Rapid Formation of Intramolecular Disulfide Bridges using Light: An Efficient Method to Control the Conformation and Function of Bioactive Peptides.

Disulfide bonds are widely found in natural peptides and play a pivotal role in stabilizing their secondary structures, which are highly associated with their biological functions. Herein, we introduce a light-mediated strategy to effectively control the formation of disulfides. Our strategy is based on 2-nitroveratryl (oNv), a widely used photolabile motif, which serves both as a photocaging group and an oxidant (after photolysis). We demonstrated that irradiation of oNv-caged thiols with UV light could release free thiols that are rapidly oxidized by locally released byproduct nitrosoarene, leading to a "break-to-bond" fashion. This strategy is highlighted by the in situ restoration of the antimicrobial peptide tachyplesin I (TPI) from its external disulfide-caged analogue TPI-1. TPI-1 exhibits a distorted structure and a diminished function. However, upon irradiation, the ß-hairpin structure and membrane activity of TPI were largely restored via rapid intramolecular disulfide formation. Our study proposes a powerful method to regulate the conformation and function of peptides in a spatiotemporal manner, which has significant potential for the design of disulfide-centered light-responsive systems.

Pentacyclic triterpene-amino acid derivatives induced apoptosis and autophagy in tumor cells, affected the JNK and PI3K/AKT/mTOR pathway.

A series of pentacyclic triterpene-amino acid derivatives were synthesized and tested for anti-proliferative activity. The results showed that most of the target compounds had good anti-proliferative activity. 2c did not contain protecting groups and hydrochloride, had excellent cytotoxicity, so it had been selected for further study in the mechanism of action in T24 cells. The data from transcriptome sequencing indicated that 2c was found to be closely related to apoptosis and autophagy. Observation of fluorescence staining and analysis from flow cytometry demonstrated that 2c induced apoptosis and cause cell cycle arrest in S/G2 phase in T24 cells. Molecular mechanism studies exhibited that 2c induced apoptosis in the intrinsic and extrinsic pathways. 2c also induced cellular autophagy in T24 cells. Results from Western Blotting showed that 2c could activate JNK pathway and inhibit PI3K/AKT/mTOR pathway. In conclusion, 2c was deserved further investigation in the field of anti-tumor.

Synthesis and in vitro/in vivo anticancer evaluation of pentacyclic triterpenoid derivatives linked with l-phenylalanine or l-proline.

Extensive research effort has been put in pentacyclic triterpenoids due to their numerous biological activities. However, their poor water solubility and low oral bioavailability limit their antitumor effects in vivo. To address these issues, 37 triterpenoid acid derivatives linked to l-phenylalanine or l-proline were designed and synthesized in this study. Structure-activity relationship (SAR) studies found two promising glycyrrhetinic acid (GA) derivatives 11 and 16. Compound 11 was obtained by C3-OH esterification and C30-COOH modification with l-phenylalanine while 16 was obtained by attaching C3-OH with l-phenylalanine. Compounds 11 and 16 exhibit up to 48- and 120-fold improvement respectively compared with the IC50 values of naturally occurring GA in the cellular assay. Fluorescence microscope and flow cytometric analysis suggested that both compounds 11 and 16 increased the content of ROS and Ca2+ in cancer cells, decreased mitochondrial membrane potential (JC-1), and activated the regulator caspase-3/8/9 to trigger cell apoptosis. RNA-seq analysis and western blot analysis indicated that compounds 11 and 16 may promote apoptosis by upregulating the functions of pro-apoptotic factors while inhibiting the proteasome activity.

Discovery of the First Selective Nanomolar Inhibitors of ERAP2 by Kinetic Target-Guided Synthesis.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a key enzyme involved in the trimming of antigenic peptides presented by Major Histocompatibility Complex class I. It is a target of growing interest for the treatment of autoimmune diseases and in cancer immunotherapy. However, the discovery of potent and selective ERAP2 inhibitors is highly challenging. Herein, we have used kinetic target-guided synthesis (KTGS) to identify such inhibitors. Co-crystallization experiments revealed the binding mode of three different inhibitors with increasing potency and selectivity over related enzymes. Selected analogues engage ERAP2 in cells and inhibit antigen presentation in a cellular context. 4 d (BDM88951) displays favorable in vitro ADME properties and in vivo exposure. In summary, KTGS allowed the discovery of the first nanomolar and selective highly promising ERAP2 inhibitors that pave the way of the exploration of the biological roles of this enzyme and provide lead compounds for drug discovery efforts.

