Structure-Activity relationship of 1-(Furan-2ylmethyl)Pyrrolidine-Based Stimulation-2 (ST2) inhibitors for treating graft versus host disease.

An elevated plasma level of soluble ST2 (sST2) is a risk biomarker for graft-versus-host disease (GVHD) and death in patients receiving hematopoietic cell transplantation (HCT). sST2 functions as a trap for IL-33 and amplifies the pro-inflammatory type 1 and 17 response while suppressing the tolerogenic type 2 and regulatory T cells activation during GVHD development. We previously identified small-molecule ST2 inhibitors particularly iST2-1 that reduces plasma sST2 levels and improved survival in two animal models. Here, we reported the structure-activity relationship of the furanylmethylpyrrolidine-based ST2 inhibitors based on iST2-1. Based on the biochemical AlphaLISA assay, we improved the activity of iST2-1 by 6-fold (∼6 µM in IC50 values) in the inhibition of ST2/IL-33 and confirmed the activities of the compounds in a cellular reporter assay. To determine the inhibition of the alloreactivity in vitro, we used the mixed lymphocyte reaction assay to demonstrate that our ST2 inhibitors decreased CD4+ and CD8+ T cells proliferation and increased Treg population. The data presented in this work are critical to the development of ST2 inhibitors in future.

Albumin Nanoparticle of Paclitaxel (Abraxane) Decreases while Taxol Increases Breast Cancer Stem Cells in Treatment of Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) has a high rate of metastasis, which is associated with breast cancer stem-like cells (CSCs). Although Taxol (micelle formulation of paclitaxel) is the first line chemotherapy to treat TNBC, it increases CSCs in residual tumors. Abraxane, albumin nanoparticle of paclitaxel, showed lower plasma concentration compared to Taxol in both human and animal models, but it is not clear why Abraxane showed superior efficacy to Taxol in treatment of metastatic breast cancer in humans. In this study, we intend to investigate if Abraxane eliminates CSCs for its better efficacy. The results showed that Abraxane showed similar cytotoxicity in SUM149 cells in comparison with Taxol. Although Abraxane showed 3- to 5-fold lower blood drug concentration compared to Taxol, it achieved similar tumor drug concentration and 10-fold higher tumor/plasma ratio in SUM149 xenograft NOD/SCID mouse model. In addition, Abraxane and Taxol showed similar efficacy to shrink the tumor size in orthotopic breast cancer NOD/SCID mouse model. However, Abraxane decreased breast CSCs frequency by 3- to 9-fold, while Taxol increased breast CSCs frequency in an orthotopic breast cancer NOD/SCID mouse model. Furthermore, Abraxane increased 3- to 15-fold intracellular uptake in both ALDH+ CSCs and differentiated ALDH- cells in comparison with Taxol, which provides a mechanism for Abraxane's superior efficacy to eliminate CSCs in comparison with Taxol. Our data suggest albumin nanoparticle Abraxane may have a broad implication to enhance drug's efficacy by eliminating breast cancer stem cells for treatment of metastatic diseases.

Neonatal Fc Receptor (FcRn) Enhances Tissue Distribution and Prevents Excretion of nab-Paclitaxel.

