Scaffold-based selective SHP2 inhibitors design using core hopping, molecular docking, biological evaluation and molecular simulation.

PTPN11 (coding the gene of SHP2), a classic non-receptor protein tyrosine phosphatase, is implicated in multiple cell signaling pathway. Abnormal activation of SHP2 has been shown to contribute to a variety of human diseases, including Juvenile myelomonocytic leukemia (JMML), Noonan syndrome and tumors. Thus, the SHP2 inhibitors have important therapeutic value. Here, based on the compound PubChem CID 8,478,960 (IC50 = 45.01 µM), a series of thiophene [2,3-d] pyrimidine derivatives (IC50 = 0.4-37.87 µM) were discovered as novel and efficient inhibitors of SHP2 through powerful "core hopping" and CDOCKER technology. Furthermore, the SHP2-PTP phosphatase activity assay indicated that Comp#5 (IC50 = 0.4 µM) was the most active SHP2 inhibitor. Subsequently, the effects of Comp#5 on the structure and function of SHP2 were investigated through molecular dynamics (MD) simulation and post-kinetic analysis. The result indicated that Comp#5 enhanced the interaction of residues THR357, ARG362, LYS366, PRO424, CYS459, SER460, ALA461, ILE463, ARG465, THR507 and GLN510 with the surrounding residues, improving the stability of the catalytic active region and the entrance of catalytic active region. In particular, the Comp#5 conjugated with residue ARG362, elevating the efficient and selectivity of SHP2 protein. The study here may pave the way for discovering the novel SHP2 inhibitors for suffering cancer patients.

Efficacy and safety of first-line chemotherapies for patients with advanced pancreatic ductal adenocarcinoma: A systematic review and network meta-analysis.

Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, often diagnosed at an advanced stage. Systemic chemotherapy is the primary treatment, but direct comparisons of different regimens are limited. This study conducted a systematic review and network meta-analysis (NMA) to compare the efficacy and safety of various chemotherapy regimens, with the unique advantage of only including Phase III randomized controlled trials (RCTs). Methods: NMA was conducted regarding the searched phase III RCTs by comparing overall survival (OS), progression-free survival (PFS), objective response rate (ORR), and adverse events (AEs) of different chemotherapy protocols. Results: The analysis included 24 studies with 11470 patients across 25 treatment modalities. Among the chemotherapy regimens evaluated, FOLFIRINOX (fluorouracil, leucovorin, irinotecan, and oxaliplatin) demonstrated the highest OS and PFS, with a risk ratio (logHR) of 4.5 (95 % confidence interval 4.32-4.68) compared to gemcitabine monotherapy. The PEFG regimen (cisplatin, epirubicin, 5-fluorouracil, and gemcitabine) exhibited the highest ORR, with an odds ratio (OR) of 6.67 (2.08-20) compared to gemcitabine monotherapy. Notably, gemcitabine plus sorafenib was associated with the lowest hematological toxicity, with an odds ratio (OR) of 0.1 (0.02-0.48). Conclusion: Combination therapies may offer greater benefits but also cause more toxic effects. However, combinations with targeted agents seem to have fewer adverse reactions.

Exploring the mechanism of C473D mutation on CDC25B causing weak binding affinity with CDK2/CyclinA by molecular dynamics study.

CDC25B belongs to the CDC25 family, and it plays an important part in regulating the activity of CDK/CyclinA. Studies have shown that CDC25B is closely related to cancer development. When CYS473 on CDC25B is mutated into ASP, the affinity between CDC25B and CDK2/CyclinA weakens, and their dissociation speed is greatly improved. However, the mechanism by which the CDC25BC473D mutant weakens its binding to CDK2/CyclinA is unclear. In order to study the effect of CDC25BC473D mutants on CDK2/CyclinA substrates, we constructed and verified the rationality of the CDC25BWT:CDK2/CyclinA system and CDC25BC473D:CDK2/CyclinA system and conducted molecular dynamics (MD) simulation analysis. In the post-analysis, the fluctuations of residues ARG488-SER499, LYS541-TRP550 on CDC25B and residues ASP206-ASP210 on CDK2 were massive in the mutant CDC25BC473D:CDK2/CyclinA system. And the interactions between residue ARG492 and residue GLU208, residue ARG544 and residue GLU42, residue ARG544 and TRP550 were weakened in the mutant CDC25BC473D:CDK2/CyclinA system. The results showed that when CYS473 on CDC25B was mutated into ASP473, the mutant CDC25BC473D:CDK2/CyclinA system was less stable than the wild-type CDC25BWT:CDK2/CyclinA system. Finally, active site CYS473 of CDC25B was speculated to be the key residue, which had great effects on the binding between CDC25BCYS473 and CDK2 in the CDC25BC473D:CDK2/CyclinA system. Consequently, overall analyses appeared in this study ultimately provided a useful understanding of the weak interactions between CDC25BCYS473D and CDK2/CyclinA.Communicated by Ramaswamy H. Sarma.