Discovery of Novel Small Molecule Inhibitors Disrupting the PCSK9-LDLR Interaction.

Proprotein convertase subtilisin kexin 9 (PCSK9) has been identified as a reliable therapeutic target for hypercholesterolemia and coronary artery heart diseases since the monoclonal antibodies of PCSK9 have launched. Disrupting the protein-protein interaction (PPI) between PCSK9 and the low-density lipoprotein receptor (LDLR) has been considered as a promising approach for developing PCSK9 inhibitors. However, PPIs have been traditionally considered difficult to target by small molecules since the PPI surface is usually large, flat, featureless, and without a "pocket" or "groove" for ligand binding. The PCSK9-LDLR PPI interface is such a typical case. In this study, a potential binding pocket was generated on the PCSK9-LDLR PPI surface of PCSK9 through induced-fit docking. On the basis of this induced binding pocket, virtual screening, molecular dynamics (MD) simulation, and biological evaluations have been applied for the identification of novel small molecule inhibitors of PCSK9-LDLR PPI. Among the selected compounds, compound 13 exhibited certain PCSK9-LDLR PPI inhibitory activity (IC50: 7.57 ± 1.40 µM). The direct binding affinity between 13 and PCSK9 was determined with a KD value of 2.50 ± 0.73 µM. The LDLR uptake function could be also restored to a certain extent by 13 in HepG2 cells. This well-characterized hit compound will facilitate the further development of novel small molecule inhibitors of PCSK9-LDLR PPI.

Design, synthesis and apoptosis-related antiproliferative activities of chelidonine derivatives.

To get chelidonine derivatives with enhanced antiproliferative activity and selectivity, a series of nitric oxide donating derivatives (10a-f and 11a-j) were designed, synthesized and biologically evaluated. Compared with chelidonine, these compounds exhibited lower IC50 values against human hepatoma cells HepG2, breast cancer cells MCF-7, colon cancer cells HCT-116, as well as leukemia cells K562. Compound 11j displayed the strongest antiproliferative activity with IC50 values of 3.91, 6.90, 4.36 and 1.12 µM against the above four cells, respectively. Nevertheless, it showed an IC50 value >40 µM against human peripheral blood mononuclear cells (PBMCs), which demonstrated high selectivity between normal and cancer blood cells. In further mechanism studies, 11j showed the capability to induce K562 cells apoptosis, S phase cell cycle arrest and mitochondrial membrane potential disorder. Besides, 11j was found to be effective in promoting the expression of proapoptotic protein Bad and suppressing the expression of anti-apoptotic proteins Bcl-xL, catalase, survivin, claspin and clusterin.

Discovery of Novel Peroxisome Proliferator-Activated Receptor α (PPARα) Agonists by Virtual Screening and Biological Evaluation.

Nonalcoholic steatohepatitis (NASH) is one of the important causes of cirrhosis and hepatocellular carcinoma worldwide. PPARα is highly expressed in the liver and plays a critical role in hepatic lipid metabolism. Our analysis of the gene expression profiles in the liver of humanized mice treated with a PPARα agonist and NASH patients suggested that PPARα might be a potential target for NASH therapy. This promoted us to find novel PPARα agonists. The results of virtual screening and biological evaluation identified compound A-4 as a selective PPARα agonist. It significantly regulated the target genes of PPARα involved in fatty acid metabolism and inflammation, exhibiting cellular anti-inflammatory activity. The key residues involved in the binding between PPARα ligand-binding domain (LBD) and compound A-4 were revealed by molecular dynamics (MD) simulation and further experimentally validated by the mutation study. Together, compound A-4 was well characterized as a novel lead compound for developing potent and selective PPARα agonists.

