We describe the biological properties of NVP-AUY922, a novel resorcinylic isoxazole amide heat shock protein 90 (HSP90) inhibitor. NVP-AUY922 potently inhibits HSP90 (K d = 1.7 nmol/L) and proliferation of human tumor cells with GI 50 values of approximately 2 to 40 nmol/L, inducing G 1 -G 2 arrest and apoptosis. Activity is independent of NQO1/DT-diaphorase, maintained in drug-resistant cells and under hypoxic conditions. The molecular signature of HSP90 inhibition, comprising induced HSP72 and depleted client proteins, was readily demonstrable. NVP-AUY922 was glucuronidated less than previously described isoxazoles, yielding higher drug levels in human cancer cells and xenografts. Daily dosing of NVP-AUY922 (50 mg/kg i.p. or i.v.) to athymic mice generated peak tumor levels at least 100-fold above cellular GI 50 . This produced statistically significant growth inhibition and/or regressions in human tumor xenografts with diverse oncogenic profiles: BT474 breast tumor treated/control, 21%; A2780 ovarian, 11%; U87MG glioblastoma, 7%; PC3 prostate, 37%; and WM266.4 melanoma, 31%. Therapeutic effects were concordant with changes in pharmacodynamic markers, including induction of HSP72 and depletion of ERBB2, CRAF, cyclin-dependent kinase 4, phospho-AKT/total AKT, and hypoxia-inducible factor-1A, determined by Western blot, electrochemiluminescent immunoassay, or immunohistochemistry. NVP-AUY922 also significantly inhibited tumor cell chemotaxis/invasion in vitro , WM266.4 melanoma lung metastases, and lymphatic metastases from orthotopically implanted PC3LN3 prostate carcinoma. NVP-AUY922 inhibited proliferation, chemomigration, and tubular differentiation of human endothelial cells and antiangiogenic activity was reflected in reduced microvessel density in tumor xenografts. Collectively, the data show that NVP-AUY922 is a potent, novel inhibitor of HSP90, acting via several processes (cytostasis, apoptosis, invasion, and angiogenesis) to inhibit tumor growth and metastasis. NVP-AUY922 has entered phase I clinical trials. [Cancer Res 2008;68(8):2850-60]

Targeted covalent inhibitors have gained widespread attention in drug discovery as a validated method to circumvent acquired resistance in oncology. This strategy exploits small-molecule/protein crystal structures to design tightly binding ligands with appropriately positioned electrophilic warheads. Whilst most focus has been on targeting binding-site cysteine residues, targeting nucleophilic lysine residues can also represent a viable approach to irreversible inhibition. However, owing to the basicity of the ϵ-amino group in lysine, this strategy generates a number of specific challenges. Herein, we review the key principles for inhibitor design, give historical examples, and present recent developments that demonstrate the potential of lysine targeting for future drug discovery.

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