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Phosphodiesterase-4 (PDE4) and PDE5 responsible for the hydrolysis of intracellular cAMP and cGMP, respectively, are promising targets for therapeutic intervention in a wide variety of diseases. Here, we report the discovery of novel, drug-like PDE4 inhibitors by performing a high-throughput drug repurposing screening of 2560 approved drugs and drug candidates in clinical trial studies. It allowed us to identify eight potent PDE4 inhibitors with IC 50 values ranging from 0.41 to 2.46 μM. Crystal structures of PDE4 in complex with four compounds, namely ethaverine hydrochloride (EH), benzbromarone (BBR), CX-4945, and CVT-313, were further solved to elucidate molecular mechanisms of action of these new inhibitors, providing a solid foundation for optimizing the inhibitors to improve their potency as well as selectivity. Unexpectedly, selectivity profiling of other PDE subfamilies followed by crystal structure determination revealed that CVT-313 was also a potent PDE5 inhibitor with a binding mode similar to that of tadalafil, a marketed PDE5 inhibitor, but distinctively different from the binding mode of CVT-313 with PDE4. Structure-guided modification of CVT-313 led to the discovery of a new inhibitor, compound 2 , with significantly improved inhibitory activity as well as selectivity towards PDE5 over PDE4. Together, these results highlight the utility of the drug repurposing in combination with structure-based drug design in identifying novel inhibitors of PDE4 and PDE5, which provides a prime example for efficient discovery of drug-like hits towards a given target protein. [Display omitted] • 8 new drug-like PDE4 inhibitors were identified by drug repurposing screening. • The binding mode of the hits and PDE4 were validated by X-ray crystallography. • CVT-313 was revealed to be a potent inhibitor of PDE4 and PDE5. • Structure-guided optimization of CVT-313 resulted in compound 2. • Compound 2 showed increased potency and selectivity toward PDE5 over PDE4. [ABSTRACT FROM AUTHOR]

Polyploid breeding can produce new species with a faster growth rate, higher disease resistance, and higher survival rate, and has achieved significant economic benefits. This study investigated the protein differences in the body wall of triploid Apostichopus japonicus and diploid A. japonicus using isotope-labeled relative and absolute quantitative Tandem Mass Tag technology. A total of 21,096 independent peptides and 4621 proteins were identified. Among them, there were 723 proteins with significant expression differences, including 413 up-regulated proteins and 310 down-regulated proteins. The differentially expressed proteins (DEPs) were enriched in 4519 Gene Ontology enrichment pathways and 320 Kyoto Encyclopedia of Genes and Genomes enrichment pathways. Twenty-two key DEPs related to important functions such as growth and immunity of triploid A. japonicus were screened from the results, among which 20 were up-regulated, such as cathepsin L2 cysteine protease and fibrinogen-like protein A. Arylsulfatase A and zonadhesin were down-regulated. The up-regulated proteins were mainly involved in oxidative stress response, innate immune response, and collagen synthesis in triploid A. japonicus , and the down-regulated proteins were mainly associated with the sterility of triploid A. japonicus. In addition, the transcriptome and proteome were analyzed jointly to support proteome data. In this study, the differences in protein composition between triploid and diploid A. japonicus were analyzed for the first time, and the results revealed the underlying reasons for the growth advantage of triploid A. japonicus. [Display omitted] • The growth and development advantages of triploid A. japonicus were discussed. • The molecular mechanisms controlling the growth traits of triploid A. japonicus were analyzed. • The function of the screened differential proteins on triploid A. japonicus was thoroughly discussed. • The protein-protein interaction (PPI) network of the identified DEPs was examined. • The relationship between the transcriptome and proteome of triploid A. japonicus was examined. [ABSTRACT FROM AUTHOR]

