In vitro antiviral activity and preclinical and clinical resistance profile of miravirsen, a novel anti-hepatitis C virus therapeutic targeting the human factor miR-122.

Miravirsen is a ß-D-oxy-locked nucleic acid-modified phosphorothioate antisense oligonucleotide targeting the liver-specific microRNA-122 (miR-122). Miravirsen demonstrated antiviral activity against hepatitis C virus (HCV) genotype 1b replicons with a mean 50% effective concentration (EC50) of 0.67 µM. No cytotoxicity was observed up to the highest concentration tested (>320 µM) in different cell culture models, yielding a therapeutic index of ≥ 297. Combination studies of miravirsen with interferon α2b, ribavirin, and nonnucleoside (VX-222) and nucleoside (2'-methylcytidine) inhibitors of NS5B, NS5A (BMS-790052), or NS3 (telaprevir) indicated additive interactions. Miravirsen demonstrated broad antiviral activity when tested against HCV replicons resistant to NS3, NS5A, and NS5B inhibitors with less than 2-fold reductions in susceptibility. In serial passage studies, an A4C nucleotide change was observed in the HCV 5' untranslated region (UTR) from cells passaged in the presence of up to 20 µM (40-fold the miravirsen EC50 concentration) at day 72 of passage but not at earlier time points (up to 39 days of passage). Likewise, a C3U nucleotide change was observed in the HCV 5'UTR from subjects with viral rebound after the completion of therapy in a miravirsen phase 2 clinical trial. An HCV variant constructed to contain the A4C change was fully susceptible to miravirsen. A C3U HCV variant demonstrated overall reductions in susceptibility to miravirsen but was fully susceptible to all other anti-HCV agents tested. In summary, miravirsen has demonstrated broad antiviral activity and a relatively high genetic barrier to resistance. The identification of nucleotide changes associated with miravirsen resistance should help further elucidate the biology of miR-122 interactions with HCV. (The clinical trial study has been registered at ClinicalTrials.gov under registration no. NCT01200420).

Unexpected renal toxicity associated with SGX523, a small molecule inhibitor of MET.

PURPOSE: SGX523 is an orally bio-available, ATP competitive, small molecule inhibitor of MET, binding the kinase domain active site in a novel mode. Two phase 1, open-label, dose-escalation studies of SGX523 were conducted to evaluate both interrupted and continuous dosing schedules. METHODS: Thirty-six patients per study were planned to be enrolled. The first study explored a 21-day cycle with SGX523 administered on an intermittent schedule at a starting dose of 60 mg PO BID for 14 days followed by 7 days of rest. The second protocol explored a continuous 28-day dosing schedule with SGX523 administered at a starting dose of 20 mg PO BID for 28 days without rest. RESULTS: A total of 10 patients were enrolled, 2 on the intermittent dosing protocol and 8 on the continuous dosing protocol. All 6 patients that received daily doses of ≥ 80 mg developed unexpected renal failure manifested by an early rise of serum blood urea nitrogen and creatinine. Human PK analysis revealed the formation of two insoluble metabolites at levels not seen in the rat or dog preclinical toxicology studies. Subsequent primate toxicology and toxicokinetic evaluation replicated human findings, and histological examination of the monkey kidneys revealed the formation of crystals both within the renal tubules and within giant cell macrophages. CONCLUSION: Two-species toxicology studies of SGX523 did not predict the occurrence of renal toxicity in the human. Subsequent primate toxicology studies suggest the cause of the renal failure seen in humans was a crystal nephropathy secondary to insoluble metabolites. SGX523 is no longer in clinical development.

Granulysin-derived peptides demonstrate antimicrobial and anti-inflammatory effects against Propionibacterium acnes.

