Non-clinical immunogenicity, biodistribution and toxicology evaluation of a chimpanzee adenovirus-based COVID-19 vaccine in rat and rhesus macaque.

Coronavirus Disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 has rapidly expanded into a serious global pandemic. Due to the high morbidity and mortality of COVID-19, there is an urgent need to develop safe and effective vaccines. AdC68-19S is an investigational chimpanzee adenovirus serotype 68 (AdC68) vector-based vaccine which encodes the full-length spike protein of SARS-CoV-2. Here, we evaluated the immunogenicity, biodistribution and safety profiles of the candidate vaccine AdC68-19S in Sprague Dawley (SD) rat and rhesus macaque under GLP conditions. To characterize the biodistribution profile of AdC68-19S, SD rats were given a single intramuscular injection of AdC68-19S 2 × 1011 VP/dose. Designated organs were collected on day 1, day 2, day 4, day 8 and day 15. Genomic DNA was extracted from all samples and was further quantified by real-time quantitative polymerase chain reaction (qPCR). To characterize the toxicology and immunogenicity profiles of AdC68-19S, the rats and rhesus macaques were injected intramuscularly with AdC68-19S up to 2 × 1011vp/dose or 4 × 1011vp/dose (2 and fourfold the proposed clinical dose of 1 × 1011vp/dose) on two or three occasions with a 14-day interval period, respectively. In addition to the conventional toxicological evaluation indexes, the antigen-specific cellular and humoral responses were evaluated. We proved that multiple intramuscular injections could elicit effective and long-lasting neutralizing antibody responses and Th1 T cell responses. AdC68-19S was mainly distributed in injection sites and no AdC68-19S related toxicological reaction was observed. In conclusion, these results have shown that AdC68-19S could induce an effective immune response with a good safety profile, and is a promising candidate vaccine against COVID-19.

Discovery of Highly Potent and Selective IRAK1 Degraders to Probe Scaffolding Functions of IRAK1 in ABC DLBCL.

MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1 (IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1 downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.

Apigenin and Temozolomide Synergistically Inhibit Glioma Growth Through the PI3K/AKT Pathway.

Background: Glioma is a devastating disease with the worst prognosis among human malignant tumors. Although temozolomide (TMZ) improves the overall survival of glioma patients, there are still many glioma patients who are resistant to TMZ. In this study, we focused on the effect of apigenin (API) and TMZ on glioma cells in vitro and in vivo, and we studied the underlying molecular mechanisms. Materials and Methods: To investigate the effect of API on glioblastoma cell proliferation, cell viability was assessed after glioma cells were incubated with various concentrations of API with or without TMZ using MTT assays. Then, we explored the synergistic effect of API and TMZ on glioma cell cycle, apoptosis, and migration. To investigate the molecular mechanism behind the synergism of API and TMZ, we examined the related genes of the major signaling pathways involved in glioma pathogenesis by Western blotting. Results: In this study, we found that API significantly suppressed the proliferation of glioma cells in a dose- and time-dependent manner. Combining API and TMZ significantly induced glioma cells arrest at the G2 phase and inhibited glioma cells proliferation compared with API or TMZ alone. In addition, API promoted the ability of TMZ to induce glioma cells apoptosis and inhibit glioma cells invasion. Furthermore, compared with treatment with individual agents, the combination of API and TMZ significantly inhibited the growth of subcutaneous tumors in mice. These results implied that API could synergistically suppress the growth of glioma cells when combined with TMZ. Combining API and TMZ significantly inhibited the protein expression of p-AKT, cyclin D1, Bcl-2, Matrix Metallopeptidase 2, and Matrix Metallopeptidase 9. Conclusion: API and TMZ synergistically inhibited glioma growth through the PI3K/AKT pathway.

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation.

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.

5-Substituted-N-pyridazinylbenzamides as potent and selective LRRK2 inhibitors: Improved brain unbound fraction enables efficacy.

