Investigation of targets and anticancer mechanisms of covalently acting natural products by functional proteomics.

Eriocalyxin B (EB), 17-hydroxy-jolkinolide B (HJB), parthenolide (PN), xanthatin (XT) and andrographolide (AG) are terpenoid natural products with a variety of promising antitumor activities, which commonly bear electrophilic groups (α,ß-unsaturated carbonyl groups and/or epoxides) capable of covalently modifying protein cysteine residues. However, their direct targets and underlying molecular mechanisms are still largely unclear, which limits the development of these compounds. In this study, we integrated activity-based protein profiling (ABPP) and quantitative proteomics approach to systematically characterize the covalent targets of these natural products and their involved cellular pathways. We first demonstrated the anti-proliferation activities of these five compounds in triple-negative breast cancer cell MDA-MB-231. Tandem mass tag (TMT)-based quantitative proteomics showed all five compounds commonly affected the ubiquitin mediated proteolysis pathways. ABPP platform identified the preferentially modified targets of EB and PN, two natural products with high anti-proliferation activity. Biochemical experiments showed that PN inhibited the cell proliferation through targeting ubiquitin carboxyl-terminal hydrolase 10 (USP10). Together, this study uncovered the covalently modified targets of these natural products and potential molecular mechanisms of their antitumor activities.

Upper Critical Solution Temperature Polyvalent Scaffolds Aggregate and Exterminate Bacteria.

Specific recognition and strong affinities of bacteria receptors with the host cell glycoconjugates pave the way to control the bacteria aggregation and kill bacteria. Herein, using aggregation-induced emission (AIE) molecules decorated upper critical solution temperature (UCST) polyvalent scaffold (PATC-GlcN), an approach toward visualizing bacteria aggregation and controlling bacteria-polyvalent scaffolds affinities under temperature stimulus is described. Polyvalent scaffolds with diblocks, one UCST block PATC of polyacrylamides showing a sharp UCST transition and typical AIE behavior, the second bacteria recognition block GlcN of hydrophilic glucosamine modified polyacrylamide, are prepared through a reversible addition and fragmentation chain transfer polymerization. Aggregated chain conformation of polyvalent scaffolds at temperature below UCST induces the aggregation of E. coli ATCC8739, because of the high density of glucosamine moieties, whereas beyond UCST, the hydrophilic state of the scaffolds dissociates the bacteria aggregation. The sweet-talking of bacteria toward the polyvalent scaffolds can be visualized by the fluorescent imaging technique, simultaneously. Due to the specific recognition of polyvalent scaffolds with bacteria, the photothermal agent IR780 loaded PATC-GlcN shows the targeted killing ability toward E. coli ATCC8739 in vitro and in vivo under NIR radiation.

Proteomic characterization of post-translational modifications in drug discovery.

Protein post-translational modifications (PTMs), which are usually enzymatically catalyzed, are major regulators of protein activity and involved in almost all celluar processes. Dysregulation of PTMs is associated with various types of diseases. Therefore, PTM regulatory enzymes represent as an attractive and important class of targets in drug research and development. Inhibitors against kinases, methyltransferases, deacetyltransferases, ubiquitin ligases have achieved remarkable success in clinical application. Mass spectrometry-based proteomics technologies serve as a powerful approach for system-wide characterization of PTMs, which facilitates the identification of drug targets, elucidation of the mechanisms of action of drugs, and discovery of biomakers in personalized therapy. In this review, we summarize recent advances of proteomics-based studies on PTM targeting drugs and discuss how proteomics strategies facilicate drug target identification, mechanism elucidation, and new therapy development in precision medicine.

Recent Progress in Bile Acid-Based Antimicrobials.

