Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

TSG101 associates with PARP1 and is essential for PARylation and DNA damage-induced NF-κB activation.

In a genome-wide screening for components of the dsDNA-break-induced IKK-NF-κB pathway, we identified scores of regulators, including tumor susceptibility gene TSG101. TSG101 is essential for DNA damage-induced formation of cellular poly(ADP-ribose) (PAR). TSG101 binds to PARP1 and is required for PARP1 activation. This function of TSG101 is independent of its role in the ESCRT-I endosomal sorting complex. In the absence of TSG101, the PAR-dependent formation of a nuclear PARP1-IKKγ signalosome, which triggers IKK activation, is impaired. According to its requirement for PARP1 and NF-κB activation, TSG101-deficient cells are defective in DNA repair and apoptosis protection. Loss of TSG101 results in PARP1 trapping at damage sites and mimics the effect of pharmacological PARP inhibition. We also show that the loss of TSG101 in connection with inactivated tumor suppressors BRCA1/2 in breast cancer cells is lethal. Our results imply TSG101 as a therapeutic target to achieve synthetic lethality in cancer treatment.

Discovery of a Novel Series of Tankyrase Inhibitors by a Hybridization Approach.

A structure-guided hybridization approach using two privileged substructures gave instant access to a new series of tankyrase inhibitors. The identified inhibitor 16 displays high target affinity on tankyrase 1 and 2 with biochemical and cellular IC50 values of 29 nM, 6.3 nM and 19 nM, respectively, and high selectivity toward other poly (ADP-ribose) polymerase enzymes. The identified inhibitor shows a favorable in vitro ADME profile as well as good oral bioavailability in mice, rats, and dogs. Critical for the approach was the utilization of an appropriate linker between 1,2,4-triazole and benzimidazolone moieties, whereby a cyclobutyl linker displayed superior affinity compared to a cyclohexane and phenyl linker.