Discovery of Novel Phenoxyaryl Pyridones as Bromodomain and Extra-Terminal Domain (BET) Inhibitors with High Selectivity for the Second Bromodomain (BD2) to Potentially Treat Acute Myeloid Leukemia.

Bromodomain-selective BET inhibition has emerged as a promising strategy to improve the safety profiles of pan-BET inhibitors. Herein, we report the discovery of potent phenoxyaryl pyridones as highly BD2-selective BET inhibitors. Compound 23 (IC50 = 2.9 nM) exhibited a comparable BRD4 BD2 inhibitory activity relative to 10 (IC50 = 1.0 nM) and remarkably improved selectivity over BRD4 BD1 (23: 2583-fold; 10: 344-fold). This lead compound significantly inhibited the proliferation of acute myeloid leukemia (AML) cell lines through induction of G0/G1 arrest and apoptosis in vitro. Excellent in vivo antitumor efficacy with 23 was achieved in an MV;411 mouse xenograft model. Pleasingly, compound 23 (hERG IC50 > 30 µM) mitigated the inhibition of the human ether-à-go-go-related gene (hERG) ion channel compared with 10 (hERG IC50 = 2.8 µM). This work provides a promising BD2-selective lead for the development of more effective and safe BET inhibitors as anticancer agents.

A Dual-Responsive Morphologically-Adaptable Nanoplatform for Targeted Delivery of Activatable Photosensitizers in Precision Photodynamic Therapy.

Photodynamic therapy (PDT) is an effective approach for treating melanoma. However, the photosensitizers employed in PDT can accumulate in healthy tissues, potentially causing harm to normal cells and resulting in side effects such as heightened photosensitivity. To address this, an activatable photosensitizer (PSD) by linking PpIX with a fluorescence quencher using a disulfide bond is designed. PSD responded to endogenous GSH, showing high selectivity for A375 cells. To enhance PSD's bioavailability and anticancer efficacy, an enzyme-responsive nanoplatform based on a lonidamine-derived self-assembling peptide is developed. Initially, PSD and the peptide self-assembled into nanoparticles, displaying potent tumor targeting of PSD in vivo. Upon cell uptake, these nanoparticles specifically responded to elevated cathepsin B, causing nanoparticle disintegration and releasing PSD and lonidamine prodrug (LND-1). PSD is selectively activated by GSH for cancer-specific fluorescence imaging and precision PDT, while LND-1 targeted mitochondria, forming a fibrous lonidamine depot in situ and intensifying photosensitizer's cytotoxicity through ROS generation, mitochondrial dysfunction, and DNA damage. Notably, intravenous administration of LND-1-PEG@PSD with light irradiation significantly suppressed A375-xenografted mouse tumor growth, with minimal systemic toxicity. Together, the synergy of activatable photosensitizer and enzyme-responsive nanoplatform elevates PDT precision and diminishes side effects, showcasing significant potential in the realm of cancer nanomedicine.

Identification of a Novel, Potent, and Orally Bioavailable Guanidine-Based SHP2 Allosteric Inhibitor from Virtual Screening and Rational Structural Optimization for the Treatment of KRAS Mutant Cancers.

Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) is a highly attractive therapeutic target for treating Kirsten rat sarcoma viral oncogene (KRAS) mutant cancers. In this work, a series of guanidine-based SHP2 allosteric inhibitors were discovered via virtual screening and rational structural optimization. Notably, lead compound 23 with potent SHP2 inhibitory activity (IC50 = 17.7 nM) effectively inhibited the proliferation, migration, and invasion of MIA PaCa-2 pancreatic cancer cells. Furthermore, compound 23 featured great in vivo pharmacokinetic properties (AUCpo = 4320 nM·h; F = 66.3%) and exhibited significant antitumor efficacy in the MIA PaCa-2 xenograft mouse model. This demonstrates that compound 23 is a potential lead compound for the development of SHP2 allosteric inhibitors to treat KRAS mutant cancers. Moreover, these guanidine-based scaffolds may provide an opportunity to mitigate the potential safety risks of the alkyl amine motif predominately incorporated in current SHP2 allosteric inhibitors.