Propionibacterium acnes is a key therapeutic target in acne, yet this bacterium has become resistant to standard antibiotic agents. We investigated whether the human antimicrobial protein granulysin is a potential candidate for the treatment of acne. Granulysin and synthetic granulysin-derived peptides possessing a helix-loop-helix motif killed P. acnes in vitro. Modification of a helix-loop-helix peptide, 31-50, by substitution of a tryptophan for the valine at amino acid 44 (peptide 31-50v44w) to increase its interaction with bacterial surfaces also increased its antimicrobial activity. Moreover, when synthesized with D- rather than L-type amino acids, this peptide (D-31-50v44w) became less susceptible to degradation by proteases and more effective in killing P. acnes. Granulysin peptides were bactericidal, demonstrating an advantage over standard bacteriostatic antibiotics in their control of P. acnes. Moreover, peptide D-31-50v44w killed P. acnes in isolated human microcomedone preparations. Importantly, peptides 31-50, 31-50v44w, and D-31-50v44w also have potential anti-inflammatory effects, as demonstrated by suppression of P. acnes-stimulated cytokine release. Taken together, these data suggest that granulysin peptides may be useful as topical therapeutic agents, providing alternatives to current acne therapies.

Gain-of-function somatic cell lines for drug discovery applications generated by homologous recombination.

Gene targeting allows for precise genomic engineering and has been used extensively to generate both loss-of-function and gain-of-function models in mice. Similar manipulation of the genome of somatic cell lines holds high value in basic and applied research, but has been hampered by low recombination frequencies and the subsequent labor-intensive analysis of a large number of cell clones. By combining gene targeting methods with fluorescence-activated cell sorting, gain-of-function cell lines were generated and identified based on a functional readout. To demonstrate the general applicability of this approach to drug discovery, we generated targeted promoter insertion cell lines for two key drug target classes -- the G protein-coupled receptor melanocortin-receptor 4 and the nuclear receptor peroxisome proliferator-activated receptor-gamma. Molecular analysis of the engineered cell clones confirmed the predicted integration of a constitutive promoter into an endogenous allele, and the appropriate pharmacology for these targets validated the use of these gain-of-function cell lines in drug discovery applications, including high-throughput compound screening.

A one-arm homologous recombination approach for developing nuclear receptor assays in somatic cells.

Drug discovery is in need of technologies that enable investigators to develop cell-based assays that accurately reflect the functional consequence of small molecule intervention on biological processes. Here, we describe a strategy that uses both one-arm homologous recombination and the beta-lactamase (BLA) reporter system, a sensitive and robust transcriptional reporter for gene activation. We demonstrate that this powerful approach can be utilized for developing cell-based assays for the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) in HEK293 somatic cells. Specifically, one-arm homologous recombination was used to introduce the GAL4 DNA-binding domain (GAL4DBD) in the GR and MR genomic loci such that a chimeric GAL4DBD-GR (ligand-binding domain) [GAL4DBD-GR(LBD)] and GAL4DBD-MR(LBD) transcript is produced from the strong CMV promoter in HEK293 cells previously stably transfected with the UAS(GAL4)-BLA reporter construct. Dexamethasone- and aldosterone-responding BLA-positive cells were isolated by fluorescence-activated cell sorting, and then further expanded into separate cell lines. The sensitivity and robustness of the resulting GR and MR assays are demonstrated by the fact that the addition of dexamethasone and aldosterone to the two transgenic clonal cell lines for 16 h results in high Z' values (>0.8) and EC(50) values of 1 and 0.3 nM, respectively. These assays illustrate the flexibility of this technology to generate high-performance cellular assays for nuclear receptor targets without the need for target-specific cDNA.

Molecular patterns of nuclear and mitochondrial microsatellite alterations in breast tumors.

