Peptide Drug Design Using Alchemical Free Energy Calculation: An Application and Validation on Agonists of Ghrelin Receptor.

With recent large-scale applications and validations, the relative binding free energy (RBFE) calculated using alchemical free energy methods has been proven to be an accurate measure to probe the binding of small-molecule drug candidates. On the other hand, given the flexibility of peptides, it is of great interest to find out whether sufficient sampling could be achieved within the typical time scale of such calculation, and a similar level of accuracy could be reached for peptide drugs. However, the systematic evaluation of such calculations on protein-peptide systems has been less reported. Most reported studies of peptides were restricted to a limited number of data points or lacking experimental support. To demonstrate the applicability of the alchemical free energy method for protein-peptide systems in a typical real-world drug discovery project, we report an application of the thermodynamic integration (TI) method to the RBFE calculation of ghrelin receptor and its peptide agonists. Along with the calculation, the synthesis and in vitro EC50 activity of relamorelin and 17 new peptide derivatives were also reported. A cost-effective criterion to determine the data collection time was proposed for peptides in the TI simulation. The average of three TI repeats yielded a mean absolute error of 0.98 kcal/mol and Pearson's correlation coefficient (R) of 0.77 against the experimental free energy derived from the in vitro EC50 activity, showing good repeatability of the proposed method and a slightly better agreement than the results obtained from the arbitrary time frames up to 20 ns. Although it is limited by having one target and a deduced binding pose, we hope that this study can add some insights into alchemical free energy calculation of protein-peptide systems, providing theoretical assistance to the development of peptide drugs.

Pasteurella multocida Pm0442 Affects Virulence Gene Expression and Targets TLR2 to Induce Inflammatory Responses.

Pasteurella multocida is an important pathogenic bacterium of domestic animals. However, the mechanisms of infection are still poorly understood. Here, we found that Pm0442 was dramatically up-regulated in infected mice among 67 predicted lipoproteins of P. multocida serotype A CQ2 strain (PmCQ2). To explore the role of Pm0442 in virulence and the potential of the mutant as a vaccine, Pm0442 mutant of PmCQ2 was successfully constructed. Then, the virulence characteristics, immune/inflammatory responses, and the survival rates of challenged mice were determined. As a result, it was found that the Pm0442 deletion of PmCQ2 significantly decreased bacterial loads and inflammatory responses of lung tissue in mice, resulting in improved survival. Mechanically, Pm0442 affects PmCQ2 capsular and lipopolysaccharide (LPS) synthesis and iron utilization-related genes expression affecting adhesion and phagocytosis. Furthermore, PM0442 bound directly to Toll-like receptor 2 (TLR2) to mediate the secretion of pro-inflammatory cytokine (IL-1ß, TNF-α, IL-6, and IL-12p40) in macrophages via activation of the NF-κB, ERK1/2 and p38 signaling pathways. Notably, PmCQ2Δ0442 could provide 70-80% protection to mice challenged with 3.08 × 107 CFU of PmCQ2. Our findings demonstrate that Pm0442 is a virulence-related gene of PmCQ2, which provides new guidance for the prevention and control of Pasteurellosis.

Glycyrrhizin Suppresses the Growth of Human NSCLC Cell Line HCC827 by Downregulating HMGB1 Level.

Lung cancer has very high mortality and glycyrrhizin was found to significantly inhibit the growth of lung cancer cells in vitro and tissues in mice. However, the detailed inhibitory role of glycyrrhizin in the growth of lung cancer is still unclear. In this study, we first found that glycyrrhizin inhibited the growth of lung tumor in PDX mice. And high level of HMGB1 promoted the migration and invasion of lung cancer cells, which was suppressed by glycyrrhizin. Moreover, glycyrrhizin reduced the activity of JAK/STAT signaling pathway, which is the upstream regulator of HMGB1. Therefore, this study revealed a potential mechanism by which glycyrrhizin can inhibit the progression of lung cancer.

High Mobility Group Box Protein 1 Serves as a Potential Prognostic Marker of Lung Cancer and Promotes Its Invasion and Metastasis by Matrix Metalloproteinase-2 in a Nuclear Factor-κB-Dependent Manner.

Several studies have reported a significant role of high mobility group box protein 1 (HMGB1) in lung cancer. Nevertheless, there is a lack of knowledge regarding the expression of HMGB1 and its correlation with the clinicopathological features of lung cancer. In addition, the potential molecular mechanisms underlying the role of HMGB1 in lung cancer are still unknown. We therefore investigated the clinicopathological and prognostic significance as well as the potential role of HMGB1 in the development and progression of lung cancer. HMGB1 expression in the tumor tissues of the cohort correlated with clinicopathological features. Moreover, lung cell migration and invasion were significantly increased after treatment with HMGB1. The matrix metalloproteinase-2 (MMP-2) expression and activity were upregulated after treatment with HMGB1, while the upregulated expression of MMP-2 stimulated by HMGB1 in lung cancer cells was significantly reduced with the blockage of si-p65. These results indicated that HMGB1 expression was significantly associated with lung cancer progression. We also showed that HMGB1 promoted lung cancer invasion and metastasis by upregulating the expression and activity of MMP-2 in an NF-κB-dependent manner. Taken together, these data suggested that HMGB1 may be a potential prognosis and therapeutic marker for lung cancer.

