Article Figures & Data
Figures
- Figure 1.
A, mercaptoketone series identified in a uHTS fluorescence-based biochemical screen. Compound 1 is readily reduced in the biochemical assay using HeLa cell nuclear extract generating two molar equivalents of active free thiol, compound 2. B, KD5170 is a mercaptoketone-based HDAC inhibitor delivered as a thioester-based prodrug. C, proposed mechanism of action. KD5170 thioester prodrug undergoes hydrolysis generating the mercaptoketone, which coordinates Zn2+ in a bidentate or monodentate fashion in the active site of HDACs.
- Figure 2.
KD5170 induces apoptosis in human tumor cell lines. HCT-116 (A) human colorectal tumor cells and HL-60 (B) human leukemia cells were treated with KD5170 at increasing concentrations for a period of 48 and 24 h, respectively, followed by staining with JC-1 dye. Cytometric analysis was done using the BD LSRII fluorescent cell analyzer, and percentage of apoptotic versus healthy (live) cells are indicated (Materials and Methods).
- Figure 3.
KD5170 induces a robust and sustained pharmacodynamic response in xenograft tumor tissues. HCT-116 tumor-bearing mice were p.o. given a single dose of KD5170 at 10, 30, or 100 mg/kg. Tumor tissue was excised and snap frozen at times indicated, and histone H3 (A and B) and α-tubulin acetylation (C) was quantified by immunoblotting and LiCor imaging technology. Total α-tubulin was used to normalize for amount of protein spotted. Points, mean; bars, SE (n = 4 tumors per time point per dose group). The T0 data points used for normalization were the same for each treatment and graphically replicated in Fig. 3A for ease of comparison.
- Figure 4.
KD5170 inhibits growth of HCT-116, NCI-H460, and PC-3 cell lines in vivo. A, HCT-116. KD5170 p.o. inhibits tumor growth in a human colon cancer (HCT-116) xenograft. Vehicle, •; KD5170 (42 mg/kg), ▪; KD5170 (84 mg/kg), ▴. Points, mean; bars, SE (n = 7 mice per dose group). B, NCI-H460. Vehicle, ▪; erlotinib (100 mg/kg), •; KD5170 (32 mg/kg), ▴; KD5170 (64 mg/kg), ▾. Points, mean; bars, SE (n = 8 mice per dose group). T/C, (treated final volume − treated initial volume) / (control final volume − control initial volume) × 100. A T/C value of 0 equals tumor stasis. *, P < 0.05. Statistical significance was determined by t test: two-sample assuming unequal variances (control versus treated on last measurement point). C, PC-3 end of treatment, day 30. No treatment, •; vehicle, ▾; KD5170 (30 mg/kg qod), ○; docetaxel (10 mg/kg i.v.), *; docetaxel (30 mg/kg i.v.), +; KD5170 (30 mg/kg qod) and docetaxel (10 mg/kg i.v.), ▴. Points, mean; bars, SE (n = 10 mice per dose group). D, PC-3 tumor regrowth posttreatment, day 90. No treatment, •; vehicle, ▾; KD5170 (30 mg/kg qod), ○; docetaxel (10 mg/kg i.v.), *; docetaxel (30 mg/kg i.v.), +; KD5170 (30 mg/kg qod) and docetaxel (10 mg/kg i.v.), ▴. Points, mean; bars, SE (n = 10 mice per dose group).
Tables
- Table 1.
KD5170 inhibits both class I and class II HDAC isoforms
Recombinant human HDAC Compound HeLa 1 2 3 4 5 6 7 8 9 10 IC50 ± SE (μmol/L) KD5170 0.045 ± 0.007 0.020 ± 0.004 2.06 ± 0.12 0.075 ± >0.01 0.026 ± 0.003 0.95 ± 0.03 0.014 ± 0.002 0.085 ± 0.009 2.50 ± 0.34 0.15 ± 0.005 0.018 ± 0.001 Compound 3 (disulfide) 0.014 ± 0.003 0.024 ± 0.007 0.74 ± 0.08 0.014 ± 0.003 0.023 ± 0.002 0.36 ± 0.02 0.002 ± 0.0002 0.07 ± 0.009 0.58 ± 0.04 0.092 ± 0.008 0.015 ± 0.0009 TSA 0.001 ± 0 0.012 ± 0.003 0.020 ± 0.0005 0.010 ± 0.005 0.022 ± 0.006 0.016 ± 0.003 0.0020 ± 0 0.081 ± 0.04 0.12 ± 0.01 0.080 ± 0.04 0.028 ± 0.01 NOTE: HDAC activity was determined indirectly by measuring the fluorescence generated by a deacetylated fluorogenic peptide product (Materials and Methods). Data presented represent mean ± SE. KD5170 and KLYP902195, n = 5 replicate assay runs for HeLa cell nuclear extract and HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, and HDAC10. HDAC9, n = 3. TSA, n = 2 for HeLa cell nuclear extract and all HDAC isoforms.
- Table 2.
