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. 2020 May 28;63(10):5501-5525.
doi: 10.1021/acs.jmedchem.0c00442. Epub 2020 May 6.

Design of Hydrazide-Bearing HDACIs Based on Panobinostat and Their p53 and FLT3-ITD Dependency in Antileukemia Activity

Xiaoyang Li  1   2 , Yuqi Jiang  1 , Yuri K Peterson  2 , Tongqiang Xu  1 , Richard A Himes  3 , Xin Luo  4 , Guilin Yin  4 , Elizabeth S Inks  2 , Nathan Dolloff  5 , Stephanie Halene  6 , Sherine S L Chan  2 , C James Chou  2
Affiliations

Design of Hydrazide-Bearing HDACIs Based on Panobinostat and Their p53 and FLT3-ITD Dependency in Antileukemia Activity

Xiaoyang Li et al. J Med Chem. .

Abstract

Here, we present a new series of hydrazide-bearing class I selective HDAC inhibitors designed based on panobinostat. The cap, linker, and zinc-binding group were derivatized to improve HDAC affinity and antileukemia efficacy. Lead inhibitor 13a shows picomolar or low nanomolar IC50 values against HDAC1 and HDAC3 and exhibits differential toxicity profiles toward multiple cancer cells with different FLT3 and p53 statuses. 13a indirectly inhibits the FLT3 signaling pathway and down-regulates master antiapoptotic proteins, resulting in the activation of pro-caspase3 in wt-p53 FLT3-ITD MV4-11 cells. While in the wt-FLT3 and p53-null cells, 13a is incapable of causing apoptosis at a therapeutic concentration. The MDM2 antagonist and the proteasome inhibitor promote 13a-triggered apoptosis by preventing p53 degradation. Furthermore, we demonstrate that apoptosis rather than autophagy is the key contributing factor for 13a-triggered cell death. When compared to panobinostat, 13a is not mutagenic and displays superior in vivo bioavailability and a higher AUC0-inf value.

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Conflict of interest statement

Competing interests

C.J.C., S.S.L.C., and R.A.H. are the co-founders of Lydex Pharmaceuticals. The other authors declare no competing financial interest.

