Discovery of quinazoline HPK1 inhibitors with high cellular potency.

Hematopoietic progenitor kinase 1 (HPK1) is regarded as a highly validated target in pre-clinical immune oncology. HPK1 has been described as regulating multiple critical signaling pathway in both adaptive and innate cells. In support of this role, HPK1 KO T cells show enhanced sensitivity to TCR activation and HPK1 KO mice display enhanced anti-tumor activity. Taken together, inhibition of HPK1 has the potential to induce enhanced anti-tumor immune response. Herein, we described the discovery of highly potent HPK1 inhibitors starting form a weak HTS hit. Using a structure-based drug design, HPK1 inhibitors exhibiting excellent cellular single-digit nanomolar potency in both proximal (pSLP76) and distal (IL-2) biomarkers along with sustained elevation of IL-2 cytokine secretion were discovered.

Discovery of 4-aminopyrimidine analogs as highly potent dual P70S6K/Akt inhibitors.

Activation of the PI3K/Akt/mTOR kinase pathway is associated with human cancers. A dual p70S6K/Akt inhibitor is sufficient to inhibit strong tumor growth and to block negative impact of the compensatory Akt feedback loop activation. A scaffold docking strategy based on an existing quinazoline carboxamide series identified 4-aminopyrimidine analog 6, which showed a single-digit nanomolar and a micromolar potencies in p70S6K and Akt enzymatic assays. SAR optimization improved Akt enzymatic and p70S6K cellular potencies, reduced hERG liability, and ultimately discovered the promising candidate 37, which exhibited with a single digit nanomolar value in both p70S6K and Akt biochemical assays, and hERG activities (IC50 = 17.4 µM). This agent demonstrated dose-dependent efficacy in inhibiting mice breast cancer tumor growth and covered more than 90% pS6 inhibition up to 24 h at a dose of 200 mg/kg po.

Synthesis and SAR development of quinoline analogs as novel P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) plays an important role in diverse conditions associated with tissue damage and inflammation, suggesting that the human P2X7R (hP2X7R) is an attractive therapeutic target. In the present study, the synthesis and structure-activity relationship (SAR) of a novel series of quinoline derivatives as P2X7R antagonists are described herein. These compounds exhibited mechanistic activity (YO PRO) in an engineered HEK293 expressing hP2X7R as well as a functional response (IL-1ß) in human THP-1 (hTHP-1) cellular assays. Compound 19 was identified as the most promising compound in this series with excellent cellular potency, low liver microsomal clearance, good permeability and low efflux ratio. In addition, this compound also displayed good pharmacokinetic properties and acceptable brain permeability (Kp,uu of 0.37).

[Expression of recombinant human ZP3 protein using the baculovirus expression system].

OBJECTIVE: To investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3. METHODS: The recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored. RESULTS: The recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm. CONCLUSION: It is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.

Impact of Nutritional Tea Polyphenols on Growth, Feed Efficiency, Biochemical Traits, Antioxidant Capacity, Haematological Parameters and Immunity in Coho Salmon (Oncorhynchus kisutch).

To evaluate the impact of nutritional tea polyphenols (TPs) on body composition, growth, biochemical markers, antioxidant capacity, and hemato-immunological levels, a ten-week feeding experiment was carried out on coho salmon (Oncorhynchus kisutch, 180.51 ± 0.15 g). The control group was fed a basal diet; the T1, T2, T3, and T4 groups were fed 0.005%, 0.01%, 0.02%, and 0.04% TPs, respectively. These results demonstrate that adding TPs significantly (p < 0.05) increased the coho salmon fish's weight gain (WG), relative growth rate (RGR), condition factor (CF), feed efficacy (FE), daily growth rate (DGR), and specific growth rate (SGR). There was no discernible difference in the body compositions of the treated TPs and the control group (p > 0.05). In addition, the T3 group showed a significant (p < 0.05) decrease in GPT, LDL, HDL, TC, and CAT. Fish given a 0.02% diet containing TPs had significantly lower levels of malondialdehyde (MDA) in their liver; yet, the TP-treated groups had higher levels of SOD and CAT than the control (p < 0.05). The data analysis shows a significant rise in lysozyme, respiratory burst activity, bactericidal activity, and blood hematological parameters in the 0.01-0.04% TP groups. According to these findings, TPs could be a useful dietary supplement for raising the antioxidant status, growth parameters, haemato-immunological response, and whole-body composition of coho salmon fish.

