Design, synthesis and anticancer activity evaluation of 4-(3-1H-indazolyl)amino quinazoline derivatives as PAK4 inhibitors.

A novel series of 4-(3-1H-indazolyl)amino quinazoline derivatives were developed as PAK4 inhibitors based on a scaffold hopping strategy. Compounds 27e, 27g, 27i and 27j were found to exhibit potent inhibitory activity against PAK4 (IC50 = 10, 13, 11 and 9 nM, respectively). Subsequent cellular assay demonstrated that compound 27e possessed the strongest antiproliferative activity against A549 cells with an IC50 value of 0.61 µM, a little bit better than PF-3758309. Further anticancer mechanistic investigation revealed that compound 27e significantly induced apoptosis of A549 cells in a concentration-dependent manner and blocked the cell cycle at phase G0/G1. A docking model between compound 27e and PAK4 was proposed to elucidate its possible binding modes. As a promising PAK4 inhibitor, compound 27e may serve as a candidate for the development of novel PAK4-targeted anticancer drug.

A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility.

Bacterial double-stranded DNA (dsDNA) cytosine deaminase DddAtox-derived cytosine base editor (DdCBE) and its evolved variant, DddA11, guided by transcription-activator-like effector (TALE) proteins, enable mitochondrial DNA (mtDNA) editing at TC or HC (H = A, C, or T) sequence contexts, while it remains relatively unattainable for GC targets. Here, we identified a dsDNA deaminase originated from a Roseburia intestinalis interbacterial toxin (riDddAtox) and generated CRISPR-mediated nuclear DdCBEs (crDdCBEs) and mitochondrial CBEs (mitoCBEs) using split riDddAtox, which catalyzed C-to-T editing at both HC and GC targets in nuclear and mitochondrial genes. Moreover, transactivator (VP64, P65, or Rta) fusion to the tail of DddAtox- or riDddAtox-mediated crDdCBEs and mitoCBEs substantially improved nuclear and mtDNA editing efficiencies by up to 3.5- and 1.7-fold, respectively. We also used riDddAtox-based and Rta-assisted mitoCBE to efficiently stimulate disease-associated mtDNA mutations in cultured cells and in mouse embryos with conversion frequencies of up to 58% at non-TC targets.

Cas9-orthologue-mediated cytosine and adenine base editors recognizing NNAAAA PAM sequences.

CRISPR/Cas9 system has been applied as an effective genome-targeting technology. By fusing deaminases with Cas9 nickase (nCas9), various cytosine and adenine base editors (CBEs and ABEs) have been successfully developed that can efficiently induce nucleotide conversions and install pathogenic single nucleotide variants (SNVs) in cultured cells and animal models. However, the applications of BEs are frequently limited by the specific protospacer adjacent motif (PAM) sequences and protein sizes. To expand the toolbox for BEs that can recognize novel PAM sequences, we cloned a Cas9 ortholog from Streptococcus sinensis (named as SsiCas9) with a smaller size and constructed it into APOBEC1- or APOBEC3A-composed CBEs and TadA or TadA*-composed ABEs, which yield high editing efficiencies, low off-targeting activities, and low indel rates in human cells. Compared to PAMless SpRY Cas9-composed BE4max, SsiCas9-mediated BE4max displayed higher editing efficiencies for targets with "NNAAAA" PAM sequences. Moreover, SsiCas9-mediated BE4max induced highly efficient C-to-T conversions in the mouse Ar gene (R841C) to introduce a human androgen resistance syndrome-related mutation (AR R820C) in early mouse embryos. Thus, we developed novel BEs mediated by SsiCas9, expanded the toolbox for base conversions, and broadened the range of editable genomes in vitro and in vivo.

Dual guide RNA-mediated concurrent C&G-to-T&A and A&T-to-G&C conversions using CRISPR base editors.

Base editing tools enable precise genome modifications, disease modeling, and promising gene therapy. However, many human genetic diseases are elicited by multi-nucleotide variants (MNVs) with heterogeneous substitutions at the same genomic locus. Based on the adenine and cytosine base editors, dual base editors that can catalyze concurrent C-to-T and A-to-G editing have been developed, while simultaneous C&G-to-T&A and A&T-to-G&C conversions on the same allele have not been achieved at the desirable site. Here we propose a strategy of combining base editors with dual guide RNAs (gRNAs) that target two overlapped neighboring loci on the opposite strands, which can induce simultaneous C&G-to-T&A and A&T-to-G&C conversions within their overlapping targeting windows. Moreover, one of the paired gRNAs is mutated to perfectly match another gRNA-edited sequence, efficiently facilitating concurrent base conversions on the same allele. To further expand the targeting scopes, PAMless SpRY Cas9-mediated base editors are combined with our optimized dual gRNAs system to induce expected concurrent base editing and to install neighboring pathogenic MNVs in TP53 in cancer cells. In addition, more complex mutation types can be achieved by integrating dual base editors and our dual gRNAs strategy. Thus, we establish a general strategy to efficiently induce MNVs in human genome, helping to dissect the functions of pathogenic MNVs with multifarious types.