Corrigendum to 'Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs)' [Genes Dis 8 (2021) 814-826].

[This corrects the article DOI: 10.1016/j.gendis.2021.01.004.].

Cascade-Activated AIEgen-Peptide Probe for Noninvasively Monitoring Chymotrypsin-like Activity of Proteasomes in Cancer Cells.

Noninvasive monitoring of chymotrypsin-like (ChT-L) activity of proteasomes is of great significance for the diagnosis and prognosis of various cancers. However, commercially available proteasome probes usually lack adequate cancer-cell selectivity. To noninvasively monitor ChT-L activity of proteasomes in living cells, we rationally designed a cascade-activated AIEgen-peptide probe (abbreviated as TPE-1p), which self-assembled in aqueous solution to exhibit bright fluorescence in response to sequential treatment of alkaline phosphatase (ALP) and ChT-L. Transmission electron microscopy, enzymatic kinetics, and in vitro fluorescence experiments validated that TPE-1p was efficiently dephosphorylated by ALP to generate TPE-1, which was recognized by ChT-L in the proteasome, and transformed to form nanofibers with strong fluorescence signals. Cell imaging experiments revealed that bright blue fluorescence was observed in TPE-1p-treated HeLa cells, whereas NIH3T3 and HepG2 cells showed less fluorescence at the same condition. The enhanced fluorescence signals in HeLa cells were attributed to the high activities of endogenous ALP and ChT-L. Moreover, TPE-1p was utilized to noninvasively assess the inhibition efficiency of a ChT-L inhibitor (bortezomib, abbreviated as Btz) in HeLa cells. Significant correlation was found between the fluorescence signals of TPE and the viabilities of Btz-treated cells in concentration ranges from 0 to 1 µM, indicating that TPE-1p could be employed to predict the activity of ChT-L inhibitors. The design of the cascade-activated AIEgen-peptide probe provides a viable approach for noninvasively monitoring the ChT-L activity of proteasomes in living cells, which facilitates high-throughput screening of ChT-L inhibitors in cancer therapy.

Development of Son of Sevenless Homologue 1 (SOS1) Modulators To Treat Cancers by Regulating RAS Signaling.

Son of sevenless homologue 1 (SOS1) protein is universally expressed in cells and plays an important role in the RAS signaling pathway. Specifically, this protein interacts with RAS in response to upstream stimuli to promote guanine nucleotide exchange in RAS and activates the downstream signaling pathways. Thus, targeting SOS1 is a new approach for treating RAS-driven cancers. In this Perspective, we briefly summarize the structural and functional aspects of SOS1 and focus on recent advances in the discovery of activators, inhibitors, and PROTACs that target SOS1. This review aims to provide a timely and updated overview on the strategies for targeting SOS1 in cancer therapy.

Design and Synthesis of Sulfonium Derivatives: A Novel Class of α-Glucosidase Inhibitors with Potent In Vivo Antihyperglycemic Activities.

We report the first attempt of double-spot structural modification on a side-chain moiety of sulfonium-type α-glucosidase inhibitors isolated from genus Salacia. A series of sulfonium salts with benzylidene acetal linkage at the C3' and C5' positions were designed and synthesized. In vitro enzyme inhibition evaluation showed that compounds with a strong electron-withdrawing group attached at the ortho position on the phenyl ring present stronger inhibitory activities. Notably, the most potent inhibitor 21b (1.0 mpk) can exhibit excellent hypoglycemic effects in mice, which can still compete with those of acarbose (20.0 mpk). Molecular docking of 21b demonstrated that besides conventional interacting patterns, the newly introduced benzylidene acetal moiety plays an important role in anchoring the whole molecule in a concave pocket of the enzyme. The successful identification of 21b as a lead compound for new drug discovery may provide a means for structure modification and diversification of the distinguished sulfonium-type α-glucosidase inhibitors.

High-Throughput Screening of Stapled Helical Peptides in Drug Discovery.

