Catalytic in vivo protein knockdown by small-molecule PROTACs.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

Characterization of two subpopulations of the PICM-19 porcine liver stem cell line for use in cell-based extracorporeal liver assistance devices.

Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent PICM-19 pig liver stem cell line and assessed for their potential application in artificial liver devices (ALD). The study included assessments of growth rate and cell density in culture, morphological features, serum protein production, gamma-glutamyltranspeptidase (GGT) activity and hepatocyte detoxification functions, i.e., inducible P450 activity, ammonia clearance, and urea production. The PICM-19H cell line was derived by temperature selection at 33-34 degrees C. After each passage, PICM-19H cells grew to a nearly confluent monolayer of cells of hepatocyte morphology, i.e., cuboidal cells with centrally located nuclei joined by biliary canaliculi. No differentiation and self-organization into multi-cellular bile ductules, as observed in the parental PICM-19 cell line, occurred within the PICM-19H cell monolayers. The PICM-19H cells contained numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles. The cells had a doubling time of 48-72 h and reached a final density of 1.5 x 10(5) cells/cm(2) at approximately10 d post-passage from a 1:6 split ratio. PICM-19H cells displayed inducible P450 activity, cleared ammonia, and produced urea in a glutamine-free medium. The PICM-19B cells were colony-cloned after spontaneous generation from the PICM-19 parental cell line. PICM-19B cells grew as a tightly knit dome-forming monolayer with no visible biliary canaliculi. Their doubling time was 48-72 h with a final cell density of 2.6 x 10(5) cells/cm(2). Ultrastructural analysis of the PICM-19B monolayers showed the roughly cuboidal cells displayed basal-apical polarization and were joined by tight junction-like complexes. Other ultrastructure features were similar to those of PICM-19H cells except that they possessed numerous cell bodies resembling mucus vacuoles. The PICM-19B cells had relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. PICM-19B cells produced markedly less serum proteins than PICM-19H cells. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an ALD.

Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance.

The androgen receptor is a major driver of prostate cancer and inhibition of its transcriptional activity using competitive antagonists, such as enzalutamide remains a frontline therapy for prostate cancer management. However, the majority of patients eventually develop drug resistance. We propose that targeting the androgen receptor for degradation via Proteolysis Targeting Chimeras (PROTACs) will be a better therapeutic strategy for targeting androgen receptor signaling in prostate cancer cells. Here we perform a head-to-head comparison between a currently approved androgen receptor antagonist enzalutamide, and its PROTAC derivative, ARCC-4, across different cellular models of prostate cancer drug resistance. ARCC-4 is a low-nanomolar androgen receptor degrader able to degrade about 95% of cellular androgen receptors. ARCC-4 inhibits prostate tumor cell proliferation, degrades clinically relevant androgen receptor point mutants and unlike enzalutamide, retains antiproliferative effect in a high androgen environment. Thus, ARCC-4 exemplifies how protein degradation can address the drug resistance hurdles of enzalutamide.

Cytochrome P450 expression profile of the PICM-19H pig liver cell line: potential application to rapid liver toxicity assays.

Liver in vitro models are needed to replace animal models for rapid assessment of drug biotransformation and toxicity. The PICM-19 pig liver stem cell line may fulfill this need since these cells have activities associated with xenobiotic phase I and II metabolism lacking in other liver cell lines. The objective of this study was to characterize phase I and II metabolic functions of a PICM-19 derivative cell line, PICM-19H, compared to the tumor-derived human HepG2 C3A cell line and primary cultures of adult porcine hepatocytes. Following exposure of PICM-19H cells to either 3-methylcholanthrene, rifampicin or phenobarbital, the induced activities of cytochrome P450 (CYP450) isozymes CYP-1A, -2, and-3A were assessed. Relative to adult porcine hepatocytes, PICM- 19H cells exhibited 30% and 43%, respectively, of CYP1A and 3A activities, while HepG2 C3A cells exhibited 7% and 0% of those activities. Fluorescent metabolites were extensively conjugated, i.e., 52% and 96% of CYP450-1A and-3A metabolites were released from medium samples following treatment with ß-glucuronidase/arylsulfatase. Rifampicin induction of CYP450 isozyme activities was confirmed by conversion of testosterone to 6ß-OH-, 2α-OH- and 2ß-OH-testosterone, as determined by mass spectrometry. Susceptibility of PICM-19H cells to acetaminophen toxicity was determined; CD50 was calculated to be 14.9±0.9 mM. Toxicity and bioactivation of aflatoxin B1 was determined in 3-methylcholanthrenetreated cultures and untreated controls; CD50 were 1.59 µM and 31 µM, respectively. These results demonstrate the potential use of PICM-19H cells in drug biotransformation and toxicity testing and further support their use in extracorporeal artificial liver device technology.