Synthesis and anticancer activity evaluation of 7-oxygen-substituted and 8,17-epoxydized 1,2-didehydro-3-ox-14-deoxyandrographolide derivatives.

Two novel series of 1,2-didehydro-7-hydroxy-3-ox-14-deoxyandrographolide and 1,2-didehydro-8,17-epoxy-3-ox-14-deoxyandrographolide derivatives were designed, synthesized and evaluated for their cytotoxic activity in vitro against two human cancer cell lines HCT-116 (human colon cancer) and MCF-7 (human breast cancer). Most tested compounds, especially those of the first series, displayed better inhibitory activity on both HCT-116 and MCF-7 cells than andrographolide. HCT-116 cells were found to be more sensitive to tested compounds than MCF-7, and compound 13b exhibited the most potent activity against HCT-116, with an IC50 value of 7.32 µM. Further anti-cancer mechanistic investigation demonstrated that compound 13b effectively suppressed the growth of HCT-116 cells by triggering early and late cellular apoptosis in a concentration-dependent manner and arresting cell cycle in S phase.

Two Compact Cas9 Ortholog-Based Cytosine Base Editors Expand the DNA Targeting Scope and Applications In Vitro and In Vivo.

CRISPR/Cas9-based base editing tools enable precise genomic installation and hold great promise for gene therapy, whereas the big size of Cas9 nucleases and its reliability on specific protospacer adjacent motif (PAM) sequences as well as target site preferences restrict the extensive applications of base editing tools. Here, we generate two cytosine base editors (CBEs) by fusing cytidine deaminases with two compact codon-optimized Cas9 orthologs from Streptococcus_gordonii_str._Challis_substr._CH1 (ancSgo-BE4) and Streptococcus_thermophilus_LMG_18311 (ancSth1a-BE4), which are much smaller than Streptococcus pyogenes (SpCas9) and recognize NNAAAG and NHGYRAA PAM sequences, respectively. Both CBEs display high activity, high fidelity, a different editing window, and low by-products for cytosine base editing with minimal DNA and RNA off-targeting activities in mammalian cells. Moreover, both editors show comparable or higher editing efficiencies than two engineered SpCas9 variant (SpCas9-NG and SpRY)-based CBEs in our tested target sites, which perfectly match the PAM sequences for ancSgo-BE4 or ancSth1a-BE4. In addition, we successfully generate two mouse models harboring clinically relevant mutations at the Ar gene via ancSgo-BE4 and ancSth1a-BE4, which display androgen insensitivity syndrome and/or developmental lethality in founder mice. Thus, the two novel CBEs broaden the base editing tool kits with expanded targeting scope and window for efficient gene modification and applications, respectively.

[Effect of Mobile Health for Standardized Management on Women with Gestational Diabetes Mellitus].

Objective To explore the performance of mobile health platform for standardized management of pregnant women with gestational diabetes mellitus(GDM). Methods A randomized controlled trial was conducted,in which 295 women with GDM were randomized into two groups(traditional management group and mobile health management group)by a computer-generated sequence.The traditional management group accepted standardized GDM management,and the mobile health management group was supplemented by mobile health management based on the standardized management.The glycemic control rate and the incidences of low birth weight,macrosomia,preterm birth,premature rupture of membranes,postpartum hemorrhage after cesarean section,neonatal asphyxia,malformation,and admission to the neonatal intensive care unit were compared between the two groups. Results The glycemic control rate in mobile health management group was significantly higher than that in the traditional management group [(67.22±22.76)% vs.(60.69±21.28)%,P=0.004].The incidences of low birth weight,macrosomia,preterm birth,premature rupture of membranes,postpartum hemorrhage after cesarean section,neonatal asphyxia,malformation,and admission to the neonatal intensive care unit demonstrated no significant differences between groups(all P > 0.05). Conclusions Mobile health applied in standardized management is conducive to the glycemic control of GDM women,whereas it does not significantly improve the pregnancy outcomes.Due to the short time of intervention,the effects of mobile health on pregnancy outcomes need further study.

