The deubiquitylase USP9X controls ribosomal stalling.

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases, respectively, through ubiquitylation of the eS10 and uS10 subunits of the ribosome. We have developed a specific small-molecule inhibitor of the deubiquitylase USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome-associated protein stability but also reveals a loss of Makorin 2 and ZNF598. We show that USP9X interacts with both these ubiquitin E3 ligases, regulating their abundance through the control of protein stability. In the absence of USP9X or following chemical inhibition of its catalytic activity, levels of Makorins and ZNF598 are diminished, and the ribosomal quality control pathway is impaired.

Optimization of benzodiazepinones as selective inhibitors of the X-linked inhibitor of apoptosis protein (XIAP) second baculovirus IAP repeat (BIR2) domain.

The IAPs are key regulators of the apoptotic pathways and are commonly overexpressed in many cancer cells. IAPs contain one to three BIR domains that are crucial for their inhibitory function. The pro-survival properties of XIAP come from binding of the BIR domains to the pro-apoptotic caspases. The BIR3 domain of XIAP binds and inhibits caspase 9, while the BIR2 domain binds and inhibits the terminal caspases 3 and 7. While XIAP BIR3 inhibitors have previously been reported, they also inhibit cIAP1/2 and promote the release of TNFα, potentially limiting their therapeutic utility. This paper will focus on the optimization of selective XIAP BIR2 inhibitors leading to the discovery of highly potent benzodiazepinone 36 (IC50 = 45 nM), which has high levels of selectivity over XIAP BIR3 and cIAP1 BIR2/3 and shows efficacy in a xenograft pharmacodynamic model monitoring caspase activity while not promoting the release of TNFα in vitro.

Benzazepinones and benzoxazepinones as antagonists of inhibitor of apoptosis proteins (IAPs) selective for the second baculovirus IAP repeat (BIR2) domain.

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

Discovery of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors.

Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1µM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.

Potent, selective MCH-1 receptor antagonists.

This paper describes the lead optimization of a new series of potent, selective, orally bioavailable, brain-penetrant MCH-1 receptor antagonists. A major focus of the work was to achieve a selectivity profile appropriate for in vivo efficacy studies and safety.

Identification of phenyl-pyridine-2-carboxylic acid derivatives as novel cell cycle inhibitors with increased selectivity for cancer cells.

Ro 41-4439, a phenyl-pyridine-2-carboxylic acid derivative, was identified by a cell-based screening approach that exploits the differences between normal and cancer cells in their sensitivity to cytotoxic agents. This compound showed low micromolar antiproliferative activity and cytotoxicity against a broad panel of human cancer cell lines in vitro, and over 10-fold selectivity to cancer cells when tested in parallel with a panel of proliferating normal human cells. Cytotoxicity of Ro 41-4439 is due to arrest of cell cycle progression in mitosis followed by induction of apoptosis. Four-week treatment of nude mice bearing established mammary tumor xenografts (MDA-MB-435) with well-tolerated doses of the compound showed 73% inhibition of tumor growth. Limited exploration of structure-activity relationships involving side chain length, and aryl and pyridine rings allowed for the identification of more potent analogs.