggesting that autophagy was upstream of apoptosis in pericytes. Both in vivo and in vitro studies indicated that the σ-1R agonist significantly decreased cell apoptosis via inhibition of autophagy with a subsequent enhancement of pericyte survival. This study identified the unique roles for σ-1R in mediating pericyte survival via the regulation of the interplay between apoptosis and autophagy, suggesting that a novel σ-1R agonist may be a promising therapeutic agent for the treatment of stroke patients.

WZ66, a novel acetyl-CoA carboxylase inhibitor, alleviates nonalcoholic steatohepatitis (NASH) in mice.

The global prevalence of nonalcoholic steatohepatitis (NASH) increases incredibly. NASH ends up to advanced liver disease, which is highly threatening to human health. Currently, treatment of NASH is very limited. Acetyl-CoA carboxylases (ACC1/ACC2) are proved as effective drug targets for NASH. We aimed to develop novel ACC inhibitors and evaluate their therapeutic value for NASH prevention. ACC inhibitors were obtained through structure-based drug design, synthesized, screened from ACC enzymatic measurement platform and elucidated in cell culture-based assays and animal models. The lipidome and microbiome analysis were integrated to assess the effects of WZ66 on lipids profiles in liver and plasma as well as gut microbiota in the intestine. WZ66 was identified as a novel ACC1/2 inhibitor. It entered systemic circulation rapidly and could accumulate in liver. WZ66 alleviated NASH-related liver features including steatosis, Kupffer cells and hepatic stellate cells activation in diet-induced obese mice. The triglycerides (TGs) and other lipids including diglycerides (DGs), phosphatidylcholine (PC) and sphingomyelin (SM) were decreased in WZ66-treated mice as evidenced by lipidome analysis in livers. The lipids profiles in plasma were also altered with WZ66 treatment. Plasma TG were moderately increased, while the activation of SREBP1c was not detected. WZ66 also downregulated the abundance of Allobaculum, Mucispirillum and Prevotella genera as well as Mucispirillum schaedleri species in gut microbiota. WZ66 is an ideal lead compound and a potential drug candidate deserving further investigation in the therapeutics of NASH.

Long noncoding RNA MALAT-1 inhibits apoptosis and matrix metabolism disorder in interleukin-1ß-induced inflammation in articular chondrocytes via the JNK signaling pathway.

Proinflammatory cytokine such as interleukin (IL)-1ß causes inflammation of articular cartilage. In this current study, we explored the chondroprotective effects of long noncoding RNA (lncRNA) MALAT-1 on cell proliferation, apoptosis, and matrix metabolism in IL-1ß-induced inflammation in articular chondrocytes. Articular chondrocytes from knee joints of normal rats were isolated and cultured, followed by identification through observation of toluidine blue and COL II immunocytochemical stainings. The proliferation of chondrocytes at passage 2 was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The inflammatory chondrocytes induced by 10 ng/mL IL-1ß were observed and identified by toluidine blue and COL II immunocytochemical stainings. pcDNA 3.1 and pcDNA-MALAT-1 were transfected in the chondrocytes. Ultrastructure of chondrocytes was observed by using a transmission electron microscope. The MTT assay was carried out to evaluate chondrocyte viability. Hoechst 33258 staining and flow cytometry were adopted to assess chondrocyte apoptosis. The chondrocytes at passage 2 with the biological characteristics of chondrocytes were used for subsequent experiments. In IL-1ß-treated chondrocytes, the growth rate of chondrocytes slowed down, the cells became narrow and long, the vacuoles were seen in the cells, and the morphology of the chondrocytes was irregular. The toluidine blue staining and the immunohistochemical staining of COL II became weaker. In response to IL-1ß induction, articular chondrocytes showed reduced MALAT-1 expression; moreover, obvious cartilage injury was observed with decreased chondrocyte viability and Col II expression and elevated chondrocyte apoptosis, MMP-13 expression, and p-JNK expression. With the treatment of pcDNA-MALAT-1, the cartilage injury was alleviated with increased chondrocyte viability and type II collagen (Col II) expression and reduced chondrocyte apoptosis, MMP-13 expression and p-JNK expression. Taken together these results, lncRNA MALAT-1 blocked the activation of the JNK signaling pathway; thereby, IL-1ß-induced inflammation in articular chondrocytes was reduced with enhanced chondrocyte proliferation and suppressed chondrocyte apoptosis and extracellular matrix degradation.

Design, synthesis and biological evaluation of novel spiro-pentacylamides as acetyl-CoA carboxylase inhibitors.