With the emergence of drug-resistant bacteria and the formation of biofilms by bacteria and fungi, microbial infections gradually threaten global health. Natural antimicrobial peptides (AMPs) have low susceptibility for developing resistance due to the membrane targeted mechanism, but instability and high manufacturing cost limit their applications in clinic. Bile acids, a group of steroids in the human body, with high stability, biocompatibility, and inherent facial amphiphilic structure similar to the characteristics of AMPs, have been applied to the biological field, such as drug delivery systems, self-healing hydrogels, antimicrobials, and so on. In this review, we mainly focus on the different classes of bile acid-based antimicrobials in recent years. Various designs and methods for the preparation of unimolecular antimicrobials with bile acid skeletons are first introduced, including coupling of primary amine, quaternary ammonium, and amino acid units with bile acid skeletons. Some representative oligomeric antimicrobials, including dimers of bile acids, are summarized. Finally, macromolecular antimicrobials bearing some positive charges at the main chain or side chain and interaction mechanisms of these bile acid-based antimicrobials are discussed.

External evaluation of published population pharmacokinetic models of polymyxin B.

OBJECTIVES: Several population pharmacokinetics (popPK) models for polymyxin B have been constructed to optimize therapeutic regimens. However, their predictive performance remains unclear when extrapolated to different clinical centers. Therefore, this study aimed to evaluate the predictive ability of polymyxin B popPK models. METHODS: A literature search was conducted, and the predictive performance was determined for each selected model using an independent dataset of 20 patients (92 concentrations) from the Third Xiangya Hospital. Prediction- and simulation-based diagnostics were used to evaluate model predictability. The influence of prior information was assessed using Bayesian forecasting. RESULTS: Eight published studies were evaluated. In prediction-based diagnostics, the prediction error within ± 30% was over 50% in two models. In simulation-based diagnostics, the prediction- and variability-corrected visual predictive check (pvcVPC) showed satisfactory predictivity in three models, while the normalized prediction distribution error (NPDE) tests indicated model misspecification in all models. Bayesian forecasting demonstrated a substantially improvement in the model predictability even with one prior observation. CONCLUSION: Not all published models were satisfactory in prediction- and simulation-based diagnostics; however, Bayesian forecasting improved the predictability considerably with priors, which can be applied to guide polymyxin B dosing recommendations and adjustments for clinicians.

A self-monitoring microneedle patch for light-controlled synergistic treatment of melanoma.

Melanoma is the most aggressive and malignant form of skin cancer. Current melanoma treatment methods generally suffer from frequent drug administration as well as difficulty in direct monitoring of drug release. Here, a self-monitoring microneedle (MN)-based drug delivery system, which integrates a dissolving MN patch with aggregation-induced emission (AIE)-active PATC microparticles, is designed to achieve light-controlled pulsatile chemo-photothermal synergistic therapy of melanoma. The PATC polymeric particles, termed D/I@PATC, encapsulate both of chemotherapeutic drug doxorubicin (DOX) and the photothermal agent indocyanine green (ICG). Upon light illumination, PATC gradually dissociates into smaller particles, causing the release of encapsulated DOX and subsequent fluorescence intensity change of PATC particles, thereby not only enabling direct observation of the drug release process under light stimuli, but also facilitating verification of drug release by fluorescence recovery after light trigger. Moreover, encapsulation of ICG in PATC particles displays significant improvement of its photothermal stability both in vitro and in vivo. In a tumor-bearing mouse, the application of one D/I@PATC MN patch combining with two cycles of light irradiation showed excellent controllable chemo-photothermal efficacy and exhibited ∼97% melanoma inhibition rate without inducing any evident systemic toxicity, suggesting a great potential for skin cancer treatment in clinics.

A scalable flood-resilience-index for measuring climate change adaptation: Munich city.

Climate change is affecting the frequency and intensity of rainfall extreme events worldwide. Despite the growing global awareness, developing flood resilient cities has proven to be a major challenge. This paper investigates the application of an event-based scalable Flood Resilience Index (FRI) for assessing climate change adaptation. Flood resilience is represented by three dimensions: physical, social and economic. A household climate adaptation is adopted consisting of a combination of a flood-proof gate with an indoor tank and a submersible pump system implemented in all houses. The climate related impact under a high-emission scenario (RCP8.5) is analysed for Munich with the CRCM5 Large-Ensemble. Results show that for Munich extreme heavy rainfall events are increasing. The FRI can successfully identify households and districts which: a) are mostly affected by heavy rainfall, b) benefit the most from the climate adaptation, and c) are the most resilient. For the most severe future scenario investigated the climate adaptation measure was able to improve 57% of all affected buildings within Maxvorstadt to an FRI equal to 1.0 during the event and recovery phase.

