Abstract
Multiple myeloma is an aggressive hematopoietic cancer of plasma cells. The recent emergence of three effective FDA-approved proteasome-inhibiting drugs, bortezomib (Velcade®), carfilzomib (Kyprolis®), and ixazomib (Ninlaro®), confirms that proteasome inhibitors are therapeutically useful against neoplastic disease, in particular refractory multiple myeloma and mantle cell lymphoma. This study describes the synthesis, computational affinity assessment, and preclinical evaluation of TIR-199, a natural product-derived syrbactin structural analog. Molecular modeling and simulation suggested that TIR-199 covalently binds each of the three catalytic subunits (β1, β2, and β5) and revealed key interaction sites. In vitro and cell culture-based proteasome activity measurements confirmed that TIR-199 inhibits the proteasome in a dose-dependent manner and induces tumor cell death in multiple myeloma and neuroblastoma cells as well as other cancer types in the NCI-60 cell panel. It is particularly effective against kidney tumor cell lines, with >250-fold higher anti-tumor activities than observed with the natural product syringolin A. In vivo studies in mice revealed a maximum tolerated dose of TIR-199 at 25 mg/kg. The anti-tumor activity of TIR-199 was confirmed in hollow fiber assays in mice. Adverse drug reaction screens in a kidney panel revealed no off-targets of concern. This is the first study to examine the efficacy of a syrbactin in animals. Taken together, the results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer.
Highlights
The results suggest that TIR-199 is a potent new proteasome inhibitor with promise for further development into a clinical drug for the treatment of multiple myeloma and other forms of cancer
We demonstrate that TIR-199 inhibits the proteasome activity and impedes MM and other tumor cell growth, with a significantly higher potency than the natural product syringolin A (SylA), both in vitro and in vivo
Structure and Synthesis of TIR-199 —The need to improve both the intrinsic potency and physicochemical properties of the syringolins led us to related natural products, the glidobactins, which were discovered based on their activity against tumor cell lines [36, 37]
Summary
Total Synthesis of Syrbactin Structural Analog TIR-199 and Reagents—The synthetic route to TIR-199 (MW 534) is provided in Scheme 1 and described in detail in the supplemental Methods. In Vivo Proteasome Activity Assay—To determine the antiproteasome activity of TIR-199 in the culture environment, the proteasome-Glo inhibition assay was performed as described previously [13, 21]. The viability of cancer cells was determined after 24 h of treatment with TIR-199 at the indicated concentrations (0 –10 M) by measuring the absorbance of the formazan product at 490 nm using a Multi-Mode SynergyTM MX microplate reader (BioTek, Inc., Winooski, VT). A total of 24 mice were used in 8 groups (n ϭ 3/group, each group representing two dose levels with 4 experiments, 3 cell lines/experiment) and treated by intraperitoneal injection with TIR-199 starting on day 3 or 4 following fiber implantation and continuing daily for 4 days. Data were prepared using Excel (Microsoft, Inc., Redmond, WA) and Prism version 6 (GraphPad Software, La Jolla, CA)
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