nab-Paclitaxel ( nab-P), an albumin-bound formulation of paclitaxel, was developed to improve the tolerability and antitumor activity of taxanes. The neonatal Fc receptor (FcRn) is a transport protein that can bind to albumin and regulate the homeostasis of circulating albumin. Therefore, the pharmacokinetics and pharmacodynamics of nab-P may be impacted by FcRn expression. This study aimed to investigate the effects of FcRn on nab-P elimination and distribution to targeted tissues. Wild-type and FcRn-knockout (FcRn-KO) mice were treated with nab-P, mouse-specific nab-P (distribution experiments only), and solvent-based paclitaxel (pac-T). Blood and tissue samples were collected for distribution analyses. Organ, urine, and fecal samples were collected for elimination analyses. The nab-P tissue penetration in the pancreas, fat pad, and kidney of wild-type mice, as reflected by the ratio of tissue/plasma concentration, was significantly higher (ranging from 5 to 80 fold) than that of FcRn-KO mice. In contrast, the tissue penetration of pac-T in these organs of FcRn-KO mice was similar to that of wild-type mice. More importantly, the excretion of nab-P in feces of FcRn-KO mice (45-68%) was significantly higher than that of wild-type mice (26-46%) from 8 to 48 h post treatment. In comparison, the difference of excretion of pac-T in feces between FcRn-KO mice and wild-type mice was smaller than that of nab-P. Furthermore, greater tissue penetration and fecal excretion were observed with nab-P than pac-T in both FcRn-KO and wild-type mice. These findings suggest that FcRn enhances the tissue distribution and penetration of nab-P in the targeted organs, while FcRn prevents excretion of nab-P to feces in the intestinal lumen. The findings support the notion that albumin nanoparticle delivery alters drug distribution and elimination through an FcRn-mediated process to impact drug efficacy and toxicity.

MEOX1 Promotes Tumor Progression and Predicts Poor Prognosis in Human Non-Small-Cell Lung Cancer.

Background: MEOX1 is a homeobox transcriptional factor, and plays essential roles in regulating somite development. Our previous study indicated that MEOX1 is a critical molecular target in mesenchymal-like cancer cells in PTEN-deficient Trastuzumab resistant breast cancer. Despite the potential implication of MEOX1 for the cancer progression, no previous studies examined its level and clinical significance in lung cancer tissues. In this study, we aimed to detect the MEOX1 expression and correlate its level with clinical outcome in non-small-cell lung cancer patients (NSCLC). Methods: MEOX1 gene expression in lung cancer was examined by using the Oncomine database. MEOX1 protein levels were evaluated by IHC using the corresponding primary antibody on two different commercial lung cancer tissue arrays. siRNA knockdown was used to elucidate the function of MEOX1. Results: Analysis of the Oncomine datasets identified that an elevation of MEOX1 in gene amplification in lung cancer tissues in comparison to normal lung tissues. Immunohistochemistical analysis demonstrated that MEOX1 was localized predominantly in the nucleus, and positive rate was 67.3% (111/165) in NSCLC samples. Statistical analysis revealed high levels of MEOX1 significantly correlated with Lymph Node Metastasis and Stage. Kaplan-Meier survival analysis showed that high levels of MEOX1 were significantly associated with unfavorable survival in NSCLC patients, and MEOX1 nucleus staining had worse survival, than did patients with overall expression in lung squamous cell carcinoma patients. Multivariate Cox's regression analysis found that MEOX1 was an independent poor prognostic predictor for patients with NSCLC. Silencing of MEOX1 by specific SiRNA significantly inhibited H460 and H1299 cell proliferation and sphere formation in serum-free medium. Conclusions: Our results firstly indentified that high levels of MEOX1 especially nuclear staining was an independent prognostic factor for NSCLC, and it served a essential roles in the regulation of cell proliferation and colony formation in vitro. It may represent a potential target for the NSCLC treatment.

Different Nanoformulations Alter the Tissue Distribution of Paclitaxel, Which Aligns with Reported Distinct Efficacy and Safety Profiles.