Piperazine-Derived α1D/1A Antagonist 1- Benzyl-N- (3-(4- (2-Methoxyphenyl) Piperazine-1-yl) Propyl) -1H- Indole-2- Carboxamide Induces Apoptosis in Benign Prostatic Hyperplasia Independently of α1-Adrenoceptor Blocking.

Previous studies have indicated that α1D/1A antagonist naftopidil (NAF) suppresses prostate growth by decreasing cell proliferation without affecting apoptosis and prostate volume in benign prostatic hyperplasia (BPH). A NAF-derived α1D/1A antagonist 1- benzyl-N-(3-(4-(2-methoxyphenyl) piperazine-1-yl) propyl)-1H-indole-2- carboxamide (HJZ-12) has been reported from our laboratory, which exhibits high subtype-selectivity to both α1D- and α1A- AR (47.9- and 19.1- fold, respectively) with respect to a1B-AR in vitro. However, no further study was conducted. In the present study, a pharmacological evaluation of HJZ-12 in BPH was performed on an estrogen/androgen-induced rat BPH model and human BPH-1 cell line. In vivo, HJZ-12 exhibited better performance than NAF in preventing the progression of rat prostatic hyperplasia by not only decreasing prostate weight and proliferation (similar to NAF) but also, shrinking prostate volume and inducing prostate apoptosis (different from NAF). In vitro, HJZ-12 exhibited significant cell viability inhibition and apoptotic induction in BPH-1 cell line, without presenting cell anti-proliferation properties. Intriguingly, the role of HJZ-12 on cell viability and apoptosis was an α1-independent action. Furthermore, RNA-Seq analysis was applied to screen out six anti-apoptotic genes (Bcl-3, B-lymphoma Mo-MLV insertion region 1 [Bmi-1], ITGA2, FGFR3, RRS1, and SGK1). Amongst them, Bmi-1 was involved in the apoptotic induction of HJZ-12 in BPH-1. Overall, HJZ-12 played a remarkable role in preventing the progression of prostatic hyperplasia through α1-independent apoptotic induction, indicating that it will be a multi-target effective candidate for BPH treatment.

Ubc9 Attenuates Myocardial Ischemic Injury Through Accelerating Autophagic Flux.

AIMS: SUMOylation is a post-translational modification that plays a crucial role in the cellular stress response. We aimed to demonstrate whether and how the SUMO E2 conjugation enzyme Ubc9 affects acute myocardial ischemic (MI) injury. METHODS AND RESULTS: Adenovirus expressing Ubc9 was administrated by multipoint injection in the border zone of heart immediately after MI in C57BL/6 mice. Neonatal rat cardiomyocytes (NRCMs) were also infected, followed by oxygen and glucose deprivation (OGD). In vivo, Ubc9 adenovirus-injected mice showed decreased cardiomyocyte apoptosis, reduced myocardial fibrosis, and improved cardiac function post-MI. In vitro, overexpression of Ubc9 decreased cardiomyocyte apoptosis, whereas silence of Ubc9 showed the opposite results during OGD. We next found that Ubc9 significantly decreased the accumulation of autophagy marker p62/SQSTM, while the LC3 II level hardly changed. When in the presence of bafilomycin A1 (BAF), the Ubc9 adenovirus plus OGD group presented a higher level of LC3 II and GFP-LC3 puncta than the OGD group. Moreover, the Ubc9 adenovirus group displayed increased numbers of yellow plus red puncta and a rising ratio of red to yellow puncta on the mRFP-GFP-LC3 fluorescence assay, indicating that Ubc9 induces an acceleration of autophagic flux from activation to degradation. Mechanistically, Ubc9 upregulated SUMOylation of the core proteins Vps34 and Beclin1 in the class III phosphatidylinositol 3-kinase (PI3K-III) complexes and boosted the protein assembly of PI3K-III complex I and II under OGD. Moreover, the colocalization of Vps34 with autophagosome marker LC3 or lysosome marker Lamp1 was augmented after Ubc9 overexpression, indicating a positive effect of Ubc9-boosted protein assembly of the PI3K-III complexes on autophagic flux enhancement. CONCLUSIONS: We uncovered a novel role of Ubc9 in protecting cardiomyocytes from ischemic stress via Ubc9-induced SUMOylation, leading to increased PI3K-III complex assembly and autophagy-positioning. These findings may indicate a potential therapeutic target, Ubc9, for treatment of myocardial ischemia.

