HDAC6 (IC50 = 0.4 nM); HDAC1 (IC50 = 8.35 nM); HDAC2 (IC50 = 15.4 nM); HDAC3 (IC50 = 51.6 nM); HDAC8 (IC50 = 1430 nM)
HDAC6 (histone deacetylase 6). Biochemical IC50: WT161 IC50 = 0.40 nM against HDAC6; for comparison, SAHA IC50 = 0.03 nM, tubacin IC50 = 1.62 nM. Selectivity: WT161 IC50 against HDAC3 = 51.61 nM [1].

WT161 has little effect on global lysine acetylation but selectively inhibits HDAC6 and dramatically increases levels of acetylated α-tubulin (Ac-α-tubulin). With IC50s ranging from 1.5 to 4.7 µM, WT161 exhibits significant toxicity in every multiple myeloma cell line tested. Combining WT161 with bortezomib causes a marked build-up of polyubiquitinated proteins as well as cell stress, which in turn causes caspase activation and apoptosis. More significantly, it has been demonstrated that bone marrow stromal cells, which have been implicated in mediating multiple myeloma cell drug resistance, and bortezomib-resistant cells both respond well to this combination treatment[1].

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WT161 is a potent and selective HDAC6 inhibitor. Biochemically, it inhibits HDAC6 with an IC50 of 0.40 nM and shows >100-fold selectivity over HDAC3 (IC50 51.61 nM) and other HDAC family members [1].
In cells, WT161 selectively increases acetylated α-tubulin (Ac-α-tubulin) levels with little effect on global lysine acetylation, as shown by high-content imaging. It induces Ac-α-tubulin accumulation in MM cell lines within 2 hours, and this induction is reversible [1].
WT161 inhibits growth of multiple myeloma (MM) cell lines with IC50 values ranging from 1.5 to 4.7 μM. In MM.1S cells, the IC50 was 3.6 μM, compared to tubacin IC50 of 9.7 μM [1].
Combination of WT161 with bortezomib (BTZ) or carfilzomib (CFZ) enhances cytotoxicity in MM cells. In RPMI8226 cells, increasing concentrations of WT161 (0, 3, 4, 5, 6, 7 μM) with BTZ (0, 5, 7.5, 10, 12.5, 15 nM) or CFZ showed synergistic growth inhibition. In MM.1S cells, combination with BTZ resulted in greater cytotoxicity than tubacin+BTZ [1].
WT161 enhances BTZ-induced accumulation of polyubiquitinated proteins, activation of JNK, upregulation of CHOP and ATF4 (ER stress markers), downregulation of IRE1α and PERK, reduction of XIAP, and induction of caspase-3, -8, -9 cleavage and PARP cleavage, leading to apoptotic cell death [1].
WT161 combined with BTZ overcomes bone marrow stromal cell (BMSC)-mediated drug resistance in MM.15/BMSC cocultures, and also overcomes acquired BTZ resistance in ANBL-6-V5R cells (BTZ-resistant) with increasing concentrations of WT161 (0, 1.25, 2.5, 5, 10, 20 μM) [1].

WT161 is well tolerated at 50 mg/kg i.p., but it exhibits toxicity at 100 mg/kg i.p. WT161 and bortezomib together show a strong antitumor effect[1].
In a xenograft mouse model of human MM (MM.15 cells injected subcutaneously into SCID mice), treatment with WT161 (50 mg/kg, i.p., every other day) in combination with bortezomib (0.5 mg/kg, i.v.) significantly suppressed tumor progression compared to vehicle control (P = 0.0078). Single-agent WT161 or bortezomib did not show significant tumor growth inhibition (P = 0.095 and P = 0.07, respectively). On-target activity was confirmed by increased Ac-α-tubulin staining in xenograft tumor tissues from WT161-treated mice [1].

WT161 is an oral bioavailable, potent, and selective HDAC6 inhibitor with IC50 values of 0.4 nM, 8.35 nM, and 15.4 nM for HDAC6, HDAC1, and HDAC2, respectively; it has been demonstrated to have selectivity over other HDACs that is >100-fold.

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The biochemical activity assay for HDAC1-9 was performed as previously reported (reference 14 in the paper). The detailed experimental protocol was not described in this manuscript. IC50 values for WT161 against HDAC6 (0.40 nM) and HDAC3 (51.61 nM) were determined using this assay [1].

WT161 (0–10 μM) is added in increasing concentrations to MM.1S cells for a 48-hour period. Using the MTT assay, cell viability is assessed[1].

