A leap into the chemical space of protein-protein interaction inhibitors.

Protein-protein interactions (PPI) are involved in vital cellular processes and are therefore associated to a growing number of diseases. But working with them as therapeutic targets comes with some major hurdles that require substantial mutations from our way to design drugs on historical targets such as enzymes and G-Protein Coupled Receptor (GPCR). Among the numerous ways we could improve our methodologies to maximize the potential of developing new chemical entities on PPI targets, is the fundamental question of what type of compounds should we use to identify the first hits and among which chemical space should we navigate to optimize them to the drug candidate stage. In this review article, we cover different aspects on PPI but with the aim to gain some insights into the specific nature of the chemical space of PPI inhibitors. We describe the work of different groups to highlight such properties and discuss their respective approach. We finally discuss a case study in which we describe the properties of a set of 115 PPI inhibitors that we compare to a reference set of 1730 enzyme inhibitors. This case study highlights interesting properties such as the unfortunate price that still needs to be paid by PPI inhibitors in terms of molecular weight, hydrophobicity, and aromaticity in order to reach a critical level of activity. But it also shows that not all PPI targets are equivalent, and that some PPI targets can demonstrate a better druggability by illustrating the better drug likeness of their associated inhibitors.

AMMOS software: method and application.

Recent advances in computational sciences enabled extensive use of in silico methods in projects at the interface between chemistry and biology. Among them virtual ligand screening, a modern set of approaches, facilitates hit identification and lead optimization in drug discovery programs. Most of these approaches require the preparation of the libraries containing small organic molecules to be screened or a refinement of the virtual screening results. Here we present an overview of the open source AMMOS software, which is a platform performing an automatic procedure that allows for a structural generation and optimization of drug-like molecules in compound collections, as well as a structural refinement of protein-ligand complexes to assist in silico screening exercises.

Identification of human cancer-related genes by naturally occurring Hepatitis B Virus DNA tagging.

Proviral tagging has been used in animals as a powerful tool for cancer genetics. We show that a similar approach is possible in patients with hepatocellular carcinoma (HCC) infected by Hepatitis B Virus (HBV), a human pararetrovirus which may act by insertional mutagenesis. In this work, the HBV genome is used as a probe to identify cancer-related genes. By using HBV-Alu-PCR, we obtained 21 HBV/cellular DNA junctions from 18 different patients. In six of 21, we found the HBV DNA integrated into a cellular gene: (1) Sarco/Endoplasmic Reticulum Calcium ATPase1 Gene; (2) Thyroid Hormone Receptor Associated Protein 150 alpha Gene; (3) Human Telomerase Reverse Transcriptase Gene; (4) Minichromosome Maintenance Protein (MCM)-Related Gene; (5) FR7, a new gene expressed in human liver and cancer tissues; and (6) Nuclear Matrix Protein p84 Gene. Seven junctions contained unique cellular sequences. In the remaining eight, the HBV DNA was next to repetitive sequences, five of them of LINE1 type. The cellular genes targeted by HBV are key regulators of cell proliferation and viability. Our results show that studies on HBV-related HCCs allow to identify cellular genes involved in cancer. We therefore propose this approach as a valuable tool for functional cancer genomic studies in humans.

SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Significance of repeatedly normal aminotransferase activities in HCV-infected patients.

The significance of repeatedly normal serum aminotransferase activities in antihepatitis C virus (anti-HCV)-positive patients is not clear. To address this issue, the authors analyzed clinical, virologic, histopathologic, and biological characteristics of such subjects. Among their active file of 1,200 anti-HCV-positive immunocompetent patients, they identified 36 subjects (3%) with repeatedly normal aminotransferase activities, as defined by at least four normal values of aminotransferase over a minimum period of 6 months without any abnormal value (mean of this period, 31 +/- 21 months). The 36 patients included 11 men and 25 women with a mean age of 45 +/- 15 years. Twenty-three of these 36 subjects (64%) had detectable HCV viremia by polymerase chain reaction. Their genotype distribution was as follows: genotype 1a or 1b, 57%; genotype 2, 26%; and genotype 3, 17%. Of the HCV ribonucleic acid (RNA)-positive and HCV RNA-negative subjects, 17 and 5 had a liver biopsy respectively. In the former, the mean Knodell score was 5.6 +/- 3.5 (range, 1 to 14), and was < 5 in 9 patients (53%) and > or = 5 in 8 (47%), including extensive fibrosis (n = 2) or cirrhosis (n = 2). In the HCV RNA-negative subjects, one patient had a Knodell score > or = 5. Comparing the 23 immunocompetent viremic subjects with repeatedly normal serum aminotransferase activities with our group (n = 564) of immunocompetent viremic patients with abnormal aminotransferase activities, there was a significant predominance of women (70% versus 44%, p < 0.05) and of genotype 2 in the former (26% versus 7%, p < 0.05), but no differences according to quantitative viremia, alcohol consumption, or distribution of risk factor were observed. Most of viremic HCV-infected patients with long-term and repeatedly normal aminotransferase values have indeed chronic active hepatitis, including extensive fibrosis or cirrhosis in as many as 20% of patients. This emphasizes the need for serum HCV RNA determination in anti-HCV-positive patients with normal aminotransferase activities. In these patients liver biopsy may be necessary and should be discussed.