SAR and evaluation of novel 5H-benzo[c][1,8]naphthyridin-6-one analogs as Aurora kinase inhibitors.

Several potent Aurora kinase inhibitors derived from 5H-benzo[c][1,8]naphthyridin-6-one scaffold were identified. A crystal structure of Aurora kinase A in complex with an initial hit revealed a binding mode of the inhibitor within the ATP binding site and provided insight for structure-guided compound optimization. Subsequent SAR campaign provided a potent and selective pan Aurora inhibitor, which demonstrated potent target modulation and antiproliferative effects in the pancreatic cell line, MIAPaCa-2. Furthermore, this compound inhibited phosphorylation of histone H3 (pHH3) in mouse bone morrow upon oral administration, which is consistent with inhibition of Aurora kinase B activity.

Optimistic effects of galaxolide and polystyrene microplastic stress on the physio-biochemical characteristics and metabolic profiles of an ornamental plant.

Galaxolide (HHCB) and polystyrene (PS) microplastics or nanoplastics have been widely recognized as emerging pollutants. However, very few efforts have been made to remove these contaminants from the environment using eco-friendly materials such as plant materials. Therefore, this study sought to investigate the physiological and biochemical effects and tolerance mechanisms of Mirabilis jalapa L. upon exposure to HHCB and PS. Our findings demonstrated that this ornamental plant was tolerant to HHCB and PS exposure. HHCB treatment increased antioxidant enzyme activity. However, superoxide dismutase (SOD) activity increased by 206.85% when the plants were treated with 0.5 mg L-1 HHCB alone, whereas co-exposure to 0.5 mg L-1 HHCB and 500 nm PS increased SOD activity by 93.82%. Contaminant exposure also affected photosynthetic parameters such as stomatal conductance and transpiration rate. In contrast, net photosynthetic rate and photosynthetic pigment content were not significantly affected. HHCB aggregated heavily in the roots of the plant. Moreover, 500 nm PS could be absorbed by the root and transported to the shoot, and 5 µm PS would be transferred to the shoot under the carrying effect of HHCB. Co-exposure to HHCB and PS significantly changed the glyoxylate and dicarboxylate metabolism, alanine, aspartate, and glutamate metabolism, and glycine, serine, and threonine metabolism pathways, thus affecting carbohydrate synthesis and energy metabolism in M. jalapa. These results provide a basis for the development of HHCB and PS remediation strategies using M. jalapa, an ornamental plant that is not only tolerant to organic contaminants but can also beautify the environment.

[Direct fumigation for freeze-thawing of human sperm: an experimental study].

