AR-LBD (Ki = 267 nM)
Androgen Receptor (AR) - amino-terminal transcriptional activation domain AF-1 (amino acids 244-360) and carboxy-terminal ligand binding domain (LBD) (no specific Ki value provided); Androgen Receptor Splice Variants (AR-SVs) [1]

UT-155 binds at a Ki of 267 nM to the AR-LBD. UT-155 has 6–10 times the potency of enzalutamide in inhibiting the wildtype AR transactivation induced by R1881. Enzalutamide is two times less effective against the W742L mutant AR than it is against the wild type AR, even though UT-155 antagonizes both wildtype and mutant ARs in a comparable way. When applied to LNCaP cells, UT-155 has 5–10 times more potency than enzalutamide in inhibiting 0.1 nM R1881-induced PSA and FKBP5 gene expression between 10 and 100 nM[1].

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Inhibited AR transactivation: HEK-293 cells were transfected with human AR cDNA, GRE-LUC, and CMV-renilla LUC. Twenty-four hours post-transfection, cells were treated with a dose response of UT-155 combined with 0.1 nM R1881, and a luciferase assay was performed 48 hours post-transfection, showing inhibitory activity with an IC50 value not explicitly provided [1]
- Cross-reacted with progesterone receptor (PR) but minimally with mineralocorticoid receptor (MR) or glucocorticoid receptor (GR): HEK-293 cells were transfected with human AR, PR, GR, or MR cDNA, along with GRE-LUC and CMV-renilla LUC. Twenty-four hours post-transfection, cells were treated with indicated doses of UT-155 combined with corresponding ligands (0.1 nM R1881 for AR, progesterone for PR, dexamethasone for GR, aldosterone for MR), and a luciferase assay was conducted 48 hours post-transfection [1]
- Potently inhibited AR-target gene expression: LNCaP or LNCaP-EnzR cells were maintained in charcoal-stripped serum-containing medium for 2 days, then treated with vehicle or UT-155 (doses: 1, 10, 100, 1000, 10,000 nM) in the presence of 0.1 nM R1881 for 24 hours. RNA was isolated, and the expression of PSA or FKBP5 was quantified by real-time PCR and normalized to GAPDH [1]
- Reduced AR expression: LNCaP cells maintained in charcoal-stripped serum-containing medium for 2 days were treated with UT-155 in the presence or absence of 0.1 nM R1881 for ~24 hours. Cells were harvested, and Western blot was performed with AR-N20 antibody (actin as loading control). AD1 cells expressing AR-FL were treated similarly, and Western blot with AR-N20 antibody confirmed reduced AR full-length expression [1]
- Did not reduce PR and ER expression: T47D cells were plated in growth medium and treated with indicated doses of UT-155. Western blot for PR, ER, and actin showed no reduction in PR or ER levels [1]
- Induced AR degradation: LNCaP cells were treated with 10 μM UT-155, 50 μM cycloheximide, or a combination for indicated times. Western blot for AR and actin (quantified from n=3 experiments) showed accelerated AR degradation. The degradation was mediated by the proteasome pathway, as co-treatment with 10 μM MG-132 or 10 μM bortezomib reversed AR reduction in LNCaP cells treated with 10 μM UT-155 (in presence of 0.1 nM R1881 for 9 hours) [1]
- Reduced AR and AR-SV expression: 22RV1 cells maintained in charcoal-stripped serum-containing medium were treated with indicated doses of UT-155 in the presence of 0.1 nM R1881 for ~24 hours. Western blot with AR-N20 antibody (actin as loading control) showed reduced AR and AR-SV levels. D567es cells (expressing AR-V567es) and LNCaP-95 cells (expressing AR and AR-SV) treated with UT-155 for 24 hours also showed decreased AR and AR-SV expression via Western blot [1]
- Inhibited AR-SV-induced target gene expression: 22RV1 cells in charcoal-stripped serum-containing medium were treated with vehicle or UT-155 (10, 100, 1000, 10,000 nM) for 48 hours. Real-time PCR showed reduced FKBP5 expression compared to vehicle-treated samples (p<0.05) [1]
- Did not alter AR mRNA levels: LNCaP cells in charcoal-stripped serum-containing medium were treated with vehicle, UT-155, or UT-69 (0.001–10,000 nM) in presence of 0.1 nM R1881 for 24 hours. Real-time PCR (normalized to GAPDH) showed no change in AR mRNA levels (n=3) [1]
- Did not inhibit AR recruitment to androgen response element (ARE): LNCaP cells serum-starved for 2 days were pre-treated with 10 μM UT-155 for 30 minutes, then 0.1 nM R1881 for 2 hours. Cross-linked DNA-protein complexes were immunoprecipitated with AR antibody, and real-time PCR showed no inhibition of AR recruitment to PSA enhancer ARE (n=3) [1]
- Inhibited proliferation of AR-positive prostate cancer cells: LNCaP, LNCaP-abl, LNCaP-EnzR cells in charcoal-stripped serum-containing medium were treated with vehicle or UT-155 (1 pM–10 μM) in presence of 0.1 nM R1881. Cells were re-treated after 3 days, and SRB assay at 6 days showed potent proliferation inhibition (n=3, mean ± S.E.). AR-negative Hela cells treated with 1–30 μM UT-155 showed minimal inhibition [1]
- Inhibited proliferation of AR-SV-expressing prostate cancer cells: 22RV1 and LNCaP-95 cells in charcoal-stripped serum-containing medium were treated with indicated concentrations of UT-155 without R1881 stimulation. SRB assay at 6 days showed proliferation inhibition [1]

