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Master Regulator proteins, controlling the transcriptional identity of infected cells, can serve as drug targets for inhibiting virus replication, here demonstrated in a SARSCoV-2 infection model.

Pathological activation of the thyroid stimulating hormone receptor (TSHR) is caused by thyroid stimulating antibodies in patients with Graves’ disease or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study we describe the identification and in-vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH inhibition bioassay. TSHR expressing CHO cells which also expressed a luciferase tagged CRE response element were optimized using bovine TSH as the activator in a 384 well plate format which had a Z score of 0.3-0.6. Using this HTS assay we screened a diverse library of ~80,000 compounds at a final concentration of 16.7μM. The selection criteria for a positive hit were based on a mean signal threshold of 50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase – confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC50 of 12.3µM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In-silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by Graves’ disease patient sera and monoclonal stimulating TSHR antibodies.In conclusion, we report the identification of a novel small molecule TSHR inhibitor which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in Graves’ disease.

Our ability to discover effective drug combinations is limited, in part by insufficient understanding of how the transcriptional response of two monotherapies results in that of their combination. We analyzed matched time course RNAseq profiling of cells treated with single drugs and their combinations and found that the transcriptional signature of the synergistic combination was unique relative to that of either constituent monotherapy. The sequential activation of transcription factors in time in the gene regulatory network was implicated. The nature of this transcriptional cascade suggests that drug synergy may ensue when the transcriptional responses elicited by two unrelated individual drugs are correlated. We used these results as the basis of a simple prediction algorithm attaining an AUROC of 0.77 in the prediction of synergistic drug combinations in an independent dataset.