Acetyl-CoA carboxylase (ACC) catalyzes the rate-determining step in de novo lipogenesis and plays an important role in the regulation of fatty acid oxidation. Therefore, ACC inhibition offers a promising option for intervention in nonalcoholic fatty liver disease (NAFLD), type 2 diabetes (T2DM) and cancer. In this paper, a series of spiropentacylamide derivatives were synthesized and evaluated for their ACC1/2 inhibitory activities and anti-proliferation effects on A549, H1975, HCT116, SW620 and Caco-2 cell lines in vitro. Compound 6o displayed potent ACC1/2 inhibitory activity (ACC1 IC50 = 0.527 µM, ACC2 IC50 = 0.397 µM) and the most potent anti-proliferation activities against A549, H1975, HCT116, SW620 and Caco-2 cell lines, with IC50 values of 1.92 µM, 0.38 µM, 1.22 µM, 2.05 µM and 5.42 µM respectively. Further molecular docking studies revealed that compound 6o maintained hydrogen bonds between the two carbonyls and protein backbone NHs (Glu-B2026 and Gly-B1958). These results indicate that compound 6o is a promising ACC1/2 inhibitor for the potent treatment of cancer.

Two cases in which 3D MRI was used to differentiate between a disc mass that mimics a tumor and neurinoma.

BACKGROUND: Since disc sequestration that mimics a tumor is rare and sometimes presents with an atypical appearance upon magnetic resonance imaging (MRI), it is often confused with other more common epidural and intradural neoplasms, particularly neurinoma. Open surgery is necessary due to the difficult of achieving a definitive diagnosis using computed tomography, MRI, and gadolinium- enhanced MRI prior to operation. Herein, we describe the use of coronal MR images of 3D fast-field echo with water selective excitation in the diagnosis of disc sequestration mimicking a tumor. CASE PRESENTATION: Two patients were admitted to our hospital with back pain, radiating pain, and hypoesthesia in the right lower limb. MRI revealed tumor-like masses in the lateral recess of L3 and posterior to the body of L4. The initial diagnosis indicated disc sequestration mimicking a tumor and neurinoma. The coronal MR images of 3D fast-field echo with water selective excitation showed a clear boundary between the tumor-like mass and the nerve root. Moreover, the mass was also completely separated from the dura. Therefore, neurinoma was excluded as a possible diagnosis prior to operation. Surgical excision to perform removal of the gross mass was performed in one patient. The histopathological diagnosis was consistent with the 3D fast-field echo with water-selective excitation MRI. Another patient was successfully treated by minimally invasive endoscopic surgery. CONCLUSIONS: Disc sequestration that mimics a tumor is difficult to diagnose preoperatively. As a non-invasive strategy, coronal MR images of 3D fast-field echo with water selective excitation is a helpful imaging tool for differentiating between diagnosis of disc sequestration that mimics a tumor and neurinoma prior to operation. If the disc fragment of mimicking tumor can be identified prior to operation, open surgery may not be necessary for all patients. Minimally invasive endoscopic surgery also is an alternative strategy.

Design, synthesis and biological evaluation of novel 2,3-dihydroquinazolin- 4(1H)-one derivatives as potential fXa inhibitors.

Coagulation factor Xa (fXa) is a particularly attractive target for the development of effective and safe anticoagulants. In this study, novel 2,3-dihydroquinazolin-4(1H)-one derivatives were designed as potential fXa inhibitors based on anthranilamide structure which has been reported in our previous research. The experimental data showed that most of the designed compounds exhibited significant in vitro potency against fXa. Among them, compound 8e displayed the strongest potency against fXa with the IC50 value of 21 nM and highly selectivity versus thrombin (IC50 = 67 µM) and excellent in vitro antithrombotic activity with its 2 × PT value of 1.2 µM and 2 × aPTT value of 0.6 µM. In addition, 8e also displayed excellent in vivo antithrombotic activity in the rat arteriovenous shunt (AV-SHUNT) model. The bleeding risk evaluation showed that 8e had a similar safety profile as that of betrixaban. All results demonstrated that compound 8e could be considered as a potential fXa inhibitor for the prevention and treatment of thromboembolic diseases.

[Early diagnosis and treatment for trauma around the knee with popliteal vascular injury].