KD5170 is a potent inhibitor of HDAC activity cell-based assays
Acetylation Compound Histone H3, EC50 ± SE (μmol/L) α-Tubulin, EC50 ± SE (μmol/L) KD5170 0.025 μmol/L (±0.004; n = 20) 0.325 μmol/L (±0.1; n = 6) NOTE: Cellular HDAC inhibitory activity in HeLa cells was assessed in a 384-well cytoblot using an antibody that recognizes acetylated histone H3 or acetylated α-tubulin. Cells were fixed and stained after a 7-h incubation with compound under standard HeLa cell culture conditions. Values shown represent a compilation of multiple independent experiments, as indicated (Materials and Methods).
- Table 3.
KD5170 dose optimization in HCT-116 xenograft tumor bearing mice
Dose (mg/kg) Regimen T/C Bodyweight Mortality 8.4 qd × 7 107% −2% 0/5 25 qd × 7 59% −9.6% 0/5 42 qd × 7 11% −22% 6/10 42 qd × 5 24% −12% 3/8 42 qod × 7 44% −2% 0/7 84 qod × 7 17% −8.7% 1/7 25 qod × 10 55% −1.5% 0/8 50 qod × 10 35% −2.4% 0/8 50 qod × 11 63% −2.6% 0/10 75 qod × 11 35% −12.9% 3/10 100 qod × 11 35% −15.7% 8/10 NOTE: Compilation of a series of HCT-116 xenograft experiments comparing efficacy and tolerability of different dose regimens of KD5170. T/C, (treated final volume − treated initial volume) / (control final volume − control initial volume) × 100. Bodyweight at day after last dose relative to that at start. Mortality, number of animals that died or were sacrificed due to overt toxicity.
- Table 4.
KD5170 inhibits tumor growth in a human prostate cancer PC-3 xenograft
Compound 1 Compound 2 T/C Bodyweight nadir (d) No treatment 0 Vehicle 1 Vehicle 2 0 KD5170 30 mg/kg qod × 14 32% 0 Docetaxel 10 mg/kg qwk × 3 9% 0 Docetaxel 30 mg/kg qwk × 3 −4% −5.7% (d23) KD5170 30 mg/kg qod × 14 Docetaxel 10 mg/kg qwk × 3 −11% 6.5% (d20) NOTE: T/C values at day 30 measurement point. Bodyweight nadir and day on which it was observed. A T/C value of 0 equals tumor stasis.
- Table 5.
KD5170 and docetaxel combination results in a significant delay in TTE
Compound 1 Compound 2 MTV (mm3), (in study; day 90) Fraction at endpoint (1,000 mm3) TTE No treatment 40 9/10 33 Vehicle 1 Vehicle 2 — 10/10 31 KD5170 30 mg/kg qod × 14 — 10/10 47* Docetaxel 10 mg/kg qwk × 3 40 9/10 60* Docetaxel 30 mg/kg qwk × 3 206 0/5 90* KD5170 30 mg/kg qod × 14 Docetaxel 10 mg/kg qwk × 3 479 1/9 90*† NOTE: Tumor growth was monitored twice weekly for 60 d post last dose of KD5170. Fraction at end point represents the fraction of mice that reached end point (1,000 mm3) by day 90. TTE represents the median time for a tumor to reach the end point in days (Materials and Methods).
↵* Denotes significant log-rank test versus vehicle and no treatment controls (P = 0.0040).
↵† Denotes significant log-rank test versus corresponding monotherapy arms [docetaxel 10 mg/kg qwk (P = 0.0056) × 3 or KD5170 30 mg/kg QOD × 14 (P = 0.0002)].
- Table 6.
KD5170 and docetaxel combination results in an increase in number of tumor regressions
Compound 1 Compound 2 Regressions PR CR TFS No treatment 0 0 0 Vehicle 1 Vehicle 2 0 0 0 KD5170 30 mg/kg qod × 14 0 0 0 Docetaxel 10 mg/kg qwk × 3 1 1 1 Docetaxel 30 mg/kg qwk × 3 2 3 0 KD5170 30 mg/kg qod × 14 Docetaxel 10 mg/kg qwk × 3 4 1 0 NOTE: Partial regression is when the tumor volume was 50% or less than that of day 1 for three consecutive measurements during the course of study and ≥13.5 mm3 for one or more of these three measurements. Complete regression is when the tumor volume was <13.5 mm3 for three consecutive measurements during course of study. An animal with a complete remission at termination of study is classified as a tumor-free survivor.
Abbreviations: PR, partial regression; CR, complete regression; TFS, tumor-free survivor.
Supplementary Material, Hassig et al
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Volume 7, Issue 5
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- Article
- Abstract
- Introduction
- Materials and Methods
- Identification of a Novel Series of HDAC Inhibitors
- Characterization of KD5170
- Tumor-Based Pharmacodynamics
- In vivo Efficacy: Monotherapy
- In vivo Efficacy: Combination Therapy
- Discussion
- Disclosure of Potential Conflicts of Interest
- Acknowledgments
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