Therapeutic peptides have revolutionized treatment for a number of human diseases. In particular, the past two decades have witnessed rapid progress of stapled helical peptides in drug discovery. Stapled helical peptides are chemically modified and constrained in their bioactive α-helical conformation. Compared to unstabilized linear peptides, stapled helical peptides exhibit superior binding affinity and selectivity, enhanced membrane permeability, and improved metabolic stability, presenting exciting promise for targeting otherwise challenging protein-protein interfaces. In this Perspective, we summarize recent applications of high-throughput screening technologies for identification of potent stapled helical peptides with optimized binding properties. We expect to provide a broad reference to accelerate the development of stapled helical peptides as the next generation of therapeutic peptides for various human diseases.

Enzymatic Synthesis of Peptide Nanofibers for Self-Delivery of Indomethacin and Tyroservatide in Cancer Therapy.

Non-steroidal anti-inflammatory drugs (NSAIDs) have drawn considerable attention in the field of cancer treatment, yet these drugs display limited potency and selectivity against cancer cells. To address these problems, we designed a peptide-based self-delivery system [Indomethacin-Phe-Phe-Tyr (H2PO3)-Ser-Val, IDM-FFpYSV] that combines an NSAID molecule (indomethacin, or IDM) and a segment of anticancer tripeptide (tyroservatide, or YSV). IDM-FFpYSV is capable of self-assembling in an aqueous solution to afford nanofibrillar hydrogels under the catalysis of alkaline phosphatases (ALPs), which are overexpressed on the plasma membrane of cancer cells. The IDM-FFpYSV + ALP hydrogel displays a continuous release profile of peptide drugs, whereas a solution mixture of pure drugs (IDM-OH + pYSV + ALP) shows burst release of drug moieties. The treatment of IDM-FFpYSV selectively inhibits the proliferation of HeLa cells in vitro, with precise regulations of intracellular targeting proteins (COX-2 and AC-H3). The enhanced potency and selectivity of IDM-FFpYSV are found to be attributed to enhanced cellular uptake of peptide drugs, which involves a caveolae-mediated endocytosis pathway. Furthermore, intravenous administration of the IDM-FFpYSV formulation significantly inhibits the tumor growth in a HeLa-xenografted mouse model, whereas treatment of solution mixtures of pure drugs (IDM-OH + pYSV) fails to do so. Taken together, the study provides a viable strategy to augment anticancer efficacies of self-delivery system through molecular integration of multiple anticancer elements with an enzyme-instructed self-assembly process.

Discovery of a Novel Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2) and Cyclin-Dependent Kinase 4 (CDK4) Dual Inhibitor for the Treatment of Triple-Negative Breast Cancer.

The treatment of triple-negative breast cancer (TNBC) remains a huge clinical challenge and dual-targeted small-molecule drugs might provide new therapeutic options for this type of breast cancer. In this work, we discovered a series of SHP2 and CDK4 dual inhibitors through a fused pharmacophore strategy and structural optimization. Notably, lead compound 10 with excellent SHP2 (IC50 = 4.3 nM) and CDK4 (IC50 = 18.2 nM) inhibitory activities effectively induced G0/G1 arrest to prevent the proliferation of TNBC cell lines. Furthermore, compound 10 showed great in vivo pharmacokinetic properties (F = 45.8%) and exerted significant antitumor efficacy in the EMT6 syngeneic mouse model. Western blotting and immunohistochemical analysis confirmed that 10 effectively targeted on both SHP2 and CDK4 and activated the immune response in tumors. These results indicate that lead compound 10, as the first SHP2 and CDK4 dual inhibitor, merits further development for treating TNBC.

Small-molecule inhibitors targeting small ubiquitin-like modifier pathway for the treatment of cancers and other diseases.

SUMOylation is a key post-translational modification that involves the covalent attachment of small ubiquitin-like modifier (SUMO) to the lysine residues of target proteins. The well-balanced SUMOylation is essential for normal cellular behaviors, while disturbance of SUMOylation is associated with various cancers and other diseases. Herein, we summarize the structures and biological functions of proteins involved in the SUMOylation process, their dysregulation in human diseases, and the discovery of small-molecular inhibitors targeting this pathway. In addition, we highlight the emerging trends in this field.