OBJECTIVE: To investigate the early diagnosis and treatment for trauma around the knee with popliteal vascular injury. METHODS: A retrospective analysis was employed to analyze the clinical data from 15 patients (9 males and 6 females were with a mean age of 39.2 years old,ranging from 26 to 62 years old) with fracture or dislocation around the knee with popliteal vascular injury from January 2007 to January 2013. Combined with clinical symptoms and signs, oxygen saturation monitors, color ultrasound, DSA angiography and interventional surgery were used to determine the vascular injury. The knee fracture and dislocation were fixed with hybrid external fixation and plate-screw fixation, respectively. Then, the blood circulation was reconstructed by thrombectomy, repair and autologous vein graft for individual injured vascular. The average total operation time, average hospitalization days, predictive salvage index (PSI), average blood transfusion amount, average medical expenses and infection cases were recorded to determine the effect of early diagnosis and treatment. RESULTS: There was one patient with death, 8 patients with amputation, and 6 patients with successful repair surgery for popliteal artery, anterior tibial and posterior tibial arteries. These six patients with surviving limbs were followed up for an average of 28.3 months (ranged, 12 to 60 months). Among the 6 successful patients, the joint function of 4 patients was good and excellent. CONCLUSION: The trauma around the knee with popliteal vascular injury is characterized by complex and serious injury, easy misdiagnosis and loss diagnosis, poor prognosis and high risk of amputation. The early diagnosis of trauma around the knee with popliteal vascular injury should depend on the mechanism of trauma, local anatomical characteristics of injury site, clinical presentations and appropriate auxiliary examinations. The appropriate indications for limb salvage and amputation should be used to achieve more effective clinical results.

[Case-control study on operative treatment for complex tibial plateau fracture].

OBJECTIVE: To explore optimal choice of surgical treatment and operative approach for closed complex tibial plateau fractures and its influencing factors. METHODS: From January 2003 to January 2011, 95 patients with closed complex tibial plateau fractures were estimated Schatzker V and Vl, and treated with three different surgical methods. The methods included single plate through anterolateral incision (Group A, 22 cases), double plates through inside and outside incisions (Group B, 36 cases), and double plates through antero-midline incisions (Group C, 37 cases). There were 56 males and 39 females, ranged the age from 19 to 57 years (averaged, 36.3 years), 50 cases in left, 45 cases in right. According to Schatzker classification, 51 cases were type V, 44 cases were VI. The data of operation time, intraoperative blood loss, complications (infectious of wound, necrosis, bad incision, collapse fracture, loosen of internal fixation, fracture failure)and recovery of function of lower limb joint were collected. RESULTS: There were no significant difference among three groups in operation time (P > 0.05); blood loss in group A was obvious better than other groups (P < 0.05); collapse of joint surface and failure rate of internal fixation in group A was higher than other groups (P > 0.05); Merchant score after 1 year were higher in group B, C than group A. For lower limb function, 10 cases got excellent results, 5 good, 4 fair and 3 poor in group A; 21 cases got excellent results, 11 good, 3 fair and 1 poor in group B; 23 cases got excellent results, 11 good,2 fair and 1 poor in group C. CONCLUSION: The blood loss in group A was least, but fracture exposure and joint surface was not satisfactory, and stable fixation could not be achieved, the long-term result was not good. For fractures with double condyles and dislocated involved, double plates through inside and outside incisions or double plates through antero-midline incisions was suggested,which benefit good reduction of joint surface, stable fixation, and erlier exercise.

[Effects of total flavones of Chrysanthemum indicum on proliferation and apoptosis of human osteosarcoma Saos-2 cells].

OBJECTIVE: To study the effects of total flavones of Chrysanthemum indicum on proliferation and apoptosis of human osteosarcoma Saos-2 cells and its mechanism. METHODS: The effect of the total flavones of Chrysanthemum indicum on the proliferation of human osteosarcoma Saos-2 cells was detected by CCK assay, and the morphological changes of cells treated with total flavones of Chrysanthemum indicum were observed using contrast microscope. Flow cytomerty was performed to analyze the apoptotic rate of the cells, and the gene expression levels of Caspase-3, BCL-2, BAX were detected by RT-PCR. RESULTS: The total flavones of Chrysanthemum indicum suppressed the proliferation of osteosarcoma cells in a dose-and time-dependent manner. Under a microscope observation of cell morphology, the volume became smaller ,the number of internal particles was increased. Cell apoptosis rate was positively related to the drug concentration. After treated for 48 hours, Caspase-3 and BAX expression were up-regulated, BCL-2 and BCL-2/BAX were decreased. CONCLUSION: The total flavones of Chrysanthemum indicum can inhibit the proliferation of osteosarcoma cell line Saos-2 by inducing cell apoptosis,the mechanism of which might be related with reducing BCL-2/BAX and activating Caspase-3.

[Research progress of protective effects of alendronate on articular cartilage in osteoarthritis].

OBJECTIVE: To summarize the recent development on chondroprotective effect of alendronate (ALN) on articular cartilage in osteoarthritis (OA). METHODS: The related literature was reviewed and the main achievements in vitro/vivo studies in the fields were summarized. RESULTS: ALN can improve the metabolic microenvironment of the articular cartilage in OA, inhibit subchondral bone remodeling, so it has potential protective effect on articular cartilage. CONCLUSION: ALN is expected to become a disease-modifying OA drug in future, but OA treatment still lack a uniform basic and clinical evaluation criteria, so it has guiding significance in development and application of ALN to develope a uniform standard and obtain the clinical data.