OBJECTIVE: To study the effect of direct fumigation on the post-thaw recovery rate of cryopreserved spermatozoa, and to search for a best method for human sperm cryopreservation. METHODS: We collected semen samples from 100 donors conforming to the normal reference values in WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), divided them into two groups, and subjected them to cryopreservation by programmable freezing (Group A) and direct fumigation (Group B), respectively. We detected the progressive motility of pre-freezing and post-thaw sperm with a computer-assisted semen analyzer, and compared the effects of the two methods on the functional integrity of sperm membrane and the rate of abnormal sperm using the percentage of hypo-osmotic swelling sperm and modified Papanicolaou staining. RESULTS: Statistically significant differences were found in post-thaw sperm progressive motility between the Groups A and B ([34.0 +/- 18.4]% vs [43.0 +/- 19.5]%, P<0.05), both remarkably decreased as compared with pre-freezing ([57.0 +/- 16.7]%, P<0.05). Such differences were also found in the post-thaw recovery rate of progressively motile sperm between the two groups ([52.2 +/- 20.6]% vs [67.1 +/- 20.0]%, P<0.05). The post-thaw percentage of hypo-osmotic swelling sperm was obviously decreased in both Groups A and B ([67.1 +/- 11.1]% and [70.6 +/- 10.0]%) in comparison with pre-freezing ([84.5 +/- 7.5]%, P<0.05), with significant differences between A and B (P<0.05). However, the rate of sperm abnormality was evidently increased in Groups A and B ([85.0 +/- 8.7% and [85.7 +/- 9.1]%), significantly higher than pre-freezing ([77.8 +/- 9.6]%, P<0.05), but with no significant differences between A and B (P>0.05). CONCLUSION: Direct fumigation is superior to programmable freezing for its easier operation, wider application, and higher sperm recovery rate.

[Reference values of semen parameters for normal fertile men in Shanghai].

OBJECTIVE: To analyze the distribution characteristics of the main semen parameters of healthy semen donors and normal fertile men in Shanghai, compare the semen quality between the two groups, and investigate the normal reference values of the semen parameters of the fertile population in Shanghai. METHODS: We obtained semen samples from 100 healthy donors and 41 fertile men, performed semen analyses according to the WHO (2010) guidelines, and determined the semen volume, sperm concentration, sperm progressive motility, total sperm count and total progressively motile sperm count. We analyzed the distribution of the semen parameters of the normal fertile men, and obtained the lower limits of their normal reference values. RESULTS: There were no statistically significant differences in the main semen parameters between the healthy donors and normal fertile men (P < 0.05). The lower reference limits for the semen parameters of normal fertile men in Shanghai (P < 0.05) were as follows: sperm concentration > or = 27.3 x 10(6)/ml, sperm progressive motility > or = 8.1%, semen volume > or = 0.82 ml, total sperm count > or = 44.73 x 10(6) per ejaculate, and total progressively motile sperm count > or = 24.68 x 10(6) per ejaculate. CONCLUSION: For the evaluation of male fecundity, total sperm count and total progressively motile sperm count may be two better predictors than others.

The G2A Receptor Deficiency Aggravates Atherosclerosis in Rats by Regulating Macrophages and Lipid Metabolism.

The orphan G protein-coupled receptor G2A has been linked to atherosclerosis development. However, available data from mouse models are controversial. Rat G2A receptor bears more similarities with its human homolog. We proposed that the atherosclerosis model established from Ldlr -/- rat, which has been reported to share more similar phenotypes with the human disease, may help to further understand this lipid receptor. G2A deletion was found markedly aggravated in the lipid disorder in the rat model, which has not been reported in mouse studies. Examination of aortas revealed exacerbated atherosclerotic plaques in G2A deficient rats, together with increased oxidative stress and macrophage accumulation. In addition, consistently promoted migration and apoptosis were noticed in G2A deficient macrophages, even in macrophages from G2A single knockout rats. Further analysis found significantly declined phosphorylation of PI3 kinase (PI3K) and AKT, together with reduced downstream genes Bcl2 and Bcl-xl, suggesting possible involvement of PI3K/AKT pathway in G2A regulation to macrophage apoptosis. These data indicate that G2A modulates atherosclerosis by regulating lipid metabolism and macrophage migration and apoptosis. Our study provides a new understanding of the role of G2A in atherosclerosis, supporting it as a potential therapeutic target.

Identification of Clinical Candidate M2698, a Dual p70S6K and Akt Inhibitor, for Treatment of PAM Pathway-Altered Cancers.