Epidemiological and virological analysis of couples infected with hepatitis C virus.

BACKGROUND: If transmission of hepatitis C virus (HCV) infection through parenteral exposure is well documented, sexual transmission of HCV is still debated. AIMS: To perform extensive epidemiological and virological analysis in 24 couples in which each spouse was anti-HCV positive in order to delineate more precisely potential sexual transmission of HCV. PATIENTS: Twenty four couples in which each partner was anti-HCV positive. These 48 spouses were recruited in a liver unit by regular screening of spouses of index patients. METHODS: All 48 spouses completed an epidemiological questionnaire on risk factors for HCV. Qualitative detection of serum HCV RNA and determination of HCV type by genotyping and serotyping were performed. Sequence analysis of HCV strains by phylogenetic analysis was carried out in seven couples with concordant genotypes. RESULTS: The mean (SD) partnership duration was 12 (10) years. Serum HCV RNA was detected in both partners in 18 of the couples (75%) and in only one partner in six of the couples (25%). HCV typing showed concordant genotypes in 12 couples (50%), discordant genotypes in seven (29%), and in the other five couples (21%) only one spouse could be genotyped. Of the 48 spouses, 33 had a major risk factor for HCV transmission such as transfusion (n = 6) and intravenous drug use (n = 27). Eleven of the 12 couples infected with the same HCV genotype had at least one parenteral risk factor for viral transmission in both spouses. Whatever the genotype concordance, in most couples (75%), both spouses showed parenteral risk factors for viral transmission. Sequence analysis of HCV strains was possible in seven of 12 couples with identical genotype and showed different and identical isolates in four and three couples respectively. CONCLUSION: The study emphasises the risk of overestimating the importance of a very low sexual HCV transmission risk as against other, mainly parenteral, risk factors.

Permissiveness of human biliary epithelial cells to infection by hepatitis C virus.

The cellular tropism of hepatitis C virus (HCV) is an important but much debated issue. Permissivity to HCV of biliary cells has never been demonstrated. In this context, we used gallbladder epithelial cells (GBEC) as a model of the more proximal biliary epithelium. These cells were isolated from HCV-positive and -negative individuals and cultured for up to 40 days. Biliary cells from HCV-negative subjects were infected in vitro with various inocula. The retention of GBEC functional characteristics was assessed by the expression of cystic fibrosis transmembrane conductance regulator (CFTR). All 12 GBEC tested from HCV-negative patients were successfully infected by HCV. This was assessed by: 1) the detection of HCV-RNA positive and negative strands; 2) the detection of the viral capsid by immunofluorescence; and 3) the combination of single-strand conformation polymorphism (SSCP) and HVR1 sequence analysis demonstrating the distinct majoritary HCV genomes in serum and in GBEC. The level of HCV RNA in cell extracts and supernatants was low, but HCV infection was highly reproducible. Our results expand those showing the cellular tropism of HCV, and demonstrate the sensitivity of biliary cells to HCV infection. This might have an important impact in terms of pathogenesis and pathological features of HCV infection. In addition, given the easy access to these cells and the high reproducibility of in vitro infection, they should constitute an important tool for studies aimed at analyzing the issue of HCV penetration and neutralizing antibodies.

Impact of HBV, HCV and GBV-C/HGV on hepatocellular carcinomas in Europe: results of a European concerted action.

BACKGROUND/AIMS: To investigate the impact of hepatitis B (HBV) and C (HCV) infections on hepatocellular carcinoma (HCC) in Europe. METHODS: Five hundred and three patients with HCC, from six liver centers, were included. All 503 sera and 80 liver samples were tested for HBV DNA and HCV RNA by polymerase chain reaction. GBV-C/HGV RNA was also tested in 57 sera. RESULTS: HBsAg and anti-HCV were detected in 19% and 40.1% of the patients, respectively. Serum and liver HBV DNA were detected in 82% and 91% of the HBsAg positive subjects. HBV DNA was also detected in the serum and liver of 33% and 47% of HBsAg negative patients. In this group, serum HBV DNA was more prevalent in anti-HBs and/or anti-HBc patients (47.9%), compared to those without any HBV marker (25.1%). HCV RNA was detected in 89% and 7% of anti-HCV positive and negative cases, respectively, HCV 1b being the most prevalent genotype (80%). Coinfection with HBV and HCV was shown in 20.4% of patients, while only 29% had neither HBV nor HCV GBV-C/HGV RNA was detected in only 4/57 sera. CONCLUSIONS: This study offers the first large analysis of HCC in Europe, based on both serology and molecular tests. It demonstrates the major impact of HBV and HCV, but not of GBV-C/HGV, in liver carcinogenesis in Northern as well as Southern Europe. It also stresses the need to use viral genome detection in epidemiological studies when serological tests are negative.