Comparative pharmacokinetic properties and antitumor activity of the marine HDACi Largazole and Largazole peptide isostere.

PURPOSE: Largazole is a potent class I-selective HDACi natural product isolated from the marine cyanobacteria Symploca sp. The purpose of this study was to test synthetic analogs of Largazole to identify potential scaffold structural modifications that would improve the drug-like properties of this clinically relevant natural product. METHODS: The impact of Largazole scaffold replacements on in vitro growth inhibition, cell cycle arrest, induction of apoptosis, pharmacokinetic properties, and in vivo activity using a xenograft model was investigated. RESULTS: In vitro studies in colon, lung, and pancreatic cancer cell lines showed that pyridyl-substituted Largazole analogs had low-nanomolar/high-picomolar antiproliferative activity, and induced apoptosis and cell cycle arrest at concentrations equivalent to or lower than the parent compound Largazole. Using IV bolus delivery at 5 mg/kg, two compartmental pharmacokinetic modeling on the peptide isostere analog of Largazole indicated improved pharmacokinetic parameters. In an A549 non-small cell lung carcinoma xenograft model using a dosage of 5 mg/kg administered intraperitoneally every other day, Largazole, Largazole thiol, and Largazole peptide isostere demonstrated tumor growth inhibition (TGI%) of 32, 44, and 66%, respectively. Largazole peptide isostere treatment was statistically superior to control (p = 0.002) and to Largazole (p = 0.006). Surprisingly, tumor growth inhibition was not observed with the potent pyridyl-based analogs. CONCLUSIONS: These results establish that replacing the depsipeptide linkage in Largazole with an amide may impart pharmacokinetic and therapeutic advantage and that alternative prodrug forms of Largazole are feasible.

Immunization with a synthetic peptide vaccine fails to protect mule deer (Odocoileus hemionus) from chronic wasting disease.

Chronic wasting disease (CWD) adversely affects both wild and captive cervid populations. A vaccine to prevent CWD would be a highly desirable tool to aid in disease management. To this end, we tested in mule deer a combination of CWD vaccines consisting of cervid prion peptide sequences 168-VDQYNNQNTFVHDC-182 and 145-NDYEDRYYRENMYRYPNQ-164 that had previously been shown to delay onset of clinical disease and increase survival in a mouse-adapted scrapie model. Thirteen captive mule deer (Odocoileus hemionus) were divided into vaccine (n=7) and control groups (n=6), and given prime and boost vaccinations intramuscularly 5 wk apart. Eight weeks postprime (3 wk postboost), all animals were challenged via natural exposure to an environment contaminated with infective CWD prions. Deer were monitored intermittently for prion infection by rectal and tonsil biopsies beginning 275 days postchallenge. All vaccinates responded to both peptide conjugates present in the combination vaccine as measured by enzyme-linked immunosorbent assay. However, all deer eventually became infected regardless of vaccine status.

Prion remains infectious after passage through digestive system of American crows (Corvus brachyrhynchos).

Avian scavengers, such as American crows (Corvus brachyrhynchos), have potential to translocate infectious agents (prions) of transmissible spongiform encephalopathy (TSE) diseases including chronic wasting disease, scrapie, and bovine spongiform encephalopathy. We inoculated mice with fecal extracts obtained from 20 American crows that were force-fed material infected with RML-strain scrapie prions. These mice all evinced severe neurological dysfunction 196-231 d postinoculation (x =198; 95% CI: 210-216) and tested positive for prion disease. Our results suggest a large proportion of crows that consume prion-positive tissue are capable of passing infectious prions in their feces (ˆp=1.0; 95% CI: 0.8-1.0). Therefore, this common, migratory North American scavenger could play a role in the geographic spread of TSE diseases.

Feasibility of infectious prion digestion using mild conditions and commercial subtilisin.

Two serine protease enzymes, subtilisin 309 and subtilisin 309-v, were used to digest brain homogenates containing high levels of prion infectivity using mildly alkaline conditions to investigate prion decontamination methods. To establish that PrP(res) infectivity was eliminated, we utilized the Rocky Mountain Laboratory (RML) mouse-adapted scrapie model system for bioassay. Only one digestion condition (subtilisin 309 at 138mAU/ml, 55 degrees C and 14h digestion time pH 7.9) was considered to be highly relevant statistically (P<0.001) compared to control, with 52% of challenged mice surviving until the end of the study period. In contrast, treatment of PrP(res) by autoclaving at 134 degrees C or treatment with hypochlorite at a concentration of 20,000 ppm completely protected mice from prionosis. Further, in vitro assays suggest that potential proteolytic based PrP(res) decontamination methods must use high enzyme concentration, pH values >9.0, and elevated temperatures to be a safely efficacious, thereby limiting applicability on delicate surgical instruments and use in the environment.

Reconstitution of nucleosome positioning, remodeling, histone acetylation, and transcriptional activation on the PHO5 promoter.

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of histone-modifying or nucleosome-remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP-dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl-CoA in addition to these other activities and cofactors. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.