Inhibitors of the Hsp90 molecular chaperone are showing considerable promise as potential chemotherapeutic agents for cancer. Here, we describe the structure-based design, synthesis, structure-activity relationships and pharmacokinetics of potent small-molecule inhibitors of Hsp90 based on the 4,5-diarylisoxazole scaffold. Analogues from this series have high affinity for Hsp90, as measured in a fluorescence polarization (FP) competitive binding assay, and are active in cancer cell lines where they inhibit proliferation and exhibit a characteristic profile of depletion of oncogenic proteins and concomitant elevation of Hsp72. Compound 40f (VER-52296/NVP-AUY922) is potent in the Hsp90 FP binding assay (IC50 = 21 nM) and inhibits proliferation of various human cancer cell lines in vitro, with GI50 averaging 9 nM. Compound 40f is retained in tumors in vivo when administered i.p., as evaluated by cassette dosing in tumor-bearing mice. In a human colon cancer xenograft model, 40f inhibits tumor growth by approximately 50%.

The serine/threonine kinase DYRK1A has been implicated in regulation of a variety of cellular processes associated with cancer progression, including cell cycle control, DNA damage repair, protection from apoptosis, cell differentiation, and metastasis. In addition, elevated-level DYRK1A activity has been associated with increased severity of symptoms in Down’s syndrome. A selective inhibitor of DYRK1A could therefore be of therapeutic benefit. We have used fragment and structure-based discovery methods to identify a highly selective, well-tolerated, brain-penetrant DYRK1A inhibitor which showed in vivo activity in a tumor model. The inhibitor provides a useful tool compound for further exploration of the effect of DYRK1A inhibition in models of disease.

The dual‐specificity tyrosine‐regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence‐proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi‐cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER‐239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure‐guided drug discovery. Inhibition of DYRK1A/B by VER‐239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re‐expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth‐promoting factors. DYRK inhibition‐induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition‐induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER‐239353 as a treatment for glioblastoma is therefore warranted.

The kinase DYRK1A is an attractive target for drug discovery programs due to its implication in multiple diseases. Through a fragment screen, we identified a simple biaryl compound that is bound to the DYRK1A ATP site with very high efficiency, although with limited selectivity. Structure-guided optimization cycles enabled us to convert this fragment hit into potent and selective DYRK1A inhibitors. Exploiting the structural differences in DYRK1A and its close homologue DYRK2, we were able to fine-tune the selectivity of our inhibitors. Our best compounds potently inhibited DYRK1A in the cell culture and in vivo and demonstrated drug-like properties. The inhibition of DYRK1A in vivo translated into dose-dependent tumor growth inhibition in a model of ovarian carcinoma.