Long short-term memory model - A deep learning approach for medical data with irregularity in cancer predication with tumor markers.

BACKGROUND: Machine learning (ML) has emerged as a superior method for the analysis of large datasets. Application of ML is often hindered by incompleteness of the data which is particularly evident when approaching disease screening data due to varied testing regimens across medical institutions. Here we explored the utility of multiple ML algorithms to predict cancer risk when trained using a large but incomplete real-world dataset of tumor marker (TM) values. METHODS: TM screening data were collected from a large asymptomatic cohort (n = 163,174) at two independent medical centers. The cohort included 785 individuals who were subsequently diagnosed with cancer. Data included levels of up to eight TMs, but for most subjects, only a subset of the biomarkers were tested. In some instances, TM values were available at multiple time points, but intervals between tests varied widely. The data were used to train and test various machine learning models to evaluate their robustness for predicting cancer risk. Multiple methods for data imputation were explored and models were developed for both single time-point as well as time-series data. RESULTS: The ML algorithm, long short-term memory (LSTM), demonstrated superiority over other models for dealing with irregular medical data. A cancer risk prediction tool was trained and validated for a single time-point test of a TM panel including up to four biomarkers (AUROC = 0.831, 95% CI: 0.827-0.835) which outperformed a single threshold method using the same biomarkers. A second model relying on time series data of up to four time-points for 5 TMs had an AUROC of 0.931. CONCLUSIONS: A cancer risk prediction tool was developed by training a LSTM model using a large but incomplete real-world dataset of TM values. The LSTM model was best able to handle irregular data compared to other ML models. The use of time-series TM data can further improve the predictive performance of LSTM models even when the intervals between tests vary widely. These risk prediction tools are useful to direct subjects to further screening sooner, resulting in earlier detection of occult tumors.

Design, synthesis, and evaluation of novel pyridone derivatives as potent BRD4 inhibitors for the potential treatment of prostate cancer.

Since androgen receptor (AR) can bind to BRD4 protein and this binding can be blocked by BRD4 inhibitors, targeting BRD4 has emerged as a promising approach for the treatment of prostate cancer (PC). Herein, we designed and synthesized a series of 5-(1-benzyl-1H-indazol-6-yl)-4-ethoxy-1-methylpyridin-2(1H)-one derivatives as novel BRD4 inhibitors for prostate cancer. Among them, compound 13 displayed the most robust BRD4 inhibitory activity with an IC50 value of 18 nM. Furthermore, 13 showed potent anti-proliferative activity against enzalutamide-resistant 22RV1 cells. The mechanism of action studies demonstrated that 13 induced cell apoptosis by regulating Bcl-2/Bax proteins and activating caspase-3 signaling pathway. In addition, the c-Myc level was significantly reduced in 22RV1 cells on the western blot assay. These findings collectively suggested that compound 13 might find potential use for the treatment of prostate cancer.

Recombinant human GLP-1 beinaglutide regulates lipid metabolism of adipose tissues in diet-induced obese mice.

GLP-1 analogs are a class of glucose-lowering agents with multiple benefits in diabetes, but its role in adipose tissues remains to be elucidated. The aim of this study was to determine the action of recombinant human GLP-1 (rhGLP-1) Beinaglutide (BN) in the insulin sensitivity and lipid metabolism of adipose tissues. We have shown that, after BN injection, obese mice displayed lower body weight, fat mass, and plasma lipid levels. In addition, BN promoted the insulin sensitivity in the white adipose tissues. Furthermore, we have found that the BN treatment caused significant changes in content and composition of different lipid classes, including glycerolipids, glycerophospholipids, and sphingolipids, as well as expression of genes in lipid metabolic pathways in the adipose tissues. Taken together, our data demonstrate that BN could resist HFD-induced obesity by targeting the composition of major lipid classes and the expression of genes in lipid metabolism of adipose tissues.