Herein, we report the discovery of a novel class of quinazoline carboxamides as dual p70S6k/Akt inhibitors for the treatment of tumors driven by alterations to the PI3K/Akt/mTOR (PAM) pathway. Through the screening of in-house proprietary kinase library, 4-benzylamino-quinazoline-8-carboxylic acid amide 1 stood out, with sub-micromolar p70S6k biochemical activity, as the starting point for a structurally enabled p70S6K/Akt dual inhibitor program that led to the discovery of M2698, a dual p70S6k/Akt inhibitor. M2698 is kinase selective, possesses favorable physical, chemical, and DMPK profiles, is orally available and well tolerated, and displayed tumor control in multiple in vivo studies of PAM pathway-driven tumors.

Synthesis and evaluation of a gamma-lactam as a highly selective EP2 and EP4 receptor agonist.

Gamma-lactam analogs (2) of EP(4) receptor agonists were identified by substitution of the pyrazolidinone ring (1) with a pyrrolidinone ring. Several compounds (such as 2a, 2h) with high potency, selectivity and acceptable PK profiles were discovered. These were assessed in animal models of ovulation induction and bronchoconstriction.

Hyperlipidemia induces typical atherosclerosis development in Ldlr and Apoe deficient rats.

BACKGROUND AND AIMS: Low-density lipoprotein receptor (Ldlr) and apolipoprotein E (Apoe) knockout (KO) mice have been widely used as animal models of atherosclerosis. However, data suggested that it is difficult to develop typical atherosclerosis in rats. To this end, Ldlr and Apoe KO rats were generated and the potential to develop novel atherosclerosis models was evaluated. METHODS: We established Apoe/Ldlr single and double KO (DKO) rats via the CRISPR/Cas9 system in the same background. Phenotypes of dyslipidemia and atherosclerosis in these KO rats were systematically characterized. RESULTS: Knockout of either gene led to severe dyslipidemia and liver steatosis. Significant atherosclerotic plaques were observed in the abdominal aorta of all mutant rats fed a normal diet for 48 weeks. Western diet greatly aggravated atherosclerosis and fatty liver. In addition, we found mononuclear cell infiltration in early lesions. Increased expression of inflammatory cytokines, as well as macrophage accumulation in lesions of mutants, was observed, indicating that mononuclear cell trafficking and endothelial inflammation affected atherogenesis. Moreover, mutant rats displayed a sex difference profile more similar to humans in which males had heavier plaque burdens than females. CONCLUSIONS: Deficiency of either Ldlr or Apoe genes induced hyperlipidemia, which promoted endothelial inflammation and led to typical atherosclerosis in rats on normal or Western diets. These models display certain advantages, which will benefit future investigations of atherosclerotic pathology and antiatherosclerotic therapeutics.

Synthesis and evaluation of novel pyrazolidinone analogs of PGE2 as EP2 and EP4 receptors agonists.

Replacement of the hydroxy cyclopentanone ring in PGE(2) with chemically more stable heterocyclic rings and substitution of the unsaturated alpha-alkenyl chain with a metabolically more stable phenethyl chain led to the development of potent and selective analogs of PGE(2). Compound 10f showed the highest potency and selectivity for EP(4) the receptor.

Steady-state kinetics and inhibition of anaerobically purified human homogentisate 1,2-dioxygenase.