We describe the discovery and optimization of 5-substituted-N-pyridazinylbenzamide derivatives as potent and selective LRRK2 inhibitors. Extensive SAR studies led to the identification of compounds 18 and 23, which demonstrated good in vitro pharmacokinetic profile and excellent selectivity over 140 other kinases. Both compounds demonstrated high unbound fractions in both blood and brain. Compound 18 proved to be brain penetrant, and the high unbound fraction of compound 18 in brain enabled its in vivo efficacy in CNS, wherein a significant inhibition of LRRK2 Ser935 phosphorylation was observed in rat brain following intravenous infusion at 5 mg/kg/h.

Discovery of 5-substituent-N-arylbenzamide derivatives as potent, selective and orally bioavailable LRRK2 inhibitors.

Leucine-rich repeat kinase 2 (LRRK2) has been suggested as a potential therapeutic target for Parkinson's disease. Herein we report the discovery of 5-substituent-N-arylbenzamide derivatives as novel LRRK2 inhibitors. Extensive SAR study led to the discovery of compounds 8e, which demonstrated potent LRRK2 inhibition activity, high selectivity across the kinome, good brain exposure, and high oral bioavailability.

Discovery of a novel 2,3,11,11a-tetrahydro-1H-pyrazino[1,2-b]isoquinoline-1,4(6H)-dione series promoting neurogenesis of human neural progenitor cells.

A novel neurogenic compound (1), discovered from a mouse neural progenitor cell (NPC) screen, showed profound neurogenic effect on human NPCs. Synthesis and SAR of this novel 2,3,11,11a-tetrahydro-1H-pyrazino[1,2-b]isoquinoline-1,4(6H)-dione series are described. Compound 20 is brain penetrable in rodents, and promotes neurogenesis in wild type mice, therefore it is a good tool molecule to study neurogenesis induction as a potential treatment for conditions associated with neurogenesis impairment diseases.

[Study on the differential expression of lipid metabolism-related genes in young LDLR knockout mice liver].

OBJECTIVE: To clarify the differential expression of the genes related to the lipid metabolism in the early stage of atherosclerosis in the young LDLR-/- mice of different ages. METHODS: A RT-PCR assay was used to analyse the gene expression patterns in the livers of LDLR-/- mice and wild type (WT) mice from 14 to 90 days. The characteristics of early lipid deposition in intima were evaluated using biochemical and pathological techniques. RESULTS: In LDLR-/- mice, when compared to WT mice, the mRNA level of the apolipoprotein A IV (apoA IV), fatty acid translocase (Fat/CD36) and carnitine palmitoyl transferase I (CPT I) changed prominently at the age of 14-days (P < 0.05). At 30 days, the mRNA level of apolipoprotein A I (apoA I) was up regulated, but apolipoprotein F (apoF), CD36 and CPT I were down regulated (P < 0.05). At 60 days, the mRNA levels of apoA I, CPT I and liver X receptor alpha (LXRalpha) were up regulated, but apoA IV was down regulated (P < 0.05). At 90 days, the level of the apoA I was higher, but the expression of the apoA IV, apoF and acyl-coenzymeA oxidase 1 (ACOX1) were down regulated (P < 0.05), whereas the expression of apolipoprotein A V (apoA V), apolipoprotein E (apoE), peroxidase proliferator-activated receptor alpha (PPARalpha) and angiopoietin-like protein 3 (angptl 3) had no significant changes (P > 0.05). The serum levels of TC (P < 0.05), TG (P < 0.05) and LDLC (P < 0.05) in LDLR-/- mice were significantly higher than those in wild type mice with the same age. CONCLUSIONS: The mRNA levels of the apoA I, apoA IV, apoF, FAT/CD36, CPT I, ACOX1 and LXRalpha of the LDLR-/- mice were significantly changed compared to the WT mice. The genes may be of some relevance to the complicated lipid metabolism network, and have effect in the early stage of atherogenesis.

[Expressions of atherosclerosis-related genes in aorta in young apoE/LDLR double knockout mice].

