Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 +/- 1.28 micromolar. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Development of a First-in-Class Small Molecule Inhibitor of the C-terminal Hsp90 Dimerization

Heat shock protein 90 (Hsp90) is a promising therapeutic target due to its involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby contributing in exerting anti-apoptotic effects, which is essential for the malignant transformation and progression of several tumor types. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain (NTD) of Hsp90. However, adverse effects, including induction of the pro-survival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded clinical approval of these Hsp90i. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR and, thus, emerge as a promising alternative approach to target Hsp90. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with this essential dimerization process by small-molecule protein-protein interaction (PPI) inhibitors is a promising strategy for anticancer drug research. We have developed the first-in-class small molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitors (TKIs) resistant leukemia cells, without inducing HSR.

9-ING-41, a Small Molecule Inhibitor of GSK-3β, Potentiates the Effects of Chemotherapy on Colorectal Cancer Cells

Colorectal cancer (CRC) is one of the most common and lethal types of cancer. Although researchers have made significant efforts to study the mechanisms underlying CRC drug resistance, our knowledge of this disease is still limited, and novel therapies are in high demand. It is urgent to find new targeted therapy considering limited chemotherapy options. KRAS mutations are the most frequent molecular alterations in CRC. However, there are no approved K-Ras targeted therapies for these tumors yet. GSK-3β is demonstrated to be a critically important kinase for the survival and proliferation of K-Ras–dependent pancreatic cancer cells. In this study, we tested combinations of standard-of-care therapy and 9-ING-41, a small molecule inhibitor of GSK-3β, in CRC cell lines and patient-derived tumor organoid models of CRC. We demonstrate that 9-ING-41 inhibits the growth of CRC cells via a distinct from chemotherapy mechanism of action. Although molecular biomarkers of 9-ING-41 efficacy are yet to be identified, the addition of 9-ING-41 to the standard-of-care drugs 5-FU and oxaliplatin could significantly enhance growth inhibition in certain CRC cells. The results of the transcriptomic analysis support our findings of cell cycle arrest and DNA repair deficiency in 9-ING-41–treated CRC cells. Notably, we find substantial similarity in the changes of the transcriptomic profile after inhibition of GSK-3β and suppression of STK33, another critically important kinase for K-Ras–dependent cells, which could be an interesting point for future research. Overall, the results of this study provide a rationale for the further investigation of GSK-3 inhibitors in combination with standard-of-care treatment of CRC.

Inhibition of SREBP-1 Activation by a Novel Small-Molecule Inhibitor Enhances the Sensitivity of Hepatocellular Carcinoma Tissue to Radiofrequency Ablation

Radiofrequency ablation (RFA) is an important strategy for treatment of advanced hepatocellular carcinoma (HCC). However, the prognostic indicators of RFA therapy are not known, and there are few strategies for RFA sensitization. The transcription factor sterol regulatory element binding protein 1 (SREBP)-1 regulates fatty-acid synthesis but also promotes the proliferation or metastasis of HCC cells. Here, the clinical importance of SREBP-1 and potential application of knockdown of SREBP-1 expression in RFA of advanced HCC was elucidated. In patients with advanced HCC receiving RFA, a high level of endogenous SREBP-1 expression correlated to poor survival. Inhibition of SREBP-1 activation using a novel small-molecule inhibitor, SI-1, not only inhibited the aerobic glycolysis of HCC cells, it also enhanced the antitumor effects of RFA on xenograft tumors. Overall, our results: (i) revealed the correlation between SREBP-1 and HCC severity; (ii) indicated that inhibition of SREBP-1 activation could be a promising approach for treatment of advanced HCC.