The widespread distribution of microsatellite sequences within the human genome has allowed researchers to identify alternative patterns of microsatellite alterations in cancer cells. Among them, aneuploid patterns of nuclear microsatellites, pseudo-diploid microsatellite instability patterns, and also patterns of microsatellite instability within the mitochondrial genome. In this context, while aneuploid patterns of multiple genomic gains and losses had already been described in breast tumors, data on nuclear microsatellite instability still remain controversial and limited data on mitochondrial instability are available. In order to clarify this, we undertook an extensive analysis of nuclear and mitochondrial microsatellite alterations in breast ductal adenocarcinomas, stratified by grades. No instability was detected in any of the 40 dinucleotide microsatellites analysed nor in bat26 and APDelta3 mononucleotide repeats, clearly concluding that microsatellite instability is not a feature of ductal breast tumorigenesis. Instead, microsatellites defined a clear pattern of aneuploid genomic gains and losses among which, losses of BRCA1 at D17S855 and gains of plakoglobin at D17S846 significantly associated to grade III tumors and poor prognosis. On the other hand, mitochondrial instability at the transcription control region was also detected in 10.8% of cases. None of the new mitochondrial variants was found in the normal tissue counterparts, confirming that these new variants arise as sporadic somatic mutations in the tumor cells. Also, no association was found between heteroplasmy in the normal tissue and mitochondrial instability in the tumors. We therefore suggest that these new variants arise in tumors as a consequence of the progressive accumulation of slippage somatic mutations and the intrinsic instability of these microsatellite sequences. Finally, our results also confirm that mitochondrial instability does not associate with nuclear MSI.

Keratin overexpression levels correlate with the extent of spontaneous pancreatic injury.

Mutation of the adult hepatocyte keratins K8 and K18 predisposes to liver disease. In contrast, exocrine pancreas K8 and K18 are dispensable and are co-expressed with limited levels of membrane-proximal K19 and K20. Overexpression of mutant K18 or genetic ablation of K8 in mouse pancreas is well tolerated whereas overexpression of K8 causes spontaneous chronic pancreatitis. To better understand the effect of exocrine pancreatic keratin overexpression, we compared transgenic mice that overexpress K18, K8, or K8/K18, associated with minimal, modest, or large increases in keratin expression, respectively, with nontransgenic wild-type (WT) mice. Overexpression of the type-II keratin K8 up-regulated type-I keratins K18, K19, and K20 and generated K19/K20-containing neocytoplasmic typical or short filaments; however, overexpression of K18 had no effect on K8 levels. K8- and K18-overexpressing pancreata were histologically similar to WT, whereas K8/K18 pancreata displayed age-enhanced vacuolization and atrophy of the exocrine pancreas and exhibited keratin hyperphosphorylation. Zymogen granules in K8/K18 pancreata were 50% smaller and more dispersed than their normal apical concentration but were twice as numerous as in WT controls. Therefore, modest keratin overexpression has minor effects on the exocrine pancreas whereas significant keratin overexpression alters zymogen granule organization and causes aging-associated exocrine atrophy. Keratin absence or mutation is well tolerated after pancreatic but not liver injury, whereas excessive overexpression is toxic to the pancreas but not the liver when induced under basal conditions.

Novel pan-neuronal Cre-transgenic line for conditional ablation of genes in the nervous system.

Tissue-specific gene ablation is accomplished by combining conventional gene targeting approaches with site-specific recombinases such as the Cre/loxP system. Despite the use of a cardiac-specific rat myosin light chain II promoter, our transgenic line (CRE3) had little or no Cre expression in the heart; however, strong Cre activity was detected in the brain as early as gestation day E11.5. This was determined by several methods including crossing our mouse line with a lacZ indicator line (ROSA26). Transgenic Cre, in this mouse line, mediated DNA recombination of loxP-flanked genes selectively in neurons throughout the gray matter of the brain, cerebellum, spinal cord, as well as retina, dorsal, and sympathetic ganglia. Cre protein was also detected by immunohistochemistry exclusively in neurons, but not in other types of cells or tissues. Thus, our transgenic CRE3 mice provide pan-neuronal expression of CRE for carrying out conditional deletion of genes in neurons and their progenitors.