As anticipated, enzalutamide has no effect on the growth of the 22RV1 tumors, but UT-155 dramatically inhibits the growth of the 22RV1 xenograft by 53%, which is consistent with the anti-proliferative effects in vitro. In animals treated with UT-155, there is a significant decrease in tumor weights, PSA, and the expression of AR and AR-SV [1].

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Inhibited LNCaP xenograft growth: Intact NSG mice (n=6–8/group) were subcutaneously implanted with 5 million LNCaP cells mixed with matrigel. When tumors reached 100–200 mm³, mice were randomized and treated with vehicle or UT-155 (100 mg/kg/day s.c.). Tumor volume was measured twice weekly, and tumor weight was recorded at sacrifice. PSA levels in tumor protein extracts were reduced, indicating inhibited tumor growth [1]
- Inhibited 22RV1 xenograft growth: Castrated NSG mice (n=6–8/group) were subcutaneously implanted with 22RV1 cells. Mice were treated with vehicle, UT-155 (100 mg/kg/day s.c.), or enzalutamide (30 mg/kg/day s.c.). Tumor volume was measured thrice weekly, and tumor weight was recorded at sacrifice. Western blot of tumor proteins showed reduced AR expression, confirming anti-tumor efficacy [1]
- Inhibited patient-derived xenograft (Pr-3001) growth: Castrated NSG mice (n=8–10/group) were subcutaneously implanted with 1 mm³ Pr-3001 fragments. Mice were treated with vehicle or UT-155 (100 mg/kg/day s.c.), and tumor volume was measured thrice weekly. Significant growth inhibition was observed compared to vehicle (p<0.05) [1]

UT-155 is a selective androgen receptor (AR) antagonist, with a Ki of 267 nM for AR-RBD. In addition to competing with one another for binding to the LBD, UT-69 and UT-155 also lower AR protein levels after 24 hours, which is similar to the reported decline in transcriptional activity. We assessed the effect of the SARDs on the pre-mRNA of the hormone-rapidly induced NDRG1 and MT2A genes to ascertain whether the reduction in expression was necessary to inhibit AR activity or whether the competitive displacement of androgen from the LBD is sufficient to inhibit transcriptional activity.