Radical-mediated vicinal addition of alkoxysulfonyl/fluorosulfonyl and trifluoromethyl groups to aryl alkyl alkynes.

The addition of sulfonyl radicals to alkenes and alkynes is a valuable method for constructing useful highly functionalized sulfonyl compounds. The underexplored alkoxy- and fluorosulfonyl radicals are easily accessed by CF3 radical addition to readily available allylsulfonic acid derivatives and then ß-fragmentation. These substituted sulfonyl radicals add to aryl alkyl alkynes to give vinyl radicals that are trapped by trifluoromethyl transfer to provide tetra-substituted alkenes bearing the privileged alkoxy- or fluorosulfonyl group on one carbon and a trifluoromethyl group on the other. This process exhibits broad functional group compatibility and allows for the late-stage functionalization of drug molecules, demonstrating its potential in drug discovery and chemical biology.

Modeling colorectal tumorigenesis using the organoids derived from conditionally immortalized mouse intestinal crypt cells (ciMICs).

Intestinal cancers are developed from intestinal epithelial stem cells (ISCs) in intestinal crypts through a multi-step process involved in genetic mutations of oncogenes and tumor suppressor genes. ISCs play a key role in maintaining the homeostasis of gut epithelium. In 2009, Sato et al established a three-dimensional culture system, which mimicked the niche microenvironment by employing the niche factors, and successfully grew crypt ISCs into organoids or Mini-guts in vitro. Since then, the intestinal organoid technology has been used to delineate cellular signaling in ISC biology. However, the cultured organoids consist of heterogeneous cell populations, and it was technically challenging to introduce genomic changes into three-dimensional organoids. Thus, there was a technical necessity to develop a two-dimensional ISC culture system for effective genomic manipulations. In this study, we established a conditionally immortalized mouse intestinal crypt (ciMIC) cell line by using a piggyBac transposon-based SV40 T antigen expression system. We showed that the ciMICs maintained long-term proliferative activity under two-dimensional niche factor-containing culture condition, retained the biological characteristics of intestinal epithelial stem cells, and could form intestinal organoids in three-dimensional culture. While in vivo cell implantation tests indicated that the ciMICs were non-tumorigenic, the ciMICs overexpressing oncogenic ß-catenin and/or KRAS exhibited high proliferative activity and developed intestinal adenoma-like pathological features in vivo. Collectively, these findings strongly suggested that the engineered ciMICs should be used as a valuable tool cell line to dissect the genetic and/or epigenetic underpinnings of intestinal tumorigenesis.

The combination of methylenehydrofolate reductase C677T polymorphism screening and gastrointestinal tumor markers detection may be an early screening method for gastrointestinal cancer related to helicobacter pylori infection.

Methyltetrahydrofolate reductase (MTHFR) is a key enzyme in folate metabolism, and its single nucleotide polymorphism (SNP) site C677T may be associated with gastrointestinal cancer. However, the relationship between MTHFR C677T polymorphism and gastrointestinal tumor markers carcinoma embryonic antigen (CEA), carbohydrate antigen 199 (CA199) and carbohydrate antigen 724 (CA724) in Helicobacter pylori (H. pylori) infection is not specified. This study aims to identify the association between MTHFR C677T polymorphism and gastrointestinal tumor markers (CEA, CA199 and CA724) in H. pylori infection. The relationship between MTHFR C677T polymorphism and gastrointestinal tumor markers in 58 patients with H. pylori infection and 94 non-infected patients was studied. We found that TT genotype was a susceptibility factor of H. pylori infection, which was also associated with increased CEA and CA724 levels. Moreover, there was a negative additive interaction between MTHFR gene C677T polymorphism and CEA levels in H.pylori infection. Meanwhile, there were significant differences in CEA levels between MTHFR C677T polymorphism and H.pylori infection. The presence of T allele led to a decrease in CEA levels when 13C urea breath test (13C-UBT) was positive, while the presence of T allele led to an increase in CEA levels when 13C-UBT was negative. Therefore, we suggest that healthy people should take MTHFR C677T polymorphism screening, combined with 13C-UBT and gastrointestinal tumor markers detection, which can screen out the susceptible population of H. pylori, and help to detect gastrointestinal cancer in the early stage.