Design, synthesis, and anticancer evaluation of novel andrographolide derivatives bearing an α,ß-unsaturated ketone moiety.

A series of 1,2-didehydro-3-ox-andrographolide derivatives based on two Michael acceptors were designed, synthesized and evaluated for their anticancer activity against two human cancer cell lines (HCT116 and MCF-7). All tested compounds exhibited significant growth inhibitory effect on HCT116 and moderate to good inhibitory effect on MCF-7 cell proliferation. Compound 10b displayed the best inhibitory activities against both HCT116 and MCF-7 cell lines, with IC50 values of 2.49 and 7.80 µM respectively. Preliminary anticancer mechanistic investigation was performed in terms of the cell cycle arrest and cell apoptosis assays of compound 10b against HCT116 using flow cytometry, and the results indicated that 10b blocked the proliferation of HCT116 cells by inducing cell apoptosis in a concentration-dependent manner and arresting cell cycle in G2/M phase.

Effect of mobile health based peripartum management of gestational diabetes mellitus on postpartum diabetes: A randomized controlled trial.

AIMS: To investigate the effects of mobile health based peripartum management of gestational diabetes mellitus (GDM) on postpartum diabetes and factors associated with postpartum diabetes. METHODS: Women with GDM (n = 309) were randomly assigned to receive standard management (SM) or mobile management (MM). 75-g OGTT was performed at 6 weeks postpartum. RESULTS: The incidence of postpartum T2DM in the MM group was much higher than that in SM group (12.36% vs. 3.88%, P =  0.0291). The fasting, 1-h and 2 h OGTT at 24-28 weeks of gestation of T2DM women were higher than those women without T2DM (fasting, 6.08 vs. 4.90, P = 0.0052; 1-h, 13.20 vs. 10.00, P < 0.0001; 11.96 vs. 8.83, P = 0.0026) in MM group. The 1-h and 2 h OGTT at 24-28 weeks of gestation of T2DM women were higher than those women without T2DM (11.54 vs. 9.78, P = 0.0484; 10.68 vs. 8.68, P = 0.0108) in SM group. Higher OGTT values at 24-28 weeks of gestation were risk factors of postpartum T2DM. CONCLUSIONS: Higher OGTT values at 24-28 weeks of gestation were risk factors to develop postpartum T2DM. Mobile health based peripartum management of GDM increased the risk of postpartum diabetes among women with GDM for lacking of postpartum management. Further studies of mobile health based postpartum management of GDM are needed. ClinicalTrials.gov registration number NCT03748576.

Glycemic qualification rate and frequency of self-monitoring blood glucose glycemic qualification rate and frequency of self-monitoring blood glucose (SMBG) in women with gestational diabetes mellitus (GDM).

AIMS: To explore the relationship between blood glucose (BG) control rate and self-monitoring blood glucose (SMBG) compliance of women with gestational diabetes mellitus (GDM). METHODS: Women with GDM (n = 309) were randomized to receive routine clinical prenatal care or additional online management. Follow-up visits were conducted every two weeks (noted here as T) from enrollment to delivery. SMBG records were used for the analysis. RESULTS: Both the intervention group and the control group had an increasing BG control rate and decreasing SMBG compliance during the whole follow-up period. Detailed data analysis on separate follow-up periods showed that the SMBG frequency was negatively correlated with the BG control rate in most Ts and that the BG control rate of Tn-1 was negatively correlated with the SMBG frequency of Tn in the adjacent T. Only in the intervention group was T2 SMBG compliance not under the influence of the T1 BG control rate. CONCLUSIONS: Our data suggested that regardless of management approach, the BG control rate increased, and the SMBG frequency decreased as gestational weeks increased in women with GDM. Even in separate follow-up periods, the SMBG frequency was negatively correlated with the BG control rate both within one follow-up period and between two adjacent follow-up periods.

Synthesis of ent-Cleistanthane Diterpenoid Spruceanol: Construction of an Aromatic C Ring via Lewis Acid-Controlled Regioselective Diels-Alder Cycloaddition.

The first synthesis of ent-cleistanthane-type diterpenoid spruceanol with significant anticancer activity is described. A chiral pool approach was employed with a linear sequence of 13 steps beginning from readily available and inexpensive andrographolide. The approach features the construction of an aromatic ring with hydroxyl and methyl groups at C-12 and C-13 of the target compound, respectively, via Lewis acid-controlled regioselective Diels-Alder cycloaddition and the regioselective removal of the primary hydroxyl group of the Diels-Alder adduct.