Previous studies have shown that different paclitaxel formulations produce distinct anticancer efficacy and safety profiles in animals and humans. This study aimed to investigate the distinct pharmacokinetics and tissue distribution of various nanoformulations of paclitaxel, which may translate into potential differences in safety and efficacy. Four nanoparticle formulations ( nab-paclitaxel, mouse albumin nab-paclitaxel [m -nab-paclitaxel], micellar paclitaxel, and polymeric nanoparticle paclitaxel) as well as solvent-based paclitaxel were intravenously administered to mice. Seventeen blood and tissue samples were collected at different time points. The total paclitaxel concentration in each tissue specimen was measured with liquid chromatography-tandem mass spectrometry. Compared with solvent-based paclitaxel, all four nanoformulations demonstrated decreased paclitaxel exposure in plasma. All nanoformulations were associated with paclitaxel blood-cell accumulation in mice; however, m- nab-paclitaxel was associated with the lowest accumulation. Five minutes after dosing, the total paclitaxel in the tissues and blood was approximately 44% to 57% of the administered dose of all paclitaxel formulations. Paclitaxel was primarily distributed to liver, muscle, intestine, kidney, skin, and bone. Compared with solvent-based paclitaxel, the different nanocarriers altered the distribution of paclitaxel in all tissues with distinct paclitaxel concentration-time profiles. nab-paclitaxel was associated with increased delivery efficiency of paclitaxel in the pancreas compared with the other formulations, consistent with the demonstrated efficacy of nab-paclitaxel in pancreatic cancer. All the nanoformulations led to high penetration in the lungs and fat pad, which potentially points to efficacy in lung and breast cancers. Micellar paclitaxel and polymeric nanoparticle paclitaxel were associated with high paclitaxel accumulation in the heart; thus, the risk of cardiovascular toxicity with these formulations may warrant further investigation. The solvent-based formulation was associated with the poorest paclitaxel penetration in all tissues and the lowest tissue-to-plasma ratio. The different nanocarriers of paclitaxel were associated with distinct pharmacokinetics and tissue distribution, which largely align with the observed efficacy and toxicity profiles in clinical trials.

Engineering exosomes as refined biological nanoplatforms for drug delivery.

Exosomes, a subgroup of extracellular vesicles (EVs), have been recognized as important mediators of long distance intercellular communication and are involved in a diverse range of biological processes. Because of their ideal native structure and characteristics, exosomes are promising nanocarriers for clinical use. Exosomes are engineered at the cellular level under natural conditions, but successful exosome modification requires further exploration. The focus of this paper is to summarize passive and active loading approaches, as well as specific exosome modifications and examples of the delivery of therapeutic and imaging molecules. Examples of exosomes derived from a variety of biological origins are also provided. The biocompatible characteristics of exosomes, with suitable modifications, can increase the stability and efficacy of imaging probes and therapeutics while enhancing cellular uptake. Challenges in clinical translation of exosome-based platforms from different cell sources and the advantages of each are also reviewed and discussed.

String (Cdc25) regulates stem cell maintenance, proliferation and aging in Drosophila testis.

Tight regulation of stem cell proliferation is fundamental to tissue homeostasis, aging and tumor suppression. Although stem cells are characterized by their high potential to proliferate throughout the life of the organism, the mechanisms that regulate the cell cycle of stem cells remain poorly understood. Here, we show that the Cdc25 homolog String (Stg) is a crucial regulator of germline stem cells (GSCs) and cyst stem cells (CySCs) in Drosophila testis. Through knockdown and overexpression experiments, we show that Stg is required for stem cell maintenance and that a decline in its expression during aging is a critical determinant of age-associated decline in stem cell function. Furthermore, we show that restoration of Stg expression reverses the age-associated decline in stem cell function but leads to late-onset tumors. We propose that Stg/Cdc25 is a crucial regulator of stem cell function during tissue homeostasis and aging.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA binding protein genetically and pathologically linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leads to the inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote the degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. Here, we show that mRNA transcripts harboring cryptic exons generated de novo proteins in TDP-43-depleted human iPSC-derived neurons in vitro, and de novo peptides were found in cerebrospinal fluid (CSF) samples from patients with ALS or FTD. Using coordinated transcriptomic and proteomic studies of TDP-43-depleted human iPSC-derived neurons, we identified 65 peptides that mapped to 12 cryptic exons. Cryptic exons identified in TDP-43-depleted human iPSC-derived neurons were predictive of cryptic exons expressed in postmortem brain tissue from patients with TDP-43 proteinopathy. These cryptic exons produced transcript variants that generated de novo proteins. We found that the inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Last, we showed that 18 de novo peptides across 13 genes were present in CSF samples from patients with ALS/FTD spectrum disorders. The demonstration of cryptic exon translation suggests new mechanisms for ALS/FTD pathophysiology downstream of TDP-43 dysfunction and may provide a potential strategy to assay TDP-43 function in patient CSF.