To systematically clarify the effects of apolipoprotein E (aopE) and low-density lipoprotein receptor (LDLR) gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis (AS), total RNA was isolated from fresh aortas of young apoE/LDLR double knockout (apoE(-/-)/LDLR(-/-)) and wild type (WT) mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. ß-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), nuclear factor-κB (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor 2 (TLR2), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet-derived growth factor-α (PDGF-α). SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-α, IL-1ß and oxidized LDL (ox-LDL) were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1ß, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE(-/-)/LDLR(-/-) mice at the age of 1 month were higher than those in age-matched WT mice (P<0.05, P<0.01), respectively. The expressions of PDGF-α and TNF-α in apoE(-/-)/LDLR(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice (P<0.05). All the expressions of target genes continued to be up-regulated (P<0.05, P<0.01) except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1b expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-α, IL-1ß and ox-LDL in apoE(-/-)/LDLR(-/-) mice at different ages were higher than those in age-matched WT mice (P<0.05, P<0.01), and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE(-/-)/LDLR(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.

[Differential expressions of lipid metabolism related genes in the liver of young apoE knockout mice].

The work was aimed to investigate the differential expressions of lipid metabolism related genes in the early stage of atherosclerosis in the young apolipoprotein E deficient (apoE(-/-)) mice at different ages with normal chow diet. The genotypes of mice were identified by using multiplex polymerase chain reaction (multi-PCR) analysis. The semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR were used to analyze the expressions of lipid metabolism related genes in the liver of apoE(-/-) and age-matched wild type (WT) mice of 14-day old, 1-month old, 2-month old, 3-month old. The serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) contents were assayed using COD-PAP and GPO-PAP methods. The serum apolipoprotein B100 (apoB100) content was quantitated by immune turbidimetry. The hearts were perfusion-fixed in 4% formaldehyde, infiltrated with 30% gum sucrose for 24 h at 4 °C, and embedded in OCT compound. The aortic sinus tissues were serially sectioned at -15 °C, stained with Sudan IV, and counterstained with light green. The results were shown as follows. Compared with that in WT mice, the mRNA levels of apoA I and apoA IV in apoE(-/-) mice aged from 14-day old to 3-month old changed prominently (P<0.05), with apoA I up-regulated and apoA IV down-regulated. At the age of 1 month, the expression of apoB100 in apoE(-/-) mice was higher than that in WT mice (P<0.05). The expression of apoA V was up-regulated (P<0.05) and there was obvious lipid deposition in the aortic intima in apoE(-/-) mice at the age of 2 months. The expressions of fatty acid translocase (Fat/CD36) and angiopoietin-like protein 3 (Angptl 3) in apoE(-/-) mice were higher than those in WT mice at the age of 3 months (P<0.05), while the expressions of peroxisome proliferator-activated receptor α (PPARα), liver X receptor α (LXRα), carnitine palmitoyl transferase I (CPT I) and acyl coenzyme A oxidase 1 (ACOX1) showed no significant changes. The serum TC, TG, LDL-C and HDL-C contents in apoE(-/-) mice aged from 14-day old to 3-month old were higher than those in age-matched WT mice. apoE(-/-) mice showed a marked increase in serum apoB100 content, consistent with the trend of serum LDL-C content and apoB100 mRNA content in the liver. The results suggest that the mRNA expressions of apoA I, apoA IV, apoA V, apoB100 and Angptl 3 in apoE(-/-) mice change significantly compared with those in WT mice, and these genes might be relevant to the complicated lipid metabolism network, and involved in the early stage of atherogenesis.

3D-pharmacophore models for selective A2A and A2B adenosine receptor antagonists.

