Inhibitors of poly-ADP-ribose polymerase (PARP) family proteins are currently in clinical trials as cancer therapeutics, yet the specificity of many of these compounds is unknown. Here we evaluated a series of 185 small-molecule inhibitors, including research reagents and compounds being tested clinically, for the ability to bind to the catalytic domains of 13 of the 17 human PARP family members including the tankyrases, TNKS1 and TNKS2. Many of the best-known inhibitors, including TIQ-A, 6(5H)-phenanthridinone, olaparib, ABT-888 and rucaparib, bound to several PARP family members, suggesting that these molecules lack specificity and have promiscuous inhibitory activity. We also determined X-ray crystal structures for five TNKS2 ligand complexes and four PARP14 ligand complexes. In addition to showing that the majority of PARP inhibitors bind multiple targets, these results provide insight into the design of new inhibitors.

Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.

Nine mutants and the wild-type human dopamine D3 receptor were expressed at high levels in BHK and CHO cells using the Semliki Forest virus system and were analysed for receptor binding with several structurally different dopamine D3 ligands. The mutation His349Leu showed a significant decrease in pKi values for raclopride, dopamine and GR218231, but an increase in affinity for GR99841. Thr369Val had an increase in pKi for both GR99841 and 7-OH-DPAT. The receptor modelling based on sequence alignment with bacteriorhodopsin indicated that Thr369 and His349 are located on the inside of the ligand binding pocket and the effect of the mutagenesis was therefore expected. The change in binding affinity for Thr369Val could be due to the location in the transmembrane domain VII close to the aspartate residue in domain III, the postulated counter ion for dopamine.

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In this work molecular modeling was applied to generate homology models of the pore region of the Na(v)1.2 and Na(v)1.8 isoforms of human voltage-gated sodium channels. The models represent the channels in the resting, open, and fast-inactivated states. The transmembrane portions of the channels were based on the equivalent domains of the closed and open conformation potassium channels KcsA and MthK, respectively. The critical selectivity loops were modeled using a structural template identified by a novel 3D-search technique and subsequently merged with the transmembrane portions. The resulting draft models were used to study the differences of tetrodotoxin binding to the tetrodotoxin-sensitive Na(v)1.2 (EC50: 0.012 microM) and -insensitive Na(v)1.8 (EC50: 60 microM) isoforms, respectively. Furthermore, we investigated binding of the local anesthetic tetracaine to Na(v)1.8 (EC50: 12.5 microM) in resting, conducting, and fast-inactivated state. In accordance with experimental mutagenesis studies, computational docking of tetrodotoxin and tetracaine provided (1) a description of site 1 toxin and local anesthetic binding sites in voltage-gated sodium channels. (2) A rationale for site 1 toxin-sensitivity versus -insensitivity in atomic detail involving interactions of the Na(v)1.2 residues F385-I and W943-II. (3) A working hypothesis of interactions between Na(v)1.8 in different conformational states and the local anesthetic tetracaine.