Strongylocentrotus intermedius is one of the most economically valuable sea urchin species in China, and its growth and survival are severely constrained by ocean warming and the hypoxia that often accompanies high water temperatures. To elucidate the molecular mechanisms of S. intermedius that regulate gene expression in response to multi-causal environmental stresses. We performed a de novo transcriptome analysis of coelomocyte from S. intermedius to heat (25 °C), hypoxia (2 mg/L), and the combined stress. We identified 35,635, 29,107, and 29,440 differentially expressed genes (DEGs) in S. intermedius cultured under high temperature, low oxygen, and combined stress, respectively. Further Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analyses revealed that these DEGs mainly enriched the functional categories of "Protein processing in endoplasmic reticulum," and "Glutathione metabolism" by heat stress, such as HSP70, GSTO1, PDIA4. After hypoxic stress, "Notch signaling pathway" and metabolism-related pathways such as "Glycerolipid metabolism", "Pyruvate metabolism" were significantly enriched. Exposure to combined stress resulted in a two-factor additive effect at the transcriptome level and have a more extensive impact on the immune correlated pathways in S. intermedius than single stress, the expression of related immune genes (C3, C5, and AIFM2) were up-regulated. Quantitative real-time PCR (qRT-PCR) analysis of the expression of 18 DEGs confirmed the RNA-Seq results. Observations in the present study will improve the understanding of the molecular mechanism of S. intermedius in response to multi-causal environmental stress. [Display omitted] • The transcriptome of S. intermedius under high temperature, hypoxia, and combined stresses was constructed. • A total of 35,635, 29,107, and 29,440 DEGs were identified under three stresses, respectively. • The transcriptome of S. intermedius showed a two-factor additive effect under combined stress. • S. intermedius demonstrated comprehensive transcriptional regulation in response to global climate change. [ABSTRACT FROM AUTHOR]

Psoriasis is a kind of chronic inflammatory skin disorder, while the long-term use of conventional therapies for this disease are limited by severe adverse effects. Novel small molecules associated with new therapeutic mechanisms are greatly needed. It is known that phosphodiesterase 4 (PDE4) plays a central role in regulating inflammatory responses through hydrolyzing intracellular cyclic adenosine monophosphate (cAMP), making PDE4 to be an important target for the treatment of inflammatory diseases (e.g. psoriasis). In our previous work, we identified a series of novel PDE4 inhibitors with a tetrahydroisoquinoline scaffold through structure-based drug design, among which compound 1 showed moderate inhibition activity against PDE4. In this study, a series of novel tetrahydroisoquinoline derivatives were developed based on the crystal structure of PDE4D in complex with compound 1. Anti-inflammatory effects of these compounds were evaluated, and compound 36, with high safety, permeability and selectivity, exhibited significant inhibitory potency against the enzymatic activity of PDE4D and the TNF-α release from the LPS-stimulated RAW 264.7 and hPBMCs. Moreover, an in vivo study demonstrated that a topical administration of 36 achieved more significant efficacy than calcipotriol to improve the features of psoriasis-like skin inflammation. Overall, our study provides a basis for further development of tetrahydroisoquinoline-based PDE4 inhibitors against psoriasis. Image 1 • 51 tetrahydroisoquinolines derivatives as novel PDE4 inhibitors were designed, synthesized and evaluated. • The optimum compound 36 exhibited high safety, potency, permeability and selectivity in vitro. • The crystal structure of PDE4D in complex with compound 36 was solved. • Compound 36 exhibited superior therapeutic efficacy than calcipotriol against the IMQ-induced murine psoriasis-like skin inflammation. [ABSTRACT FROM AUTHOR]

Phosphodiesterase-4 (PDE4) functions as a critical intracellular enzyme in immune cells and keratinocytes through the hydrolysis of cAMP. Inhibition of PDE4 has been considered as an effective therapeutic strategy in multiple inflammatory diseases. This study was intended to assess the anti-inflammatory effects of the PDE4 inhibitor, DC591017, both in vitro and in vivo. Murine RAW264.7 cells, BMDMs, BMDCs, and human NHEKs were incubated with DC591017 and then inflammatory mediators, intracellular cAMP and cAMP-mediated signaling pathways were analyzed. Carrageenan-induced acute inflammation in murine air pouches and rat paws, as well as imiquimod (IMQ)-induced psoriasis-like skin lesions were conducted to explore the therapeutic effects and underlying mechanisms of DC591017. We demonstrated herein that DC591017 suppressed the inflammatory responses of macrophages and DCs through promoting cAMP-dependent PKA-CREB signaling. Addition of forskolin functioned synergistically with DC591017, which could be blocked following H89 intervention or knockdown of PKA expression by siRNA transfection. In vivo , DC591017 treatment alleviated the leukocytes infiltration and secretion of inflammatory cytokines in murine air pouches and significantly attenuated carrageenan-induced paw swelling in rats. Moreover, we also illustrated that topical application of DC591017 ointment ameliorated IMQ-caused experimental psoriatic skin lesions, as evidenced by decreasing epidermal thickening and inflammatory infiltrations to inflamed skins. Consistently, DC591017 decreased expression of PDE4 isoforms and subsequently regulated PKA-CREB and NF-κB signaling. In brief, our study brought out a patent PDE4 inhibitor with robust anti-inflammation and provided the credible evidence in the treatment of patients with psoriasis. [ABSTRACT FROM AUTHOR]