Discovery and evaluation of new compounds targeting ribosomal protein S1 in antibiotic-resistant Mycobacterium Tuberculosis.

The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide.

Synthesis and anti-fibrosis activity study of 14-deoxyandrographolide-19-oic acid and 14-deoxydidehydroandrographolide-19-oic acid derivatives.

A series of 14-deoxyandrographolide-19-oic acid and 14-deoxy-11,12 (or 14,15)-didehydroandrographolide-19-oic acid derivatives were designed, synthesized and screened in vitro against the mouse fibroblast cell lines NIH-3T3. Thirteen compounds 8a-f, 14a-c, 14e-f, and 18a-b were found to exhibit better anti-fibrotic activities than andrographolide, with compounds 8b and 14e displaying best activity with IC50 values of 12.86 and 13.57 µM against NIH-3T3 respectively. Further anti-fibrotic investigation was performed in terms of PCR and western bolt analysis. Our study demonstrated that compounds 8b and 14e suppressed effectively the expression of α-smooth muscle actin, fibronectin and collagen in NIH-3T3. Preliminary structure-activity analysis revealed that 14-deoxygenation and 19-carboxylation of andrographolide could significantly improve its anti-fibrotic effect, which made 14-deoxyandrographolide-19-oic acid and 14-deoxy-11,12-didehydroandrographolide-19-oic acid promising leads for the development of new anti-fibrotic agents.

A potent peptide as adiponectin receptor 1 agonist to against fibrosis.

Fibrotic diseases have become a major cause of death in the developed world. AdipoR1 agonists are potent inhibitors of fibrotic responses. Here, we focused on the in silico identification of novel AdipoR1 peptide agonists. A homology model was constructed to predict the 3D structure of AdipoR1. By docking to known active peptides, the putative active site of the model was further explored. A virtual screening study was then carried out with a set of manually designed peptides using molecular docking. Peptides with high docking scores were then evaluated for their anti-fibrotic properties. The data indicated that the novel peptide Pep70 significantly inhibited the proliferation of hepatic stellate cells (HSC) and NIH-3T3 cells (18.33% and 27.80%) and resulted in favouring cell-cycle arrest through increasing the accumulation of cells in the G0/G1 phase by 17.08% and 15.86%, thereby reducing the cell population in the G2/M phase by 11.25% and 15.95%, respectively. Additionally, Pep70 exhibited the most marked suppression on the expression of α-smooth muscle actin (α-SMA), collagen type I alpha1 (COL1A1) and TGF-ß1. Therefore, the peptide Pep70 was ultimately identified as an inhibitor of fibrotic responses and as a potential AdipoR1 agonist.

Hepatoprotective effects of AdipoRon against d-galactosamine-induced liver injury in mice.

Adiponectin is an antidiabetic and antiatherogenic adipokine, which plays distinct roles in the balance of energy homoeostasis. As an insulin sensitizing hormone, adiponectin exerts multiple biological effects by the specific receptors (AdipoR1 and AdipoR2), through activation of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor (PPAR)α pathways. AdipoRon, an orally active synthetic small-molecule AdipoR agonist, shows very similar effects to adiponectin in vitro and in vivo, which could be a promising therapeutic approach for obesity-related disorders. In view of the regulatory effects of adiponectin or AdipoRon on inflammatory response and energy metabolism, they might be endowed a curative potential for tissue damage. Hence, its effects and possible mechanism were investigated. In vitro studies on hepatocytes (L02) and macrophages (RAW264.7) suggested a protective and anti-inflammatory potential of AdipoRon. The effects were verified in acute hepatic injury mice induced by d-galactosamine (D-GalN): hepatic lesions were restored by AdipoRon or bicyclol (positive reference drug) pretreatment, which were characterized by a significant increase in serological and hepatic biomarkers (AST, ALT, MDA and NOSs). Besides, AdipoRon attenuated the inflammation in the liver, characterized by the dwindling proinflammatory macrophage infiltration, as well as the shrinkage of tumor necrosis factor-α (TNF-α), transforming growth factor beta 1 (TGF-ß1), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6); meanwhile conversely promoted AMPK activation by phosphorylation. Combined with liver histopathology, these results demonstrated the hepatoprotective effects of AdipoRon against D-GalN-induced damage, which might be ascribed to the attenuation of inflammation, inhibition of free radical reactions, as well as enhancement of liver energy metabolism.

Synthesis of CDDO-amino acid-nitric oxide donor trihybrids as potential antitumor agents against both drug-sensitive and drug-resistant colon cancer.