Molecular dynamics study of CDC25BR492L mutant causing the activity decrease of CDC25B.

Cell division cycle 25B (CDC25B) was responsible for regulating the various stages of cell division in the cell cycle. R492L was one of the common types of CDC25B mutants. Researches showed that compared to CDC25BWT, CDC25BR492L mutant had a ∼100-fold reduction in the rate constant for forming phosphatase intermediate (k2). However, the molecular basis of how the CDC25BR492L mutant influenced the process of binding between CDC25B and CDK2/CyclinA was not yet known. Therefore, the optimizations of three-dimensional structure of the CDC25BWT-CDK2/CyclinA system and the CDC25BR492L-CDK2/CyclinA system were constructed by ZDOCK and RDOCK, and five methods were employed to verify the reasonability of the docking structure. Then the molecular dynamics simulations on the two systems were performed to explore the reason why CDC25BR492L mutant caused the weak interactions between CDC25BR492L and CDK2/CyclinA, respectively. The remote docking site (Arg488-Tyr497) and the second active site (Lys538-Arg544) of CDC25B were observed to have high fluctuations in the CDC25BR492L-CDK2/CyclinA system with post-analysis, where the high fluctuation of these two regions resulted in weak interactions between CD25B and CDK2. In addition, Asp38-Glu42 and Asp206-Asp210 of CDK2 showed the slightly descending fluctuation, and CDK2 revealed an enhanced the self-interaction, which made CDK2 keep a relatively stable state in the CDC25BR492L-CDK2/CyclinA system. Finally, Leu492 of CDC25B was speculated to be the key residue, which had great effects on the binding between CDC25BR492L and CDK2 in the CDC25BR492L-CDK2/CyclinA system. Consequently, overall analyses appeared in this study ultimately offered a helpful understanding of the weak interactions between CDC25BR492L and CDK2.

Protective efficacy against Chlamydophila psittaci by oral immunization based on transgenic rice expressing MOMP in mice.

Avian chlamydiosis is caused by Chlamydophila psittaci (Cp. psittaci) and major outer membrane protein (MOMP) of Cp. psittaci is an excellent vaccine candidate. In this study, the MOMP gene was expressed in rice callus by the Agrobacterium tumefaciens vector. The production of protein in transgenic rice seeds was confirmed and quantified by Western-blot and ELISA, the results demonstrating that the antigen was expressed stably. The transgenic rice seeds expressing the MOMP protein were administered by the oral route to BALB/c mice, which developed MOMP-specific serum IgG and fecal IgA antibodies and a splenocyte MOMP-specific proliferative response and significant levels of IFN-γ, IL-2, IL-4, IL-5 and TGF-ß production. Immunization with MOMP transgenic seeds induced partial protection (50%) against a lethal challenge with the highly virulent Cp. psittaci 6BC strain. Lung function after challenge was less affected compared non-MOMP immunized animals. The results demonstrate the feasibility of using transgenic rice seeds as an oral vaccine to generate protective immunity and reduce the lung lesions in mice against virulent Cp. psittaci 6BC strain. This finding has implications for further development of an oral vaccine against avian chlamydiosis.