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AR-LBD binding assay: Purified GST-tagged human AR-LBD protein was incubated with 1 nM ³H mibolerone. Various concentrations of UT-155 were added to compete for binding, and the binding affinity (Ki value) was determined by measuring radioactive signal displacement [1]
- AR-AF-1 steady-state fluorescence assay: Purified AR-AF-1 protein (1 μM) was pre-incubated with UT-155 for at least 30 minutes. Steady-state fluorescence emission spectra were measured, with corrections for buffer alone or buffer containing UT-155 to assess binding [1]
- AR-AF-1 NMR binding assay: UT-155 (500 μM) dissolved in deuterated-d₆ DMSO was added to NMR tubes alone or in combination with 5 μM GST (negative control) or GST-AF-1 purified protein. Nuclear spin intensity was measured at different magnetic fields (δ ppm), and peaks between 7 and 8 ppm (corresponding to aromatic rings of UT-155) were analyzed to confirm binding [1]
- AR-AF-1 WaterLOGSY binding assay: UT-155 (200 μM) was tested alone or in combination with 2 μM purified GST-AR-AF-1 in a WaterLOGSY experiment to confirm specific binding to AR-AF-1 [1]
- AR N-terminal domain fragment binding assay: Various N-terminal domain fragments of AR were cloned, expressed, and purified (molecular weight adjusted for GST tag of 26 kDa). UT-155 (500 μM) was incubated with 5 μM of each fragment, and NMR experiments were performed to map the binding region to amino acids 244–360 of AR-AF-1 [1]

Cells were cultured in 96-well plates according to the cell line, with different densities and serum-containing media. Viability was assessed using the cell-titer glo assay (Promega, Madison, WI) or sulforhodamine B (SRB), as shown in the figures.

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AR transactivation luciferase assay: HEK-293 cells were transfected with human AR cDNA, GRE-LUC reporter plasmid, and CMV-renilla LUC internal control plasmid. Twenty-four hours post-transfection, cells were treated with a dose gradient of UT-155 combined with 0.1 nM R1881. After 48 hours of incubation, luciferase activity was measured to evaluate AR transactivation inhibition and calculate IC50 [1]
- Receptor cross-reactivity luciferase assay: HEK-293 cells were separately transfected with AR, PR, GR, or MR cDNA, along with GRE-LUC and CMV-renilla LUC plasmids. Twenty-four hours post-transfection, cells were treated with indicated doses of UT-155 combined with specific ligands (0.1 nM R1881 for AR, progesterone for PR, dexamethasone for GR, aldosterone for MR). Luciferase activity was measured after 48 hours to assess cross-reactivity with other steroid receptors [1]
- AR target gene expression real-time PCR assay: LNCaP or LNCaP-EnzR cells were cultured in charcoal-stripped serum-containing medium for 2 days, then treated with 0.1 nM R1881 and UT-155 (1, 10, 100, 1000, 10,000 nM) for 24 hours. Total RNA was isolated, and real-time PCR was performed to quantify PSA and FKBP5 mRNA levels, with normalization to GAPDH [1]
- AR/AR-SV protein expression Western blot assay: LNCaP, AD1, 22RV1, D567es, LNCaP-95, and T47D cells were treated as specified, then harvested for protein extraction. Western blot was performed using AR-N20, AR-C-19 (for AR), PR, ER, actin, or GAPDH antibodies. Densitometric quantification was conducted to determine relative protein expression levels [1]
- AR degradation kinetics assay: LNCaP or 22RV1 cells were treated with 10 μM UT-155, 50 μM cycloheximide, or their combination for different time points. Proteins were extracted and subjected to Western blot for AR and actin. Blot quantification (n=3) was plotted on a semi-logarithmic scale with a best-fit line to analyze AR degradation rate [1]
- AR degradation pathway assay: LNCaP cells were cultured in charcoal-stripped serum-containing medium for 2 days, then treated with vehicle, 10 μM UT-155 alone, or combined with 10 μM MG-132 or 10 μM bortezomib in the presence of 0.1 nM R1881 for 9 hours. Proteins were extracted, and Western blot for AR and GAPDH was performed. Densitometric quantification was used to assess the effect of proteasome inhibitors on AR degradation [1]
- Cell proliferation SRB assay: LNCaP, LNCaP-abl, LNCaP-EnzR, Hela, 22RV1, and LNCaP-95 cells were seeded in appropriate media, then treated with UT-155 at specified concentrations (with or without 0.1 nM R1881). Medium was changed and cells were re-treated after 3 days. After 6 days of total culture, SRB assay was performed to measure cell viability [1]
- AR recruitment ChIP-PCR assay: LNCaP cells were serum-starved for 2 days, pre-treated with 10 μM UT-155 for 30 minutes, then stimulated with 0.1 nM R1881 for 2 hours. DNA-protein complexes were cross-linked, and AR was immunoprecipitated. Real-time PCR was used to detect the recruitment of AR to the PSA enhancer ARE region [1]