Regulation of cyclin A localization downstream of Par-1 function is critical for the centrosome orientation checkpoint in Drosophila male germline stem cells.

Male germline stem cells (GSCs) in Drosophila melanogaster divide asymmetrically by orienting the mitotic spindle with respect to the niche, a microenvironment that specifies stem cell identity. The spindle orientation is prepared during interphase through stereotypical positioning of the centrosomes. We recently demonstrated that GSCs possess a checkpoint ("the centrosome orientation checkpoint") that monitors correct centrosome orientation prior to mitosis to ensure an oriented spindle and thus asymmetric outcome of the division. Here, we show that Par-1, a serine/threonine kinase that regulates polarity in many systems, is involved in this checkpoint. Par-1 shows a cell cycle-dependent localization to the spectrosome, a germline-specific, endoplasmic reticulum-like organelle. Furthermore, the localization of cyclin A, which is normally localized to the spectrosome, is perturbed in par-1 mutant GSCs. Interestingly, overexpression of mutant cyclin A that does not localize to the spectrosome and mutation in hts, a core component of the spectrosome, both lead to defects in the centrosome orientation checkpoint. We propose that the regulation of cyclin A localization via Par-1 function plays a critical role in the centrosome orientation checkpoint.

A steamed broccoli sprout diet preparation that reduces colitis via the gut microbiota.

Sulforaphane is a bioactive metabolite with anti-inflammatory activity and is derived from the glucosinolate glucoraphanin, which is highly abundant in broccoli sprouts. However, due to its inherent instability its use as a therapeutic against inflammatory diseases has been limited. There are few studies to investigate a whole food approach to increase sulforaphane levels with therapeutic effect and reduce inflammation. In the current study, using a mouse model of inflammatory bowel disease, we investigated the ability of steamed broccoli sprouts to ameliorate colitis and the role of the gut microbiota in mediating any effects. We observed that despite inactivation of the plant myrosinase enzyme responsible for the generation of sulforaphane via steaming, measurable levels of sulforaphane were detectable in the colon tissue and feces of mice after ingestion of steamed broccoli sprouts. In addition, this preparation of broccoli sprouts was also capable of reducing chemically-induced colitis. This protective effect was dependent on the presence of an intact microbiota, highlighting an important role for the gut microbiota in the metabolism of cruciferous vegetables to generate bioactive metabolites and promote their anti-inflammatory effects.

Prophylactic Mitigation of Acute Graft versus Host Disease by Novel 2-(Pyrrolidin-1-ylmethyl)pyrrole-Based Stimulation-2 (ST2) Inhibitors.

Hematopoietic cell transplantation (HCT) is a proven and potentially curable therapy for hematological malignancies and inherited hematological disease. The main risk of HCT is the development of graft versus host disease (GVHD) acquired in up to 50% of patients. Upregulation of soluble ST2 (sST2) is a key clinical biomarker for GVHD prognosis and was shown to be a potential therapeutic target for GVHD. Agents targeting sST2 to reduce the sST2 level after HCT have the potential to mitigate GVHD progression. Here, we report 32 (or XY52) as the lead ST2 inhibitor from our optimization campaign. XY52 had improved inhibitory activity and metabolic stability in vitro and in vivo. XY52 suppressed proinflammatory T-cell proliferation while increasing regulatory T cells in vitro. In a clinically relevant GVHD model, a 21-day prophylactic regimen of XY52 reduced plasma sST2 and IFN-γ levels and GVHD score and extended survival in mice. XY52 represented a significant improvement over our previous compound, iST2-1, and further optimization of XY52 is warranted. The small-molecule ST2 inhibitors can potentially be used as a biomarker-guided therapy for mitigating GVHD in future clinical applications.

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD.

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development.

The role of GRASP65 in Golgi cisternal stacking and cell cycle progression.