Inhibitory effect of α1D/1A antagonist 2-(1H-indol-3-yl)-N-[3-(4-(2-methoxyphenyl) piperazinyl) propyl] acetamide on estrogen/androgen-induced rat benign prostatic hyperplasia model in vivo.

Benign prostatic hyperplasia (BPH) is a common disorder of the urinary system in aging men. 2-(1H-indol-3-yl)-N-[3-(4-(2-methoxyphenyl) piperazinyl) propyl] acetamide (HJZ-3), which is derived from naftopidil, exhibited 97.7- and 64.6-fold greater inhibitory effects for a1D adrenoceptor than for a1B- and a1A-adrenoceptors in vitro, respectively. To investigate the therapeutic potential for treating BPH, we evaluated the pharmacological activity of HJZ-3. Specifically, we evaluated through estrogen/androgen-induced rat benign prostatic hyperplasia model in vivo. HJZ-3 effectively prevented the progression of rat prostatic hyperplasia by suppressing the increase in prostate index and reducing the quantitative analysis of the relative acinus volume, relative stroma, epithelial volume and epithelial thickness and expression of proliferating cell nuclear antigen and α-smooth muscle actin. HJZ-3 decreased α1A- and α1D-adrenoceptor protein expressions in prostate tissue. HJZ-3 is a good alternative for α1A- and α1D-adrenoceptor blocker. It may relax smooth muscle tone and relieve symptoms of BPH.

Increased expression of Sonic hedgehog restores diabetic endothelial progenitor cells and improves cardiac repair after acute myocardial infarction in diabetic mice.

Damaged endothelial progenitor cells (EPCs) are associated with poor prognosis in diabetic myocardial infarction (DMI). Our previous studies revealed that an impaired Sonic hedgehog (Shh) pathway contributes to insufficient function in diabetic EPCs; however, the roles of the Shh pathway in diabetic EPC apoptosis under basal and hypoxic/ischemic conditions remain unknown. Therefore, the present study investigated whether Shh revitalized diabetic EPCs and consequently improved the deteriorative status of DMI. For this purpose, streptozotocin injection was used in male C57/BL6 mice to induce type­1 diabetes, and diabetic EPCs were isolated from the bone marrow. Apoptosis, cell function, and protein expression were investigated in EPCs in vitro. Mouse hearts were injected with adenovirus Shh­modified diabetic EPCs (DM­EPCShh) or control DM­EPCNull immediately after coronary artery ligation in vivo. Cardiac function, capillary numbers, fibrosis, and cell apoptosis were then detected. First, the in vitro results demonstrated that the apoptosis of diabetic EPCs was reduced following treatment with Shh protein for 24 h, under normal or hypoxic conditions. BMI1 proto­oncogene (Bmi1), an antiapoptotic protein found in several cells, was reduced in diabetic EPCs under normal or hypoxic conditions, but was upregulated after Shh protein stimulation. When Bmi1­siRNA was administered, the antiapoptotic effect of Shh protein was significantly reversed. In addition, p53, a Bmi1­targeted gene, was demonstrated to mediate the antiapoptotic effect of the Shh/Bmi1 pathway in diabetic EPCs. The Shh/Bmi1/p53 axis also enhanced the diabetic EPC function. In vivo, Shh­modified diabetic EPCs exhibited increased EPC retention and decreased apoptosis at 3 days post­DMI. At 14 days post­DMI, these cells presented enhanced capillary density, reduced myocardial fibrosis and improved cardiac function. In conclusion, the present results demonstrated that the Shh pathway restored diabetic EPCs through the Shh/Bmi1/p53 axis, suppressed myocardial apoptosis and improved myocardial angiogenesis, thus reducing cardiac fibrosis and finally restoring myocardial repair and cardiac function in DMI. Thus, the Shh pathway may serve as a potential target for autologous cell therapy in diabetic myocardial ischemia.