[1]. Novel Selective Agents for the Degradation of Androgen Receptor Variants to Treat Castration-Resistant Prostate Cancer. Cancer Res. 2017 Nov 15;77(22):6282-6298.

UT-155 is a potent and selective androgen receptor degrader (SARD) designed to treat castration-resistant prostate cancer by targeting the wild-type androgen receptor (AR) and AR splice variants (AR-SV)[1]. UT-155 binds to two distinct regions of the AR: the N-terminal transcriptional activation domain AF-1 (amino acids 244–360) and the C-terminal ligand-binding domain (LBD). Since AF-1 has not been previously used as a target for AR degradation, the mechanism of action of UT-155 is novel[1]. UT-155-induced AR degradation is mediated via the proteasome pathway, which has been demonstrated to be reversed in the presence of proteasome inhibitors (MG-132, bortezomib)[1]. UT-155 has a stronger inhibitory effect on AR function compared to approved AR antagonists. For example, enzalutamide and bicalutamide [1]
UT-155 cross-reacts with progesterone receptor (PR), but has minimal cross-reactance with mineralocorticoid receptor (MR) or glucocorticoid receptor (GR), and does not reduce estrogen receptor (ER) expression [1]
UT-155's (R)-isomer binds only to the AF-1 domain of AR (not LBD), but can still inhibit AR transcriptional activation, promote AR degradation, inhibit LNCaP cell proliferation, and reduce R1881-induced FKBP5 gene expression at concentrations comparable to the parent compound [1]
UT-155 does not change AR mRNA levels, indicating that its effect on AR is post-transcriptional (protein degradation) rather than transcriptional repression [1]

C20H15F4N3O2

405.345618486404

405.11

C, 59.26; H, 3.73; F, 18.75; N, 10.37; O, 7.89

2031161-35-8

(R)-UT-155;2031161-54-1

122640156

White to off-white solid powder

3.1

2

7

4

29

664

1

C[C@](CN1C=CC2=C1C=CC(=C2)F)(C(=O)NC3=CC(=C(C=C3)C#N)C(F)(F)F)O

CFSAYQVTXBMPRF-IBGZPJMESA-N

InChI=1S/C20H15F4N3O2/c1-19(29,11-27-7-6-12-8-14(21)3-5-17(12)27)18(28)26-15-4-2-13(10-25)16(9-15)20(22,23)24/h2-9,29H,11H2,1H3,(H,26,28)/t19-/m0/s1

(2S)-N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(5-fluoroindol-1-yl)-2-hydroxy-2-methylpropanamide

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: ≥ 2.17 mg/mL (5.35 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 21.7 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: ≥ 2.17 mg/mL (5.35 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 21.7 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: ≥ 2.17 mg/mL (5.35 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 21.7 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.)