In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans-oligomers. These conclusions, however, require further confirmation in the cell. In this study, we showed that the first 112 amino acids at the N-terminus (including the first PDZ domain, PDZ1) of the protein are sufficient for oligomerization. Systematic electron microscopic analysis showed that the expression of non-regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. These results provided evidence and a molecular mechanism by which GRASP65 stacks Golgi cisternal membranes. Further experiments revealed that inhibition of mitotic Golgi disassembly by expressing non-regulatable GRASP65 mutants did not affect equal partitioning of the Golgi membranes into the daughter cells. However, it delayed mitotic entry and suppressed cell growth; this effect was diminished by dispersing the Golgi apparatus with Brefeldin A treatment prior to mitosis, suggesting that Golgi disassembly at the onset of mitosis plays a role in cell cycle progression.

Structure‒tissue exposure/selectivity relationship (STR) correlates with clinical efficacy/safety.

Drug optimization, which improves drug potency/specificity by structure‒activity relationship (SAR) and drug-like properties, is rigorously performed to select drug candidates for clinical trials. However, the current drug optimization may overlook the structure‒tissue exposure/selectivity-relationship (STR) in disease-targeted tissues vs. normal tissues, which may mislead the drug candidate selection and impact the balance of clinical efficacy/toxicity. In this study, we investigated the STR in correlation with observed clinical efficacy/toxicity using seven selective estrogen receptor modulators (SERMs) that have similar structures, same molecular target, and similar/different pharmacokinetics. The results showed that drug's plasma exposure was not correlated with drug's exposures in the target tissues (tumor, fat pad, bone, uterus), while tissue exposure/selectivity of SERMs was correlated with clinical efficacy/safety. Slight structure modifications of four SERMs did not change drug's plasma exposure but altered drug's tissue exposure/selectivity. Seven SERMs with high protein binding showed higher accumulation in tumors compared to surrounding normal tissues, which is likely due to tumor EPR effect of protein-bound drugs. These suggest that STR alters drug's tissue exposure/selectivity in disease-targeted tissues vs. normal tissues impacting clinical efficacy/toxicity. Drug optimization needs to balance the SAR and STR in selecting drug candidate for clinical trial to improve success of clinical drug development.

Albumin nanoparticle containing a PI3Kγ inhibitor and paclitaxel in combination with α-PD1 induces tumor remission of breast cancer in mice.

Immunomodulators that remodel the tumor immunosuppressive microenvironment have been combined with anti-programmed death 1 (α-PD1) or anti-programmed death ligand 1 (α-PDL1) immunotherapy but have shown limited success in clinical trials. However, therapeutic strategies to modulate the immunosuppressive microenvironment of lymph nodes have been largely overlooked. Here, we designed an albumin nanoparticle, Nano-PI, containing the immunomodulators PI3Kγ inhibitor (IPI-549) and paclitaxel (PTX). We treated two breast cancer mouse models with Nano-PI in combination with α-PD1, which remodeled the tumor microenvironment in both lymph nodes and tumors. This combination achieved long-term tumor remission in mouse models and eliminated lung metastases. PTX combined with IPI-549 enabled the formation of a stable nanoparticle and enhanced the repolarization of M2 to M1 macrophages. Nano-PI not only enhanced the delivery of both immunomodulators to lymph nodes and tumors but also improved the drug accumulation in the macrophages of these two tissues. Immune cell profiling revealed that the combination of Nano-PI with α-PD1 remodeled the immune microenvironment by polarizing M2 to M1 macrophages, increasing CD4+ and CD8+ T cells, B cells, and dendritic cells, decreasing regulatory T cells, and preventing T cell exhaustion. Our data suggest that Nano-PI in combination with α-PD1 modulates the immune microenvironment in both lymph nodes and tumors to achieve long-term remission in mice with metastatic breast cancer, and represents a promising candidate for future clinical trials.

Optimization of the Prodrug Moiety of Remdesivir to Improve Lung Exposure/Selectivity and Enhance Anti-SARS-CoV-2 Activity.