Seventeen CDDO-amino acid-NO donor trihybrids (4a-q) were designed and synthesized. Biological evaluation indicated that the most active compound 4c produced high levels of NO and inhibited the proliferation of drug-sensitive (HCT-8, IC50 = 0.294 µM) and drug-resistant (HCT-8/5-FU, IC50 = 0.232 µM) colon cancer cells, which were attenuated by an NO scavenger or typical substrate of PepT1. Furthermore, 4c triggered HCT-8 and HCT-8/5-FU cell apoptosis more strongly than CDDO-Me, inhibited the HIF-1α, Stat3, AKT, and ERK signaling, and induced the nitration of P-gp, MRP1, and BCRP proteins in HCT-8/5-FU cells. Finally, 4c had 4.36-5.53-fold less inhibitory activity against nontumor colon epithelial-like cells (CCD841, IC50 = 1.282 µM) in vitro and inhibited the growth of implanted human drug-resistant colon cancers in mice more potently than CDDO-Me. Together, 4c is a novel trihybrid with potent antitumor activity and may be a promising candidate for the treatment of drug-resistant colon cancer.

Synthesis and anticancer activity of some novel indolo[3,2-b]andrographolide derivatives as apoptosis-inducing agents.

A series of novel indolo[3,2-b]andrographolide derivatives were designed, synthesized and screened in vitro against three human cancer cell lines MCF7 (human breast cancer), HCT116 (human colon cancer), and DU145 (human prostate cancer). Fourteen compounds 6b, 6e, 6i, 6j, 6l, 6m, 6n, 12a, 12b, 13a, 13b, 15a, 17a, and 17b exhibited better anti-cancer activities than andrographolide for all three human cancer lines, with compound 6l displaying best activity with IC50 values of 1.85, 1.22 and 1.24 µM against MCF7, HCT116 and DU145 respectively. Preliminary anti-cancer mechanistic investigation was performed in terms of the cell cycle arrest and cell apoptosis assays of compound 6l against HCT116 using flow cytometry, and the results suggested that compound 6l inhibited tumor proliferation through inducing early and late cellular apoptosis in a concentration-dependent manner and causing cell cycle arrest in the S-phase.

Synthesis and in vitro cytotoxicity of andrographolide-19-oic acid analogues as anti-cancer agents.

The synthesis of a series of andrographolide-19-oic acid derivatives was described and their in vitro anti-tumor activity against two human cell lines was evaluated. Most compounds were found to exhibit significant cytotoxicity, better than andrographolide, and compounds 9d and 9b were identified as the most potent with IC50 values of 1.18 and 6.28 µm against HCT-116 and MCF-7 cell lines, respectively. The preliminary results indicated that the oxidation of C-19-hydroxyl group of andrographolide to corresponding carboxyl group and the subsequent esterification of the formed carboxylic acid led to considerable improvement in cytotoxicity against the cancer cells.

Design, synthesis, and biological evaluation of novel histone deacetylase 1 inhibitors through click chemistry.

We report the design, synthesis, and biological evaluation of a new series of HDAC1 inhibitors using click chemistry. Compound 17 bearing a phenyl ring at meta-position was identified to show much better selectivity for HDAC1 over HDAC7 than SAHA. The compond 17 also showed better in vitro anticancer activities against several cancer cell lines than that of SAHA. This work could serve as a foundation for further exploration of selective HDAC inhibitors using the compound 17 molecular scaffold.

Enthalpy (DeltaH) and entropy (DeltaS) for pi-stacking interactions in near-sandwich configurations: relative importance of electrostatic, dispersive, and charge-transfer effects.

Interactions between two aromatic rings with various substituents in a near-sandwich configuration have been quantitatively studied by using the triptycene derived molecular models. This model system allows a stacking arrangement of two arenes to assume a near-perfect face-to-face configuration in its ground state conformation. Comparing to our previous study of the parallel displaced configuration, repulsive interactions are predominant for most arenes currently studied. However, if one arene is strongly electron deficient (Ar2=pentafluorobenzoate), attractive interactions were observed regardless of the character of the other arene (Ar1). For stacking interactions between Me2NC6H4 and C6F5CO groups, a DeltaH of -1.84+/-0.2 kcal/mol and a DeltaS of -2.9+/-0.8 cal/(mol.K) were determined. The general trend in the attractive stacking interaction toward a pentafluorobenzoate is Me2NC6H4>Me3C6H2>Me2C6H3>MeC6H4>MeOC6H4>C6H5>O2NC6H4. The observed trend is consistent with a donor-acceptor relationship and the acceptor is a C6F5CO group.