A paper from the laboratory of Prof. John Blenis describes the development of a high throughput screening platform for identifying inhibitors of mTORC1 signaling.

The mTOR signaling pathway plays a causative role in multiple human diseases ranging from cancer and the tumor syndromes TSC and LAM to metabolic disorders including diabetes and obesity. A recent paper describes the development of a new cell based assay designed for high throughput screening small molecule and siRNAs that modulate the mTORC1 signaling pathway (Hoffman GR, Moerke NJ, Hsia M, Shamu CE, Blenis J.Assay Drug Dev Technol. 2010). The assay relies on immunofluorescence based detection of the rpS6-phosphorylation downstream of mTORC1 activation. The fluorescence signal is quantified using the “In Cell Western” (ICW) technique on the LI-COR Aerius infrared imaging system. An important feature of the assay development described in the manuscript is the novel application of the lysine reactive probe Alexa-680 succinamidyl ester for the quantitative measurement of cell number in formaldehyde fixed cells. The ICW approach has a number of unique advantages that make this an attractive platform for screening the mTORC1 pathway. In particular, the ICW method is significantly higher throughput than traditional microscopy based screening methods used for quantitative immunofluorescence based screens. The paper presents the results of pilot screens using the ICW technique to screen both small molecule and siRNA libraries. Importantly, the unbiased small molecule screen identified known small molecule inhibitors of mTORC1 signaling while the siRNA screen identified genes with well characterized functions in the mTORC1 pathway. The characterization of novel genes identified in the screening effort is part of ongoing efforts in the Blenis laboratory.

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A paper from the Rapoport lab reports the X-ray structure of a bacterial homolog of the vitamin K epoxide reductase (VKOR), an enzyme that is crucial for blood coagulation (collaboration with the group of J. Beckwith, HMS, Department of Microbiology).

Several blood coagulation factors are g-carboxylated at select glutamic acids, a modification that is required for their Ca²⁺-dependent activation at sites of injury. The g-carboxylase is an endoplasmic reticulum (ER) enzyme, which requires vitamin K hydroquinone as a cofactor; during the reaction the hydroquinone is oxidized to an epoxide. VKOR is necessary to regenerate the hydroquinone; it reduces the epoxide in two steps, first to the quinone and then to the hydroquinone. The reducing equivalents ultimately come from cysteines in newly synthesized proteins, which are oxidized to disulfides. VKOR is the target of warfarin, the most commonly used oral anticoagulant.

Several bacteria have homologs of VKOR, which cooperate with a thioredoxin (Trx)-like redox partner in disulfide bridge formation of newly synthesized proteins in the periplasm (results of the Beckwith group). The X-ray structure was determined for a bacterial VKOR homolog from Synechococcus, which is naturally fused to its Trx-like redox partner. The structure corresponds to an arrested state of electron transfer in which a cysteine from the Trx-domain is disulfide bonded with a cysteine of VKOR. The core of VKOR is a four-helix bundle that surrounds the quinone, a feature that is generally found in disulfide-generating enzymes. The structure indicates how the electrons flow from cysteines in newly synthesized proteins, through the Trx-domain, all the way to the quinone, a mechanism that was confirmed by the in vitro reconstitution of vitamin K-dependent disulfide bridge formation. Many mutations that cause warfarin resistance in mammals map to the region surrounding the quinone, indicating that warfarin inhibits VKOR by replacing vitamin K.

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