Betulinic acid (BA) is a natural pentacyclic triterpenoid that has a wide range of biological and pharmacological effects. Here, computational methods such as pharmacophore screening and reverse docking were used to predict the potential target for BA. Retinoic acid receptor-related orphan receptor gamma (RORγ) was confirmed as its target by several molecular assays as well as crystal complex structure determination. RORγ has been the focus of metabolic regulation, but its potential role in cancer treatment has only recently come to the fore. In this study, rationale optimization of BA was performed and several new derivatives were generated. Among them, the compound 22 showed stronger binding affinity with RORγ (K D = 180 nM), good anti-proliferative activity against cancer cell lines, and potent anti-tumor efficacy with a TGI value of 71.6% (at a dose of 15 mg/kg) in the HPAF-II pancreatic cancer xenograft model. Further RNA-seq analysis and cellular validation experiments supported that RORγ antagonism was closely related to the antitumor activity of BA and 22 , resulting in suppression of the RAS/MAPK and AKT/mTORC1 pathway and inducing caspase-dependent apoptosis in pancreatic cancer cells. RORγ was highly expressed in cancer cells and tissues and positively correlated with the poor prognosis of cancer patients. These results suggest that BA derivatives are potential RORγ antagonists worthy of further exploration. RORγ has been identified as a natural anti-tumor product targeting betulinic acid. A series of new, more potent betulinic acid derivatives were designed, synthesized, and evaluated. 22 proved to be the best lead. [Display omitted] • RORγ was identified as a target of betulinic acid combining the strategy of reverse docking and X-ray crystallography. • Utilizing structure and QSAR-based approach, a series of BA derivatives as RORγ antagonists were synthesized and evaluated. • The compound 22 exhibited good anti-proliferation activity and the pharmacological mechanism of anti-tumor was studied. • Compound 22 had potent anti-tumor efficacy in the HPAF-II pancreatic cancer xenograft model. [ABSTRACT FROM AUTHOR]

The 3C-like protease (3CLpro) is essential for the replication and transcription of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), making it a promising target for the treatment of corona virus disease 2019 (COVID-19). In this study, a series of 2,3,5-substituted [1,2,4]-thiadiazole analogs were discovered to be able to inhibit 3CLpro as non-peptidomimetic covalent binders at submicromolar levels, with IC 50 values ranging from 0.118 to 0.582 μM. Interestingly, these compounds were also shown to inhibit PLpro with the same level of IC 50 values, but had negligible effect on proteases such as chymotrypsin, cathepsin B, and cathepsin L. Subsequently, the antiviral abilities of these compounds were evaluated in cell-based assays, and compound 6g showed potent antiviral activity with an EC 50 value of 7.249 μM. It was proposed that these compounds covalently bind to the catalytic cysteine 145 via a ring-opening metathesis reaction mechanism. To understand this covalent-binding reaction, we chose compound 6a , one of the identified hit compounds, as a representative to investigate the reaction mechanism in detail by combing several computational predictions and experimental validation. The process of ring-opening metathesis was theoretically studied using quantum chemistry calculations according to the transition state theory. Our study revealed that the 2,3,5-substituted [1,2,4]-thiadiazole group could covalently modify the catalytic cysteine in the binding pocket of 3CLpro as a potential warhead. Moreover, 6a was a known GPCR modulator, and our study is also a successful computational method-based drug-repurposing study. 2,3,5-substituted [1,2,4]-thiadiazoles as potent inhibitors targeting 3C-Like protease were proposed to covalently bind with the catalytic cysteine via a ring-opening metathesis reaction mechanism. [Display omitted] • Discovery of a new type of non-peptidomimetic covalent inhibitor against 3CLpro of SARS-CoV-2 with inhibition as strong as sub-micromolar level. • A molecular mechanism of the ring-opening metathesis was explored by combining types of computational and experimental technologies. • A multi-technologies integrated research paradigm for lead identification, validation and molecular mechanism study was explored. [ABSTRACT FROM AUTHOR]