Discovery of Ubiquitin-Specific Protease 7 (USP7) Inhibitors with Novel Scaffold Structures by Virtual Screening, Molecular Dynamics Simulation, and Biological Evaluation.

USP7 has been regarded as a potential therapeutic target for cancer. In this study, virtual screening, molecular dynamics (MD) simulation, and biological evaluation have been applied for the discovery of novel USP7 inhibitors targeting the catalytic active site. Among the obtained compounds, compound 12 with a novel scaffold structure exhibited certain USP7 inhibitory activity (Ub-AMC assay IC50 = 18.40 ± 1.75 µM, Ub-Rho assay IC50 = 7.75 µM). The binding affinity between USP7CD (USP7 catalytic domain) and this hit compound was confirmed with a KD value of 4.46 ± 0.86 µM. Preliminary in vitro studies disclosed its antiproliferative activity on human prostate cancer cell line LNCaP with an IC50 value of 15.43 ± 3.49 µM. MD simulation revealed the detailed differences of protein-ligand interactions between USP7CD and the ligands, including the reference compound ALM4 and compound 12, providing some important information for improving the bioactivity of 12. This hit compound will serve as a promising starting point for facilitating the further discovery of novel USP7 inhibitors.

Discovery of novel quinazolines as potential anti-tubulin agents occupying three zones of colchicine domain.

A series of novel quinazolines as tubulin inhibitors occupying three zones of colchicine domain have been designed and synthesized inspired by the recently disclosed crystal structure of verubulin analogue 6 with tubulin. Among the newly synthesized compounds, 19c showed noteworthy potency against K562, HepG2, KB, HCT-8 and MDB-MB-231 cancer cells. In vitro microtubule polymerization assays identified 19c as a potent tubulin assembly inhibitor, the binding mode of which with tubulin was confirmed by molecular modeling studies to occupy three zones of tubulin domain. Furthermore, 19c disrupted the intracellular microtubule network, caused G2/M phase arrest, induced cell apoptosis and depolarized mitochondria of K562 cells. 19c also reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 19c significantly and dose dependently inhibited tumor growth in H22 liver cancer xenograft mouse model. All these results suggested that 19c deserves further research as a novel and potential anti-tubulin agent for the treatment of cancers.

Estimation of elimination half-lives of organic chemicals in humans using gradient boosting machine.

BACKGROUND: Elimination half-life is an important pharmacokinetic parameter that determines exposure duration to approach steady state of drugs and regulates drug administration. The experimental evaluation of half-life is time-consuming and costly. Thus, it is attractive to build an accurate prediction model for half-life. METHODS: In this study, several machine learning methods, including gradient boosting machine (GBM), support vector regressions (RBF-SVR and Linear-SVR), local lazy regression (LLR), SA, SR, and GP, were employed to build high-quality prediction models. Two strategies of building consensus models were explored to improve the accuracy of prediction. Moreover, the applicability domains (ADs) of the models were determined by using the distance-based threshold. RESULTS: Among seven individual models, GBM showed the best performance (R(2)=0.820 and RMSE=0.555 for the test set), and Linear-SVR produced the inferior prediction accuracy (R(2)=0.738 and RMSE=0.672). The use of distance-based ADs effectively determined the scope of QSAR models. However, the consensus models by combing the individual models could not improve the prediction performance. Some essential descriptors relevant to half-life were identified and analyzed. CONCLUSIONS: An accurate prediction model for elimination half-life was built by GBM, which was superior to the reference model (R(2)=0.723 and RMSE=0.698). GENERAL SIGNIFICANCE: Encouraged by the promising results, we expect that the GBM model for elimination half-life would have potential applications for the early pharmacokinetic evaluations, and provide guidance for designing drug candidates with favorable in vivo exposure profile. This article is part of a Special Issue entitled "System Genetics" Guest Editor: Dr. Yudong Cai and Dr. Tao Huang.

Antiproliferative activity and apoptosis inducing effects of nitric oxide donating derivatives of evodiamine.

