Safe and Efficient Sigma1 Ligand: A Potential Drug Candidate for Multiple Sclerosis.

Multiple Sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease of the central nervous system (CNS). Current management strategies suppress or modulate immune function, all with consequences and known side effects. They demonstrate a high level of success in limiting new relapses. However, the neurodegenerative process still affects both grey and white matter in the central nervous system. The sigma1 (S1R) ligand-regulated chaperone is implicated in many biological processes in various CNS-targeted diseases, acting on neural plasticity, myelination and neuroinflammation. Among the proteins involved in MS, S1R has therefore emerged as a promising new target. Standard and robust methods have been adopted to analyze the adsorption, distribution, metabolism, excretion (ADME) properties, safety pharmacology and toxicology of a previously synthetized simple benzamide-derived compound with nanomolar affinity for S1R, high selectivity, no cytotoxicity and good metabolic stability. The compound was also characterized as an agonist based on well-validated assays prior to in vivo investigations. Interestingly, we found that the oral administration of this compound resulted in an overall significant reduction in clinical progression in an MS experimental model. This effect is mediated through S1R action. Our results further suggest the potential use of this compound in the treatment of MS.

Synthesis and pharmacological evaluation of benzamide derivatives as potent and selective sigma-1 protein ligands.

A series of novel benzamide-derived compounds was designed, synthesized and pharmacologically evaluated. Among all 37 synthesized compounds, two series were developed with the modulation of the nature, the position of atoms or groups on the benzamide scaffold, but also the nature of the amine group separated from the benzamide with 2, 3 or 4 methylene groups. In vitro competition binding assays against sigma proteins (sigma-1 S1R and sigma-2 S2R) revealed that most of them conferred S2R/S1R selectivity toward without cytotoxic effects on SY5Y cells, especially with the first series with compounds 7a-z. Some selected compounds were also evaluated for their agonist and antagonist activities on a panel of 40 receptors. Results showed the importance of the nature and the position with halogeno atom on the benzamide scaffold, the length chain but also the contribution of the hydrophobic part on the amine group. Among them, compounds 7i, w, y with Cl, CN or NO2 groups at the 4-position of the benzamide scaffold showed excellent affinity for S1R (Ki = 1.2-3.6 nM), selectivity for S2R (Ki up to 1400 nM) and high selectivity index (IC50(SY5Y)/Ki(S1R) ratio from 28 000 to 83 000). Futhermore, these compounds presented an excellent safety profile over 40 other receptors. These derivatives will be selected for further biological investigations.

Critical role of charged residues in helix 7 of the ligand binding domain in Hepatocyte Nuclear Factor 4alpha dimerisation and transcriptional activity.

Hepatocyte Nuclear Factor 4alpha (HNF4alpha, NR2A1) is central to hepatocyte and pancreatic beta-cell functions. Along with retinoid X receptor alpha (RXRalpha), HNF4alpha belongs to the nuclear receptor subfamily 2 (NR2), characterised by a conserved arginyl residue and a glutamate residue insert in helix 7 (H7) of the ligand binding domain (LBD). Crystallographic studies indicate that R348 and E352 residues in RXRalpha H7 are involved in charge-driven interactions that improve dimerisation. Consistent with these findings, we showed that removing the charge of the corresponding residues in HNF4alpha H7, R258 and E262, impaired dimerisation in solution. Moreover, our results provide a new concept according to which helices of the HNF4alpha LBD dimerisation interface contribute differently to dimerisation required for DNA binding; unlike H9 and H10, H7 is not involved in DNA binding. Substitutions of E262 decreased the repression of HNF4alpha transcriptional activity by a dominant-negative HNF4alpha mutant, highlighting the importance of this residue for dimerisation in the cell context. The E262 insert is crucial for HNF4alpha function since its deletion abolished HNF4alpha transcriptional activity and coactivator recruitment. The glutamate residue insert and the conserved arginyl residue in H7 most probably represent a signature of the NR2 subfamily of nuclear receptors.

Mutations in hepatocyte nuclear factor 4alpha (HNF4alpha) gene associated with diabetes result in greater loss of HNF4alpha function in pancreatic beta-cells than in nonpancreatic beta-cells and in reduced activation of the apolipoprotein CIII promoter in hepatic cells.

Mutations in the HNF4alpha gene have been correlated with maturity-onset diabetes of the young, which is characterized mainly by pancreatic beta-cell dysfunction and is also associated with mild liver abnormalities. HNF4alpha D126Y and D126H mutations were found in a patient with early-onset type 2 diabetes, and the R324H mutation was found in a common type 2 diabetic nephropathic patient. We investigated whether these mutations, which have not yet been functionally characterized, impair HNF4alpha function in three cell models: HEK 293 embryonal kidney cells, HepG2 hepatoma cells, and betaTC3 pancreatic beta-cells. The R324H mutation had no effect on HNF4alpha function with either the HNF1alpha and L-type pyruvate kinase (LPK) promoters, but the D126Y and D126H mutations impaired HNF4alpha transcriptional activities in all tested cell lines. These impairments by D126Y and D126H mutations, which are located in the T box, are not due to a loss of dimerization but to a loss of DNA binding. Interestingly, the strongest functional consequences of these mutations were observed on the HNF1alpha promoter in betaTC3 cells. Given the key role of the transcription factor HNF1alpha in pancreatic beta-cell function, it can be inferred that impairment of HNF4alpha function by these mutations affects metabolic pathways in pancreatic beta-cells and contributes to development of diabetes. Moreover, the HNF4alpha-mediated activation of the apolipoprotein CIII promoter in HepG2 cells was significantly impaired by D126Y and D126H mutations. These results support clinical findings that liver function can also be impaired in diabetic patients having HNF4alpha mutations.

The G115S mutation associated with maturity-onset diabetes of the young impairs hepatocyte nuclear factor 4alpha activities and introduces a PKA phosphorylation site in its DNA-binding domain.

HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function.

A charter for biomedical research ethics in a progressive, caring society.

BACKGROUND: Given that advances in research continuously raise new ethical issues, a multidisciplinary working group of investigators involved in biomedical research has gathered to discuss and compare ethical viewpoints in their daily practice. METHODS: The working group has drafted a Charter for Ethics in Biomedical Research that encompasses all the steps in the research process, i.e. from the initial idea to analysis and publication of the results. RESULTS: Based on key principles for ethically responsible research, the Charter may serve as a tool for performing research, discussing research issues and training researchers. CONCLUSIONS: The Charter should stimulate researchers to think about their responsibility for research in a progressive, caring society.

Susceptibility to experimental autoimmune encephalomyelitis is associated with altered B-cell subsets distribution and decreased serum BAFF levels.

B cells possess the ability to regulate either pathogenic or protective events in several autoimmune diseases such as multiple sclerosis (MS) and its experimental model, experimental autoimmune encephalomyelitis (EAE). Given the extensive use of B-cell-targeting treatments, it appears crucial to more precisely define the dual role of B cells in the progression of the disease. In the present study, we explored the impact of EAE induction on the distribution of potential regulatory B-cell subsets (CD5(+) B1a, marginal zone and transitional 2 B cells) over critical time points in the relapsing-remitting EAE model, SJL/J (H2s). The same approach was carried out in B10.S mice that are resistant to EAE induction, (H2s). The comparative data obtained from these experiments showed that the homeostasis of the regulatory B-cell subsets is altered during the EAE preclinical and acute phases. These observations were associated with a distortion of the BAFF response. All these data suggest the existence of a close relationship between B-cell homeostasis, BAFF response and the susceptibility to develop EAE.