Three-dimensional pharmacophore models were generated for A2A and A2B adenosine receptors (ARs) based on highly selective A2A and A2B antagonists using the Catalyst program. The best pharmacophore model for selective A2A antagonists (Hypo-A2A) was obtained through a careful validation process. Four features contained in Hypo-A2A (one ring aromatic feature (R), one positively ionizable feature (P), one hydrogen bond acceptor lipid feature (L), and one hydrophobic feature (H)) seem to be essential for antagonists in terms of binding activity and A2A AR selectivity. The best pharmacophore model for selective A2B antagonists (Hypo-A2B) was elaborated by modifying the Catalyst common features (HipHop) hypotheses generated from the selective A2B antagonists training set. Hypo-A2B also consists of four features: one ring aromatic feature (R), one hydrophobic aliphatic feature (Z), and two hydrogen bond acceptor lipid features (L). All features play an important role in A2B AR binding affinity and are essential for A2B selectivity. Both A2A and A2B pharmacophore models have been validated toward a wide set of test molecules containing structurally diverse selective antagonists of all AR subtypes. They are capable of identifying correspondingly high potent antagonists and differentiating antagonists between subtypes. The results of our study will act as a valuable tool for retrieving structurally diverse compounds with desired biological activities and designing novel selective adenosine receptor ligands.

[Expression profiles of lipid metabolism-related genes in liver of apoE(-/-)/LDLR(-/-) mice].

OBJECTIVE: To explore the relationship between the expression characteristics of lipid metabolism-related genes in the liver and early atherosclerotic lesions in apolipoprotein E and low density lipoprotein receptor gene double knockout (apoE(-/-)/LDLR(-/-)) mice. METHODS: RT-PCR was used to detect the differential expression of lipid metabolism-related genes in the liver of apoE(-/-)/LDLR(-/-) and wild type (WT) mice. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) level as well as aortic morphology were also analyzed. RESULTS: Among the 11 lipid metabolism-related genes, apolipoprotein B100 (apoB100) mRNA levels were significantly higher in apoE(-/-)/LDLR(-/-)mice compared with WT mice. At 14 days, 1, 2 and 3 months of age, the level of mRNA expression were 1.55, 1.47, 1.50 and 2.42 folds of those of the age matched WT mice respectively. The fatty acid transporter (FAT/CD36) mRNA expression levels were higher in 14-day and 3-month old mice at 1.30 and 1.35 folds of those of the age matched WT mice, respectively. Apolipoprotein A IV (apoA IV) and Apolipoprotein AV (apoAV) mRNA levels were significantly down-regulated (0.89 fold decrease in 14-day, and 0.90 folds decrease in 3-month, respectively). The mRNA expression levels of apolipoprotein AI (apo AI), apolipoprotein F (apo F), peroxidase proliferator-activated receptor alpha (PPAR-alpha), liver X receptor alpha (LXRalpha), angiopoietin-like protein 3 (ANGPTL3), acyl-coenzymeA oxidase 1 (ACOX1) and carnitine palmitoyl transferase 1 (CPT1) had no significant changes. Serum TC, TG and LDL-C were higher than those of age matched WT mice at 7, 2 and 30 folds, respectively. Furthermore, apoE(-/-)/LDLR(-/-) mice demonstrated typical early atherosclerotic lesions at sinus and root regions of aorta in an age dependent manner. CONCLUSION: Alterations of the expression of lipid metabolism-related genes in liver play important roles in the development of AS in the apoE(-/-)/LDLR(-/-) mice at early ages.

Synthesis and Resolution of Quinazolinone Atropisomeric Phosphine Ligands(,).

The syntheses of 2-methyl-3-[2'-(diphenylphosphino)phenyl]-4(3H)-quinazolinone (MPQ, 1a) and methyl-substituted analogues were achieved in good yield by coupling N-acetylanthranilic acid with the corresponding phosphinoanilines. Resolution of ligand 1bwas achieved using (-)-di-&mgr;-chlorobis[(S)-dimethyl-(1-phenylethyl)aminato-C(2),N]dipalladium(II) (5). The resulting crystalline complex (S,R)-6 served to unambiguously assign the absolute configuration of antipode (R)-(-)-1b. A practical resolution of this series of ligands 1a-c was accomplished using the (benzenesulfonyl)hydrazone derivative of camphorsulfonic acid (7) as a resolving agent.