The first series of nitric oxide donating derivatives of evodiamine were designed and prepared. NO releasing ability of all target derivatives was evaluated in BGC-823, Bel-7402 and L-02 cells. The cytotoxicity was evaluated against three human tumor cell lines (Bel-7402, A549 and BGC-823) and normal human liver cells L-02. The nitrate derivatives 11a and 11b only exhibited moderate activity and furoxan-based derivatives 13a-c, 14a and 14b showed promising activity. 13c showed good cytotoxic selectivity between tumor and normal liver cells and was further investigated for its apoptotic properties on human hepatocarcinoma Bel-7402 cells. The molecular mode of action revealed that 13c caused cell-cycle arrest at S phase and induced apoptosis in Bel-7402 cells through mitochondria-related caspase-dependent pathways.

Synthesis, Biological Activity, and Apoptotic Properties of NO-Donor/Enmein-Type ent-Kauranoid Hybrids.

Herein, we reported on a series of synthetic nitric oxide-releasing enmein-type diterpenoid hybrids (9a-i). All the target compounds showed potent antibacterial activity against selected Gram-positive bacteria S. aureus and B. subtilis. The antiproliferative activity against human tumor K562, MGC-803, CaEs-17 and Bel-7402 cells, and human normal liver cells L-02 was tested and the structure activity relationships (SARs) were also concluded. Compounds 9b and 9d showed the best activity against S. aureus and B. subtilis with the same minimal inhibitory concentrations (MICs) of 4 and 2 µg/mL, respectively. The derivative 9f displayed IC50 values of 1.68, 1.11, 3.60 and 0.72 µM against the four cancer cell lines above and 18.80 µM against normal liver cells L-02; meanwhile, 9f also released a high level of NO at the time point of 60 min of 22.24 µmol/L. Furthermore, it was also found that 9f induced apoptosis via the mitochondria-related pathway and arrested cell cycle of Bel-7402 cells at S phase. These findings might be important to explore new chemical entities for the main causes of in-hospital mortality of S. aureus infection, combined with a solid tumor.

Oridonin, a Promising ent-Kaurane Diterpenoid Lead Compound.

Oridonin belongs to ent-kaurane tetracyclic diterpenoid and was first isolated from Isodon species. It exhibits inhibitory activities against a variety of tumor cells, and pharmacological study shows that oridonin could inhibit cell proliferation, DNA, RNA and protein synthesis of cancer cells, induce apoptosis and exhibit an antimutagenic effect. In addition, the large amount of the commercially-available supply is also very important for the natural lead oridonin. Moreover, the good stability, suitable molecular weight and drug-like property guarantee its further generation of a natural-like compound library. Oridonin has become the hot molecule in recent years, and from the year 2010, more than 200 publications can be found. In this review, we summarize the synthetic medicinal chemistry work of oridonin from the first publication 40 years ago and share our research experience of oridonin for about 10 years, which may provide useful information to those who are interested in this research field.

Antitumor and Antibacterial Derivatives of Oridonin: A Main Composition of Dong-Ling-Cao.

Isodon rubescens has been used as a traditional green tea for more than 1000 years and many medicinal functions of I. rubescens are also very useful, such as its well-known antitumor and antibacterial activities. Oridonin, a bioactive ent-kaurane diterpenoid, is the major ingredient of this medicinal tea. Herein, 22 novel oridonin derivatives were designed and synthesized. The antibacterial activity was evaluated for the first time. Compound 12 was the most promising one with MIC of 2.0 µg/mL against B. subtilis, which was nearly 3-fold stronger than positive control chloromycetin. The antiproliferative property was also assayed and compound 19 showed stronger activity than taxol. The apoptosis-inducing ability, cell cycle arrest effect at S phase and influence of mitochondrial membrane potential by 19 in CaEs-17 cancer cells were first disclosed. Based on the above results, the cell apoptosis induced by compound 19 in CaEs-17 cells was most probably involved in the intrinsic apoptotic pathway.

Radical Acylfluoroalkylation of 1,3-Enynes via N-Heterocyclic Carbene/Photoredox Cooperative Catalysis.