HGO (homogentisate 1,2-dioxygenase; EC 1.13.11.5) catalyses the O2-dependent cleavage of HGA (homogentisate) to maleylacetoacetate in the catabolism of tyrosine. Anaerobic purification of heterologously expressed Fe(II)-containing human HGO yielded an enzyme preparation with a specific activity of 28.3+/- 0.6 micromol x min(-1) x mg(-1) (20 mM Mes, 80 mM NaCl, pH 6.2, 25 degrees C), which is almost twice that of the most active preparation described to date. Moreover, the addition of reducing agents or other additives did not increase the specific activity, in contrast with previous reports. The apparent specificity of HGO for HGA was highest at pH 6.2 and the steady-state cleavage of HGA fit a compulsory-order ternary-complex mechanism (K(m) value of 28.6+/-6.2 microM for HGA, K(m) value of 1240+/-160 microM for O2). Free HGO was subject to inactivation in the presence of O2 and during the steady-state cleavage of HGA. Both cases involved the oxidation of the active site Fe(II). 3-Cl HGA, a potential inhibitor of HGO, and its isosteric analogue, 3-Me HGO, were synthesized. At saturating substrate concentrations, HGO cleaved 3-Me and 3-Cl HGA 10 and 100 times slower than HGA respectively. The apparent specificity of HGO for HGA was approx. two orders of magnitude higher than for either 3-Me or 3-Cl HGA. Interestingly, 3-Cl HGA inactivated HGO only twice as rapidly as HGA. This contrasts with what has been observed in mechanistically related dioxygenases, which are rapidly inactivated by chlorinated substrate analogues, such as 3-hydroxyanthranilate dioxygenase by 4-Cl 3-hydroxyanthranilate.

[Study on the influence of medical abortion and surgical abortion on subsequent pregnancy].

OBJECTIVE: To evaluate and compare medical abortion versus surgical abortion in respect of their influence on the safety of the mother and baby in the subsequent pregnancy and parturition. METHODS: Based on the principle of informed consent of subjects, 150 healthy pregnant women with a past history of having experienced medical abortion once were included in the study group (also called medical abortion group), and in the same period, 150 healthy pregnant women with a past history of having experienced surgical abortion once were enrolled into the comparison group (also called surgical abortion group). From then on, all the pregnant women in the two groups were followed up till a week after labor. The baseline data of the two groups were comparable (P>0.05). The rates of complications observed in these women during pregnancy and labor were evaluated. RESULTS: The incidence rates of miscarriage, placental abnormality, premature delivery and postpartum hemorrhage in the study group were significantly lower than those in the comparison group (P<0.05). No significant differences on other variables were observed between the two groups. CONCLUSION: Medical abortion is probably safer than surgical abortion in respect of their influence on subsequent pregnancy. So, provided there is less contraindication, medical abortion may be the choice for terminating unwanted pregnancy, especially for those women without a child.

Direct determination of trace cadmium in environmental samples by dynamic reaction cell inductively coupled plasma mass spectrometry.

Cadmium is subject to significant Zr and Mo based oxide/hydroxide interferences in ICP-MS analysis of environmental samples. In this work, a dynamic reaction cell (DRC) technology was used to eliminate these metal oxide/hydroxide interferences. The potentially interfering ions (94)Zr(16)OH(+), (94)Mo(16)OH(+) and (95)Mo(16)O(+) on (111)Cd(+) were oxidized to higher oxides (94)ZrO(2)H(+)/(94)ZrO(3)H(+), (94)MoO(2)H(+)/(94)MoO(3)H(+), and (95)MoO(2)(+)/(95)MoO(3)(+) by O(2) as the reaction gas in DRC. Under the optimized O(2) flow rate (2.0 mLmin(-1)) and DRC rejection parameter q (Rpq, 0.75), the background signal was reduced by up to 100-fold at m/z 111 and the limit of quantitation (LOQ, 10σ) of 0.1 ngg(-1) was obtained. The proposed method was applied to direct analysis of trace Cd in a series of soil and sediment standard reference materials and the satisfactory results showed that it has great potential for the direct determination of trace or ultra-trace levels of cadmium in environmental samples.

Discovery of novel prostaglandin analogs of PGE2 as potent and selective EP2 and EP4 receptor agonists.

Analogs of PGE(2) with introduction of diene groups at the omega-side chain have been synthesized and evaluated for their binding affinity for EP(2) and EP(4) receptors. An optimized analog (compound 9b) showed high potency and selectivity for the EP(4) receptor over other known receptors.