Development of a simplified and inexpensive RNA depletion method for plasmid DNA purification using size selection magnetic beads (SSMBs).

Plasmid DNA (pDNA) isolation from bacterial cells is one of the most common and critical steps in molecular cloning and biomedical research. Almost all pDNA purification involves disruption of bacteria, removal of membrane lipids, proteins and genomic DNA, purification of pDNA from bulk lysate, and concentration of pDNA for downstream applications. While many liquid-phase and solid-phase pDNA purification methods are used, the final pDNA preparations are usually contaminated with varied degrees of host RNA, which cannot be completely digested by RNase A. To develop a simple, cost-effective, and yet effective method for RNA depletion, we investigated whether commercially available size selection magnetic beads (SSMBs), such as Mag-Bind® TotalPure NGS Kit (or Mag-Bind), can completely deplete bacterial RNA in pDNA preparations. In this proof-of-principle study, we demonstrated that, compared with RNase A digestion and two commercial plasmid affinity purification kits, the SSMB method was highly efficient in depleting contaminating RNA from pDNA minipreps. Gene transfection and bacterial colony formation assays revealed that pDNA purified from SSMB method had superior quality and integrity to pDNA samples cleaned up by RNase A digestion and/or commercial plasmid purification kits. We further demonstrated that the SSMB method completely depleted contaminating RNA in large-scale pDNA samples. Furthermore, the Mag-bind-based SSMB method costs only 5-10% of most commercial plasmid purification kits on a per sample basis. Thus, the reported SSMB method can be a valuable and inexpensive tool for the removal of bacterial RNA for routine pDNA preparations.

Notch signaling: Its essential roles in bone and craniofacial development.

Notch is a cell-cell signaling pathway that is involved in a host of activities including development, oncogenesis, skeletal homeostasis, and much more. More specifically, recent research has demonstrated the importance of Notch signaling in osteogenic differentiation, bone healing, and in the development of the skeleton. The craniofacial skeleton is complex and understanding its development has remained an important focus in biology. In this review we briefly summarize what recent research has revealed about Notch signaling and the current understanding of how the skeleton, skull, and face develop. We then discuss the crucial role that Notch plays in both craniofacial development and the skeletal system, and what importance it may play in the future.

FAMSi: A Synthetic Biology Approach to the Fast Assembly of Multiplex siRNAs for Silencing Gene Expression in Mammalian Cells.

RNA interference (RNAi) is mediated by an ∼21-nt double-stranded small interfering RNA (siRNA) and shows great promise in delineating gene functions and in developing therapeutics for human diseases. However, effective gene silencing usually requires the delivery of multiple siRNAs for a given gene, which is often technically challenging and time-consuming. In this study, by exploiting the type IIS restriction endonuclease-based synthetic biology methodology, we developed the fast assembly of multiplex siRNAs (FAMSi) system. In our proof-of-concept experiments, we demonstrated that multiple fragments containing three, four, or five siRNA sites targeting common Smad4 and/or BMPR-specific Smad1, Smad5, and Smad8 required for BMP9 signaling could be assembled efficiently. The constructed multiplex siRNAs effectively knocked down the expression of Smad4 and/or Smad1, Smad5, and Smad8 in mesenchymal stem cells (MSCs), and they inhibited all aspects of BMP9-induced osteogenic differentiation in bone marrow MSCs (BMSCs), including decreased expression of osteogenic regulators/markers, reduced osteogenic marker alkaline phosphatase (ALP) activity, and diminished in vitro matrix mineralization and in vivo ectopic bone formation. Collectively, we demonstrate that the engineered FAMSi system provides a fast-track platform for assembling multiplexed siRNAs in a single vector, and thus it may be a valuable tool to study gene functions or to develop novel siRNA-based therapeutics.