We report a novel three-component radical acylfluoroalkylation of 1,3-enynes by synergistic N-heterocyclic carbene (NHC)/photoredox catalysis toward various fluorinated allenic aryl ketones. This protocol features a broad substrate scope and excellent functional group tolerability, with examples of late-stage modification of drug molecules and natural products. Notably, seven different fluoroalkyl motifs can be introduced to 1,3-enynes, further demonstrating the robustness and generality of this method. The generation of the fluoroalkyl radical from each sulfinate reagent was individually supported by EPR experiments.

Research progress on GPX4 targeted compounds.

Blocking the System Xc-_ GSH_GPX4 pathway to induce ferroptosis in tumor cells is a novel strategy for cancer treatment. GPX4 serves as the core of the System Xc-/GSH/GPX4 pathway and is a predominant target for inducing ferroptosis in tumor cells. This article summarizes compounds identified in current research that directly target the GPX4 protein, including inhibitors, activators, small molecule degraders, chimeric degraders, and the application of combination therapies with other drugs, aiming to promote further research on the target and related diseases.

6-O-angeloylplenolin inhibits osteoclastogenesis in vitro via suppressing c-Src/NF-κB/NFATc1 pathways and ameliorates bone resorption in collagen-induced arthritis mouse model.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

N-Heterocyclic Carbene and Manganese Synergistic Catalysis: A Three-Component Radical Acylmonofluoroalkylation of Alkenes.

Despite the importance of monofluoroalkyl groups in pharmaceutically relevant molecules, catalytic protocols for their incorporation into alkenes remain limited. We describe herein a three-component acylmonofluoroalkylation of alkenes for the introduction of such moieties through an unprecedented cooperativity between the N-heterocyclic carbene catalyst and earth-abundant Mn(II) complex. This general method can be applied to a variety of alkenes, including styrenes, 1,3-enynes, and allenes, as well as complex substrates containing natural product and drug motifs.

(4-Picolylamino)-17ß-Estradiol derivative and analogues induce apoptosis with death receptor trail R2/DR5 in MCF-7.

In order to discover more effective and less toxic drugs in the field of anti-tumor, the backbone structure of 17ß-estradiol was modified, and 11 target compounds were synthesized. Compounds 5 and 10, which exhibited better anti-tumor activity and higher selectivity (more than 10-fold), were chosen for further biological investigation. Flow cytometry results indicated that 5 and 10 could arrest MCF-7 cells in the G2 phase and induce apoptosis. Immunohistochemical analysis revealed that 5 and 10 could bind to the estradiol receptor alpha in MCF-7 cells. Western blotting and real-time PCR assays were performed to detect the effects of compounds on apoptosis-related targets at the protein and gene levels. These results showed that both 5 and 10 could dosed-dependently increase the expression of Apaf-1, Bax, caspase-3,8,9 and reduce the expression levels of the anti-apoptotic factors Bcl-2 and Bcl-xL. Besides, the Human apoptosis array assay demonstrated the expression level of death receptor Trail R2/DR5 was upregulated obviously while the expression of TNF R1, IAPs and Hsp27/60/70 were downregulated. On the whole, 5 induced MCF-7 cell death through the endogenous pathway in mitochondria and the exogenous pathway with death receptor Trail R2/DR5.

Pyrazolone derivatives as potent and selective small-molecule SIRT5 inhibitors.

Sirtiun 5 (SIRT5) is a NAD+-dependent protein lysine deacylase. It is emerging as a promising target for the development of drugs to treat cancer and metabolism-related diseases. In this study, we screened 5000 compounds and identified a hit compound 14 bearing a pyrazolone functional group as a novel SIRT5-selective inhibitor. Structure-based optimization of 14 resulted in compound 47 with an IC50 value of 0.21 ± 0.02 µM and a 100-fold improved potency. Compound 47 showed substantial selectivity for SIRT5 over SIRT1-3 and SIRT6. Biochemical studies suggest that 47 does not occupy the NAD + -binding pocket and acts as a substrate-competitive inhibitor. The identified potent and selective SIRT5 inhibitors allow further studies as research tools and therapeutic agents.