Figures

Figure 1.
Figure 1.
Design of hydrazide-bearing HDACIs based on the structure of panobinostat.
Figure 2A and B:
Figure 2A and B:
Lineweaver-Burke plots of enzyme kinetics data in the presence of inhibitors. Y-axes units: (pmoles acetylated substrate cleaved/min)−1, Xaxes units: (μmoles)−1. Compound 13a for HDAC1, and 3, respectively. Intersection on Xaxes are indicative of non-competative inhibition (HDAC1, 3A), while intersections in 2nd quadrant are indicative of mixed and competitive inhibition (HDAC3, 3B). Representative plots of n ≥ 3 experiments. 2C. Molecular docking probe of the catalytic and allosteric binding pockets of HDAC1 (PDB: 5ICN) and HDAC3 (PDB: 4A69). Data is represented as whisker plot using the top 5 docking poses for each condition. The catalytic site was defined as the catalytic pocket that incorporated the Zn metal iron, and the allosteric site was defined as the allosteric pocket created by the interface between the HDAC heterodimer. 2D. Potential binding mode of 13a with catalytic site and allosteric site of HDAC3 in silico. Top 5 docking poses for each site are displayed.
Figure 3A:
Figure 3A:
IC50 curves of 13a and panobinostat for HDACs 1, 2, and 3 after pre-incubation of 10 min, 30 min, 60 min, and 90 min, respectively. 13a shows time-dependent inhibition toward HDAC1 and 3, which indicates slow-on inhibition; while panobinostat reaches a steady-state with 10 min pre-incubation, indicating fast-on kinetics. Dose response curves for 13a and panobinostat performed in triplicates were generated using GraphPad Prism software. 3B: Histone acetylation caused by 13a does not reduce within 6 h of drug removal, while panobinostat reduces acetylation within 30 min after removal of drug. MV4–11 cells are treated with 13a or panobinostat (Pano) for 3 h, control cells are collected for the 0 min point, the drugs are washed out and the cells are cultured for various lengths of time.
Figure 4A.
Figure 4A.
NCI 60 cells panel screen of 13a, panobinostat and SAHA. Compared to panobinostat, 13a shows better selectivity among all cell lines, and most of the cell lines sensitive to 13a is p53 wild type. 4B. In leukemia cells, 13a is altered due to different p53 and FLT3 status. 3C. 13a causes wt-p53 primary cell death. Concentration response curves performed in triplicates were generated using GraphPad Prism software.
Figure 5A.
Figure 5A.
Treatment of 13a, LP411, panobinostat (Pan), vorinostat (Vor) and entinostat (Ent) in wt-p53, FLT3-ITD MV4–11 cell line for 24 h. 5B. (top) Treatment of 200 nM 13a with 3 h, 6 h, 9 h, 12 h, and 24 h, respectively. LC3-II accumulated within 12 h and is fully degraded in 24 h; (bottom) Quantification of LC3-II values were normalized to actin levels. 5C. Treatment of 13a, LP411, panobinostat (Pan), vorinostat (Vor) and entinostat (Ent) in wt-p53, wt-FLT3 RS4;11 cell line for 24 h. 5D. Treatment of 13a, LP411, panobinostat (Pan), vorinostat (Vor) and entinostat (Ent) in p53-null, wt-FLT3 HL60 cell line for 24 h.
Figure 6A.
Figure 6A.
Treatment of 13a (100 nM) or 13a (100 nM) in combination with p53-MDM2 inhibitor RG7388 (300 nM) and p53 activator Prima-1met (1000 nM) for 24h, respectively. RG7388 can prevent p53 degradation and promote 13a-triggered apoptosis obviously; 6B. Treatment of 13a (200 nM) and 13a (200 nM) in combination with RG7388 (300 nM) for 3h, 6h, 9h, 12h and 24h, respectively. p53 begins to degrade after treatment by 13a for 6h-9h, and the degradation of p53 can be fully recovered by RG7388 within 12h; 6C. Combination Index (CI) for 13a and RG7388 after treatment 0f 24 h. Data was analyzed using CompuSyn Software. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively.
Figure 7A.
Figure 7A.
Treatment of 13a (200 nM) or 13a (200 nM) in combination with bortezomib (5 nM) for 3 h, 6 h and 9 h, respectively. p53 begins to degrade in treatment of 13a at 6 h, and the degradation can be rescued by bortezomib. 7B. Treatment of 13a 50 nM, 100 nM or in combination with bortezomib (5 nM). Bortezomib can promote 13a-triggered apoptosis. 7C. Combination Index (CI) for 13a and Bortezomib after treatment 0f 24 h. Data was analyzed using CompuSyn Software. CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively.
Figure 8A.
Figure 8A.
Treatment of 13a (200 nM) with z-VAD (50 μM), chloroquine (CQ 5 μM) or wortmannin (Wort 100 nM) for 24 h incubation (*p < 0.001, n = 3). The pan-caspase inhibitor z-VAD is capable of attenuating cell death rather than the autophagy inhibitors wortmannin and chloroquine, suggesting although both of apoptosis and autophagy occur after treatment of 13a, apoptosis is the key factor leading to cell death. 8B. Treatment of 13a (200 nM) with z-VAD (50 μM), chloroquine (CQ 5 μM) or wortmannin (Wort 100 nM) for 24 h incubation (*p < 0.001, n = 3). z-VAD rescues procaspase activation, and chloroquine blocks the degradation of LC3-II in 24 h and prevent the progression of autophagy.
Figure 9.
Figure 9.
Mini-Ames tests with liver S9 fraction activation (EBPI, Canada). Mutagen controls 2-aminoanthracene (2-AA) and HDAC inhibitor panobinostat (*p < 0.01 versus Ctrl) are Ames positive. 13a is not mutagenic as compared to panobinostat and the known mutagen 2-aminoanthracene.
Scheme 1.
Scheme 1.
Synthesis of 3, 6a-6c, 10a and 10b. Reagents and conditions: (a) Ethanol, NaOH, reflux, 47%; (b) NaBH3CN, CH3COOH, methanol, 70–80% yield; (c) Boc2O, TEA, DCM, 80–85% yield; (d) Propionaldehyde or benzaldehyde, NaBH3CN, HCl, Ethanol, 60–70%.
Scheme 2.
Scheme 2.
Synthesis of 13a-13d. Reagents and conditions: (a) hydrazine monohydrate, TBTU, TEA, DMF, yield 50%; (b) propionaldehyde or N-Boc-2-aminoacetaldehyde, MgSO4, ethanol, yield 60–70%; (c) NaBH3CN, HCl, methanol, H2O, methyl orange, yield 60–70%; (d) acrylonitrile or methyl acrylate, ethanol, refluxed, yield 40–50%; (e) TFA, DCM, yield 60%.
Scheme 3.
Scheme 3.
Synthesis of 16a-16c. Reagents and conditions: (a) hydrazine monohydrate, methanol, reflux, yield 90–95%; (b) propionaldehyde, MgSO4, ethanol, yield 60–70%; (c) NaBH3CN, HCl, methanol, H2O, methyl orange, yield 60–70%; (d) TFA, DCM, yield 60–70%.
Scheme 4.
Scheme 4.
Synthesis of 24a-24c. Reagents and conditions: (a) DCM, TEA, DMF, yield 80%; (b) PPh3, DEAD, anhydrous THF, yield 53%; (c) hydrazine monohydrate, methanol, reflux, yield 95%; (d) propionaldehyde, MgSO4, ethanol, yield 60–70%; (e) NaBH3CN, HCl, methanol, H2O, methyl orange, yield 60–70%.
Scheme 5.
Scheme 5.
Synthesis of 26a-26c, 28a-28e. Reagents and conditions: (a) 1,1-dimethylhydrazine, pyrrolidin-1-amine, piperidin-1-amine or pyrazolidine, TBTU, TEA, DMF, yield 50–60%; (b) TFA, DCM, yield 60–65%. (c) ethanal, propanal, n-butanal, n-pentanal, n-hexanal, NaBH3CN, CH3COOH, methanol, yield 50–60%.
Scheme 6.
Scheme 6.
Synthesis of 30. Reagents and conditions: (a) 3-Bromo-1-propanol, K2CO3, ethanol, reflux, yield 70%；(b) TFA, DCM, yield 60%.
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