Vaccination with a DNA vaccine coding for perforin-like protein 1 and MIC6 induces significant protective immunity against Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is tightly related to microneme protein 6 (MIC6) and T. gondii perforin-like protein 1 (TgPLP1). In this study, we constructed a DNA vaccine expressing a TgPLP1/MIC6 fusion protein using the pIRESneo vector, and we evaluated the immune response induced by this vaccine in Kunming mice. Levels of IgG antibody, gamma interferon (IFN-γ), interleukin 2 (IL-2), IL-12, IL-4, and IL-10 were examined. Five mice were chosen randomly from every group (vaccinated groups or the nonvaccinated control group) and were challenged intragastrically with 80 cysts of T. gondii strain PRU (genotype II) in order to observe mortality daily. To analyze protection against a less-virulent challenge, eight mice of each group were orally infected with 20 cysts of strain PRU at the 14th day after the last immunization. The brain parasite load was evaluated 6 weeks after infection. The results demonstrated that immunization with pIRESneo/MIC6/PLP1 resulted in the lowest brain cyst count and prolonged the survival time of immunized mice. The levels of Toxoplasma-specific IgG, IFN-γ, IL-2, and IL-12 increased significantly, and the numbers of cysts in brains decreased more obviously, in the group immunized with plasmid pIRESneo/MIC6/PLP1 than in the other groups (P < 0.05). Compared with pIRESneo/MIC6/PLP1, coimmunization with pIRESneo/MIC6/PLP1 and adjuvant murine IL-18 promoted cellular and humoral immune responses but did not contribute significantly to cyst reduction (65.43% versus 61.60%) or the survival of immunized mice (45.0 ± 2.9 days versus 42.8 ± 2.9 days) (P > 0.05). Furthermore, the study also showed that the immune efficacy induced by pIRESneo/MIC6/PLP1 was better than that induced by pVAX/PLP1 or pVAX/MIC6 alone.

Protective efficacy of a Toxoplasma gondii rhoptry protein 13 plasmid DNA vaccine in mice.

Toxoplasma gondii is an obligate intracellular parasite infecting humans and other warm-blooded animals, resulting in serious public health problems and economic losses worldwide. Rhoptries are involved in T. gondii invasion and host cell interaction and have been implicated as important virulence factors. In the present study, a DNA vaccine expressing rhoptry protein 13 (ROP13) of T. gondii inserted into eukaryotic expression vector pVAX I was constructed, and the immune protection it induced in Kunming mice was evaluated. Kunming mice were immunized intramuscularly with pVAX-ROP13 and/or with interleukin-18 (IL-18). Then, we evaluated the immune response using a lymphoproliferative assay, cytokine and antibody measurements, and the survival times of mice challenged with the virulent T. gondii RH strain (type I) and the cyst-forming PRU strain (type II). The results showed that pVAX-ROP13 alone or with pVAX/IL-18 induced a high level of specific anti-T. gondii antibodies and specific lymphocyte proliferative responses. Coinjection of pVAX/IL-18 significantly increased the production of gamma interferon (IFN-γ), IL-2, IL-4, and IL-10. Further, challenge experiments showed that coimmunization of pVAX-ROP13 with pVAX/IL-18 significantly (P < 0.05) increased survival time (32.3 ± 2.7 days) compared with pVAX-ROP13 alone (24.9 ± 2.3 days). Immunized mice challenged with T. gondii cysts (strain PRU) had a significant reduction in the number of brain cysts, suggesting that ROP13 could trigger a strong humoral and cellular response against T. gondii cyst infection and that it is a potential vaccine candidate against toxoplasmosis, which provided the foundation for further development of effective vaccines against T. gondii.

Protective effect against toxoplasmosis in mice induced by DNA immunization with gene encoding Toxoplasma gondii ROP18.

Toxoplasma gondii is an obligate intracellular protozoan parasite infecting mammals and birds including humans. Rhoptry protein 18 has been implicated as an important virulence factor. In this study, we constructed a DNA vaccine expressing rhoptry protein 18 (ROP18) of T. gondii, and evaluated the immune response and protective immunity in Kunming mice. The gene sequence encoding ROP18 was inserted into the eukaryotic expression vector pVAX I. Intramuscular immunization of mice with pVAX-ROP18 elicited specific humoral responses and stimulated lymphoproliferation (P<0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P<0.05). By the expression of the CD69, an activation marker of CD4+ and CD8+ T cells, we found that pVAX-ROP18 enhanced the activation of CD4+ and CD8+ T cells in lymphoid in mice. After lethal challenge, the mice immunized with the pVAX-ROP18 showed a significantly increased survival time (27.9±15.1 days) compared with control mice which died within 7 days of challenge (P<0.05). Our results show for the first time, that a ROP18 vaccine construct can enhance the T. gondii-specific CTL. Th1 responses and increased survival suggested that ROP18 is a promising vaccine candidate against infection with T. gondii.