The immunological function of GABAergic system.

As a well-known inhibitory neurotransmitter in the central nervous system, gamma-aminobutyric acid also has critical roles in immune system. Immune cells (e.g., lymphocytes, macrophages) express the components of GABAergic system, including GABA receptors, GABA transporters, and GABA metabolic enzymes. The functions of immune cells are highly impacted on GABA signaling. GABAergic components negatively regulate the immune responses, particularly the T cell-mediated immunity, via their effects on production of pro-inflammatory cytokines and activation of signal pathways, like mitogen-activated protein kinase and nuclear factor-kappaB pathways. These results may indicate that GABAergic components provide a new therapeutic approach for inflammatory and autoimmune diseases, such as experimental autoimmune encephalomyelitis, multiple sclerosis, and inflammatory bowel diseases.

Transcriptomic Analysis on Responses of Murine Lungs to Pasteurella multocida Infection.

Pasteurella multocida infection in cattle causes serious epidemic diseases and leads to great economic losses in livestock industry; however, little is known about the interaction between host and P. multocida in the lungs. To explore a fully insight into the host responses in the lungs during P. multocida infection, a mouse model of Pasteurella pneumonia was established by intraperitoneal infection, and then transcriptomic analysis of infected lungs was performed. P. multocida localized and grew in murine lungs, and induced inflammation in the lungs, as well as mice death. With transcriptomic analysis, approximately 107 clean reads were acquired. 4236 differently expressed genes (DEGs) were detected during P. multocida infection, of which 1924 DEGs were up-regulated. By gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichments, 5,303 GO enrichments and 116 KEGG pathways were significantly enriched in the context of P. multocida infection. Interestingly, genes related to immune responses, such as pattern recognition receptors (PRRs), chemokines and inflammatory cytokines, were significantly up-regulated, suggesting the key roles of these genes in P. multocida infection. Transcriptomic data showed that IFN-γ/IL-17-related genes were increased, which were validated by qRT-PCR, ELISA, and immunoblotting. Our study characterized the transcriptomic profile of the lungs in mice upon Pasteurella infection, and our findings could provide valuable information with respect to better understanding the responses in mice during P. multocida infection.

[Lumbar interbody fusion impacted bone grafts combined with regrafting in situ with spinous process and vertebral plate complex and pedicle screw fixation for lumbar degenerative instability].

OBJECTIVE: To evaluate the effectiveness of lumbar interbody fusion impacted bone grafts combined with regrafting in situ with spinous process and vertebral plate complex and pedicle screw fixation for lumbar degenerative instability. METHODS: Between January 1998 and October 2010, 48 patients with lumbar degenerative instability were treated by posterior decompression, lumbar interbody fusion impacted bone grafts combined with regrafting in situ with spinous process and vertebral plate complex and pedicle screw fixation. There were 26 males and 22 females, aged 52-76 years (mean, 62.4 years). The disease duration was 7 months to 25 years (mean, 6.5 years). One segmental instability was located at L(3, 4) in 1 case, at L(4, 5) in 10 cases, and at L5, S1 in 11 cases; multi-segmental instability was located at L(3, 4), L(4,5), and L5, S1 in 5 cases, at L(2,3) and L(3,4) in 2 cases, at L(3, 4) and L(4, 5) in 10 cases, and at L(4, 5) and L(5), S1 in 9 cases. Of 48 patients, 32 complicated by lumbar disc herniation, 46 by lumbar spinal stenosis, and 16 by degenerative scoliosis. The clinical results were evaluated by the Japanese Orthopaedic Association (JOA) score, recovery rate, disc height, and lumbar lordosis angles. RESULTS: The incisions obtained healing by first intention after operation. No nerve injury, rod or screw breakage, and infection occurred during and after operation. All 48 patients were followed up 1 to 6 years. The fusion time was 12-18 weeks (mean, 16.2 weeks). Vertebra slipping or degenerative scoliosis was corrected, and spinal column series became normal. At preoperation, 6 months after operation, and last follow-up, the disc heights were (5.2 +/- 2.3), (11.9 +/- 2.0), and (11.6 +/- 2.1) mm, respectively; the JOA scores were 3.2 +/- 2.1, 12.8 +/- 1.6, and 13.6 +/- 1.2, respectively; and the lumbar lordosis angles were (-20.5 +/- 10.5), (30.5 +/- 8.5), and (31.2 +/- 5.6) degrees, respectively. The JOA scores, disc heights, and lumbar lordosis angles were significantly improved at 6 months after operation and last follow-up when compared with preoperative ones (P < 0.05), but no significant difference was found between 6 months after operation and last follow-up (P > 0.05). The recovery rate of JOA was excellent in 36 cases, good in 10 cases, and fair in 2 cases at 6 months after operation, with an excellent and good rate of 95.8%. CONCLUSION: Lumbar interbody fusion impacted bone grafts combined with regrafting in situ with spinous process and vertebral plate complex and pedicle screw fixation for lumbar degenerative instability can restore and maintain the intervertebral disc height effectively with high fusion rate. It is a plasty close to anatomic reconstruction.