This study aimed to explore the effects of trans -anethole (TA) on lipopolysaccharide (LPS)-induced acute jejunal inflammation model of broilers. A total of 160 one-day-old broilers (male; Arbor Acres) were randomly allocated into four treatment groups with 8 replicates of 5 birds each. On d 20, the dose of 5 mg/kg body weight LPS solution and the equal amount of sterile saline were intraperitoneally injected into LPS-challenged and unchallenged broilers, respectively. Compared with the control group, LPS decreased (P < 0.05) the villus height (VH) and the ratio of villus height to crypt depth (VCR) but increased (P < 0.05) the crypt depth (CD), meanwhile, enhanced (P < 0.01) the levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) but decreased (P < 0.01) the level of interleukin-10 (IL-10). The group supplemented with 600 mg/kg of TA had lower (P < 0.01) CD and higher (P < 0.01) VCR than the LPS group. TA increased (P < 0.01) the level of IL-10 and decreased (P < 0.01) the level of IL-1β. The mRNA expression levels of IL-6 , nuclear factor kappa B ( NF-κB ), TNF-α were up-regulated (P < 0.05) and the levels of IL-10 and inhibitor of NF-κB alpha ( IκBα ) were down-regulated (P < 0.05) by LPS as compared with the control group. TA down-regulated (P < 0.05) the increased mRNA expression levels of genes caused by LPS, as well as up-regulated (P < 0.05) the levels of IL-10 and IκBα. Furthermore, LPS down-regulated (P < 0.05) and up-regulated (P < 0.05) the protein expression levels of IκBα and NF-κB p65, respectively. TA up-regulated (P < 0.05) the level of IκBα and down-regulated (P < 0.05) the level of NF-κB p65. The conclusion of this study is that TA could exert protective effect on the LPS-induced acute jejunal inflammation of broilers via repressing the activation of NF-κB and the 600 mg/kg is the optimal dose against LPS-induced acute jejunal inflammation of broilers. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the protective effect of trans -anethole (TA) on lipopolysaccharide-induced acute liver inflammation model of chickens by determining the levels of inflammatory mediators in serum and liver, relative mRNA expression and protein expression of inflammation-related genes in NF-κB signaling pathway. A total of 160 one-day-old male chickens (Arbor Acres) were assigned into 4 treatments with 8 replicates of 5 birds each. On d 20, the control group was intraperitoneally injected with sterile saline and the other groups were injected with lipopolysaccharide (LPS ; 5 mg/kg body weight). There were no significant differences in average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (FCR) among groups. However, compared with the control group, the LPS group significantly increased (P < 0.01) the serum levels of interleukin-6 (IL-6), interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and decreased (P < 0.01) the interleukin-10 (IL-10) level. TA attenuated (P < 0.01) these increases in IL-1β, TNF-α, ALT, and AST levels and improved (P < 0.01) the IL-10 level. In liver, the groups fed with TA had lower (P < 0.01) concentrations of IL-6 and TNF-α as well as higher (P < 0.05) concentration of IL-10. Furthermore, TA downregulated (P < 0.05) the mRNA expression levels of nuclear factor kappa B p65 ( NF-κB p65 ) and TNF-α , also upregulated (P < 0.05) IL-10 and inhibitor of NF-κB alpha ( IκBα ) upon LPS challenge. In protein level, supplementation of 600 mg/kg of TA downregulated (P < 0.05) and upregulated (P < 0.05) the protein expression of NF-κB p65 and IκBα, respectively. The present findings suggest that TA could alleviate the acute liver inflammation induced by LPS via blocking the activation of NF-κB and the 600 mg/kg of TA plays more fruitful role in protecting broilers against LPS stimulus. [ABSTRACT FROM AUTHOR]

• A modified contact detection judgment in MPM for coining process is proposed. • The improved method overcomes small time step and abnormal termination of simulation. • The proposed method avoids virtual contact distance and obtains fine patterns. • The novel method reduces amount of data storage and computations. • Predicted insufficient filling agrees well with experimental result. [Display omitted] Extremely small time step caused by large deformation in coining process significantly increases computational time in dynamic explicit finite element method (FEM). Furthermore, negative volumes of distortion elements in the rim of coin lead to abnormal termination of simulation. In order to overcome these problems, dynamic explicit material point method (MPM) is employed for coining process. However, most of the point-to-point contact algorithms in MPM cause fake contacting detection. In addition, abundant tiny patterns on die surfaces put forward high requirement for the contact algorithm to calculate the normal vectors. A point-to-segment based contact algorithm is proposed to obtain accurate normal vectors at real physical contact point and embedded into the framework of modified update stress last (MUSL) for simulating coining. Amount of data storage and computations are reduced since the background domain only contains the deformable workpiece and one velocity field is required. The improved MPM is applied to predict insufficient filling during minting, and its performance is validated by experimental results. [ABSTRACT FROM AUTHOR]