COVID-19 patients with severe symptoms still lack antiviral treatment options. Although remdesivir is the only FDA-approved drug for those patients, its efficacy is limited by premature hydrolysis to nucleoside (NUC), low accumulation in the disease-targeted tissue (lungs), and low antiviral potency. In this study, we synthesized a new series of remdesivir analogues by modifying the ProTide moiety. In comparison with remdesivir, the lead compound MMT5-14 showed 2- to 7-fold higher antiviral activity in four variants of SARS-CoV-2. By reducing premature hydrolysis in hamsters, MMT5-14 increased the prodrug concentration by 200- to 300-fold in the plasma and lungs but also enhanced lung accumulation of the active metabolite triphosphate nucleosides (NTP) by 5-fold. Compared to remdesivir, MMT5-14 also increased the intracellular uptake and activation in lung epithelial cells by 4- to 25-fold. These data suggest that MMT5-14 could be a potential antiviral drug to treat COVID-19 patients with severe symptoms.

Reappraisal of anticancer nanomedicine design criteria in three types of preclinical cancer models for better clinical translation.

Anticancer nanomedicines are designed to improve anticancer efficacy by increasing drug accumulation in tumors through enhanced permeability retention (EPR) effect, and to reduce toxicity by decreasing drug accumulation in normal organs through long systemic circulation. However, the inconsistent efficacy/safety of nanomedicines in cancer patients versus preclinical cancer models have provoked debate for nanomedicine design criteria. In this study, we investigate nanomedicine design criteria in three types of preclinical cancer models using five clinically used nanomedicines, which identifies the factors for better clinical translations of their observed clinical efficacy/safety compared to free drug or clinical micelle formulation. When those nanomedicines were compared with drug solution or clinical micelle formulation in breast tumors, long and short-circulating nanomedicines did not enhance tumor accumulation by EPR effect in transgenic spontaneous breast cancer model regardless of their size or composition, although they improved tumor accumulations in subcutaneous and orthotopic breast cancer models. However, when tumors were compared to normal breast tissue, nanomedicines, drug solution and clinical micelle formulation showed enhanced tumor accumulation regardless of the breast cancer models. In addition, long-circulating nanomedicines did not further increase tumor accumulation in transgenic mouse spontaneous breast cancer nor universally decrease drug accumulations in normal organs; they decreased or increased accumulation in different organs, potentially changing the clinical efficacy/safety. In contrast, short-circulating nanomedicines decreased blood concentration and altered drug distribution in normal organs, which are correlated with their clinical efficacy/safety. A reappraisal of current nanomedicine design criteria is needed to ensure consistent clinical translation for improvement of their clinical efficacy/safety in cancer patients.

Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping.

In the current post-genomic era, large scale efforts are underway to functionally explore the proteome by assembling large antibody libraries. However, because many proteins are modified post-translationally to regulate their function, collections of modification-specific sensors are also needed. Here we applied a novel approach to select monoclonal phosphospecific antibodies directly from the full-length protein and without up-front phosphoamino acid identification. We chose as antigen GRASP65, a well studied Golgi phosphoprotein. Bacterially produced full-length protein was first incubated with mitotic cytosol, thus allowing modification by naturally occurring kinases, and then used directly for affinity-based antibody selection using a single chain variable fragment phagemid library. In less than 1 week, three distinct and highly functional monoclonal phosphospecific antibodies against two GRASP65 epitopes were obtained and subsequently characterized. The presented approach is carried out fully in vitro, requires no prior knowledge of the phosphoamino acid identity, and is fast and inexpensive. It therefore has great potential to be an attractive alternative to classic animal-based protocols for the selection of post-translation modification sensors and thus to become an invaluable tool in our quest to understand the proteome in all its complexity.

The peptide PROTAC modality: a novel strategy for targeted protein ubiquitination.