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Cell viability was assessed by MTT assay. MM.1S cells were cultured with increasing concentrations of WT161 or tubacin for 48 hours, and then MTT was added to measure cell proliferation. The IC50 values were calculated from dose-response curves [1].
Western blotting: MM cells or patient-derived cells were treated with WT161 (0.3–3 μM) alone or with bortezomib (3–5 nM) for indicated times (2–24 h). Whole-cell lysates were separated by SDS-PAGE and immunoblotted with antibodies against Ac-α-tubulin, acetylated lysine, polyubiquitinated proteins, JNK, CHOP, ATF4, IRE1α, PERK, XIAP, cleaved caspases, and PARP [1].
Apoptosis was measured by Annexin V positivity using flow cytometry after treatment with WT161 and/or bortezomib [1].
Cell proliferation in coculture with BMSCs was measured by 3H-thymidine uptake. MM.15 cells were cocultured with BMSCs in the presence of WT161 and increasing concentrations of bortezomib for 24 h [1].
In BTZ-resistant ANBL-6-V5R cells, cells were cultured with increasing concentrations of WT161 (0–20 μM) and bortezomib for 24 h, and cell growth was assessed by MTT assay [1].

Mice: The tumor xenograft mice are divided into cohorts that receive BTZ (0.5 mg/kg, i.v. ), WT161 (50 mg/kg, i.p. ), BTZ+WT161, or vehicle (control). Twice weekly, BTZ is administered, while WT161 is given for five days in a row. Estimating the tumor volume involves measuring the longest perpendicular tumor diameters with a calibre on alternate days[1].

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Female SCID mice were subcutaneously injected with 5×10^6 MM.15 cells. When tumors reached an average volume of 80 mm^3, mice were randomized into four groups (n=9 per group). WT161 was formulated for intraperitoneal (i.p.) administration at 50 mg/kg every other day (q.o.d.). Bortezomib (BTZ) was given intravenously (i.v.) at 0.5 mg/kg daily. The combination group received both treatments. Tumor volumes were measured every two days and calculated as (length × width)^2 / 2. Treatment continued for 26 days [1].

Pharmacokinetic (PK) properties of WT161 were evaluated in mice. The compound had a reasonable half-life of 1.4 hours and a maximum concentration (Cmax) of 18 mg/L after intraperitoneal administration. Based on in vitro activity (IC50 1.5–4.7 μM in MM cells) and PK data, an i.p. dose of 50–100 mg/kg was empirically determined [1].

Toxicity was observed with WT161 at 100 mg/kg/day i.p. (daily dosing). However, at 50 mg/kg every other day (q.o.d.) i.p., WT161 was well tolerated as a single agent and in combination with bortezomib [1].

[1]. Discovery of selective small-molecule HDAC6 inhibitor for overcoming proteasome inhibitor resistance in multiple myeloma. Proc Natl Acad Sci U S A. 2016 Nov 15;113(46):13162-13167.

[2]. Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target. Nat Chem Biol. 2022 Apr 28.

LSM-6269 is a tertiary amine.

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WT161 was developed from a hydrazone library of ~400 molecules. It features a simplified synthesis (three steps, 40% overall yield) with no stereocenters, enabling large-scale production. The structural basis for HDAC6 selectivity is the Y-shaped lipophilic cap that fits into a unique hydrophobic pocket formed by Phe200, Phe201, and Leu270 in HDAC6. WT161 served as a precursor to the clinical HDAC6 inhibitor ricolinostat (ACY-1215) [1].

C₂₇H₃₀N₄O₃

458.55

458.231

C, 70.72; H, 6.59; N, 12.22; O, 10.47

1206731-57-8

44601455

White to off-white solid powder

1.2±0.1 g/cm3

1.594

5.34

3

5

12

34

603

0

O=C(N/N=C/C1=CC=C(N(C2=CC=CC=C2)C3=CC=CC=C3)C=C1)CCCCCCC(NO)=O

KXWWYFKVBFUVIZ-SGWCAAJKSA-N

InChI=1S/C27H30N4O3/c32-26(15-9-1-2-10-16-27(33)30-34)29-28-21-22-17-19-25(20-18-22)31(23-11-5-3-6-12-23)24-13-7-4-8-14-24/h3-8,11-14,17-21,34H,1-2,9-10,15-16H2,(H,29,32)(H,30,33)/b28-21+

N'-hydroxy-N-[(E)-[4-(N-phenylanilino)phenyl]methylideneamino]octanediamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.45 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)