Despite dramatic advances in drug discovery over the decades, effective therapeutic strategies for cancers treatment are still in urgent demands. PROteolysis TArgeting Chimera (PROTAC), a novel therapeutic modality, has been vigorously promoted in preclinical and clinical applications. Unlike small molecule PROTAC, peptide PROTAC (p-PROTAC) with advantages of high specificity and low toxicity, while avoiding the limitations of shallow binding pockets through large interacting surfaces, provides promising substitutions for E3 ubiquitin ligase complex-mediated ubiquitination of "undruggable proteins". It is worth noting that successful applications of p-PROTAC still have some obstacles, including low stability and poor membrane permeability. Hence, we highlight that p-PROTAC combined with cell-penetrating peptides, constrained conformation technique, and targeted delivery systems could be the future efforts for potential translational research.

Lei-gong-gen formula granule attenuates hyperlipidemia in rats via cGMP-PKG signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Lei-gong-gen formula granule (LFG) is a folk prescription derived from Zhuang nationality, the largest ethnic minority among the 56 nationalities in China. It is composed of three herbs, namely Centella asiatica (L.) Urb., Eclipta prostrata (L.) L., Smilax glabra Roxb. It has been widely used as health protection tea for many years to prevent cardiovascular and cerebrovascular diseases such as hyperlipidemia and hypertension. AIM OF THE STUDY: This study validated the lipid-lowering effect of LFG in a hyperlipidemia rat model. Then we employed network pharmacology and molecular biological approach to identify the active ingredients of LFG, corresponding targets, and its anti-hyperlipidemia mechanisms. MATERIALS AND METHODS: Hyperlipidemia rat model was established by feeding male Sprague-Dawley rats with high-fat diet for two weeks. LFG (two doses of 10 and 20 g/kg) was administered orally to hyperlipidemia rat model for 4 weeks, twice per day. Serum levels of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) were monitored in rats pre and post-treatment. Hematoxylin-eosin staining was applied to observe the pathology and lipid accumulation of liver. We then performed network pharmacology analysis to predict the ingredients, their associated targets, and hyperlipidemia associated targets. Pathway analysis with significant genes was carried out using KEGG pathway. These genes and proteins intersectioned between compound targets and hyperlipidemia targets were further verified with samples from hyperlipidemia rats treated with LFG using Real-time RT-PCR and Western Blot. RESULTS: LFG attenuated hyperlipidemia in rat model, and this was characterized with decreased serum levels of TC, LDL-C, liver wet weight, and liver index. LFG alleviated the hepatic steatosis in hyperlipidemia rats. Network pharmacology analysis identified 53 bioactive ingredients from LFG formula (three herbs), which link to 765 potential targets. 53 hyperlipidemia associated genes were retrieved from public databases. There were 10 common genes between ingredients-targets and hyperlipidemia associated genes, which linked to 20 bioactive ingredients. Among these 10 genes, 3 of them were validated to be involved in LFG's anti-hyperlipidemia effect using Real-time RT-PCR, namely ADRB2 encoding beta-2 adrenergic receptor, NOS3 encoding nitric oxide synthase 3, LDLR encoding low-density lipoprotein receptor. The cGMP-PKG signaling pathway was enriched for hyperlipidemia after pharmacology network analysis with ADRB2, NOS3, and LDLR. Interestingly, expression of cGMP-dependent protein kinase (PKG) was downregulated in hyperlipidemia rat after LFG treatment. Molecular docking study further supported that ferulic acid, histidine, p-hydroxybenzoic acid, and linalool were potential active ingredients for LFG's anti-hyperlipidemia effect. LC-MS/MS analysis confirmed that ferulic acid and p-hydroxybenzoic acid were active ingredients of LFG. CONCLUSION: LFG exhibited the lipid-lowering effect, which might be attributed to downregulating ADRB2 and NOS3, and upregulating LDLR through the cGMP-PKG signaling pathway in hyperlipidemia rat. Ferulic acid and p-hydroxybenzoic acid might be the underlying active ingredients which affect the potential targets for their anti-hyperlipidemia effect.