A-485 treatment of prostate cancer PC-3 cells for three hours causes a dose-dependent reduction in H3K27Ac, with a half maximum effective concentration (EC50) of 73 nM. A-485 therapy has no effect on the levels of the proteins p300 or CBP. A-485 shows strong action in the majority of multiple myeloma cell lines, as well as in a minority of acute myeloid leukemia and non-Hodgkin's lymphoma lines, in haematological cancers, where the largest sensitivity is found. In all five prostate cancer cell lines, A-485 causes a similar reduction in H3K27Ac[1].

Twice daily intraperitoneal injections of A-485 result in 54% tumor growth suppression after 21 days of therapy (P<0.005 compared to vehicle control) after tumors are grown in male SCID mice. Furthermore, a seven-day dose of A-485 causes a reduction in the mRNA levels of MYC and the AR-dependent gene SLC45A3 three hours after the dose, as well as a decrease in the protein level of MYC, suggesting that A-485 inhibits p300-mediated transcriptional activity in vivo. These results are obtained in tumour-bearing animals. On the seventh day, however, the levels of the medication A-485 in the plasma and tumor are lower at 16 hours after dosage compared to 3 hours. After finishing the A-485 dosage regimen, the animals experience a slight 9% reduction in body weight and quickly recover[1].

p300 and CBP biochemical activity assay [1]
Acetyltransferase activity assays for p300-BHC and CBP-BHC domains were performed by detecting the acetylation of Lysine residues of a histone H4 synthetic-peptide using a TR-FRET assay. Reactions were performed in a 10 μL volume using an assay buffer containing 100 mM HEPES; pH 7.9, 80 μM EDTA, 40 μg/mL BSA, 100 mM KCl, 1 mM DTT, 0.01% triton X-100. Given the use of EDTA in this assay, it is possible that the structural integrity of the BHC protein used could be affected due to the presence of multiple Zinc containing domains (C/H3, PHD, and RING) in this protein. Thus, the p300-BHC activity assay was also performed with p300-BHC purified in the complete absence of EDTA and in assay buffer in the absence of EDTA. Each compound of interest was dissolved in DMSO and dispensed at 50 nL by a Labcyte Echo into white 384 well low- volume plates in 3-fold dilutions from 50 μM to 0.00075 μM. p300-BHC or CBP-BHC protein at 0.6 nM was pre-incubated with A-485 or A-486 for 30 minutes. The reaction was initiated by adding 5 μL of a biotinylated synthetic Histone-H4 peptide at 2 μM and acetyl coenzyme A at 0.5 μM. Following incubation for 1 hour at room temperature in a humidified chamber, the reaction was terminated with 10 μL of 3 nM LANCE Ultra Europium-anti-acetyl-Histone H4 Lysine antibody, 900 nM LANCE Ultra ULight-Streptavidin in LANCE Detection Buffer. TR-FRET measurements were obtained using a Perkin Elmer Envision with laser excitation at 335 nm and emission at 665 nm and 620 1 nm. For acetyl-CoA competition experiments, the assay was run as above except the acetyl-CoA concentration was varied from 0.078 to 10 μM. IC50 values for inhibition were calculated using a sigmoidal fit of the concentration/inhibition response curves using Prism GraphPad 5.

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p300 AlphaLISA Peptide Binding Assay [1]
Binding of P300-BHC to a histone H4 synthetic-peptide was assessed using AlphaLISA technology. Assays were performed in a 40 μL volume in white 384 well assay plates with an assay buffer containing 100 mM HEPES, pH 7.9, 80 μM EDTA, 40ug/mL BSA, 100 mM KCl, 1 mM DTT, 0.01% triton X-100. Ten μL of a biotin-labeled H4 peptide was added to 10 μL of P300-BHC (for a final concentration of 15 nM and 115 nM respectively) and incubated for one hour at room temperature. To demonstrate a decrease in AlphaLISA signal by competing with the biotin-labeled peptide, a 10 μL 2-fold mix of an unlabeled H4 peptide and biotin labeled H4 peptide (final concentration of 300 μM and 115 nM respectively) was added to 20 μL of P300-BHC (final concentration of 15nM) and incubated for 1 hour at room temperature. 20 μL of a 2- fold mix containing Nickel- chelate AlphaLISA acceptor beads and AlphaScreen streptavidin donor beads was added (final concentration of 20 μg/mL each) to the enzyme-peptide complex and incubated at room temperature for 1.5 hours. AlphaLISA counts were obtained using a Perkin Elmer Envision with laser excitation at 680 nm and emission at 615 nm.

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Thermal shift assay [1]
Thermal shift assays were performed on the Roche LightCycler 480 instrument using p300-BHC purified with EDTA in the lysis buffer (above). Sypro Orange dye was purchased from Invitrogen as 5000X stock. The assay was performed in 20mM HEPES, pH 7.5, 50mM NaCl, 1mM TCEP, 2% DMSO, and 1:500 dilution of the dye at a protein concentration of 1.5 uM. 50x stock DMSO 1 samples of Lys CoA, and A-485 were prepared so the final DMSO concentration was 2%, (v/v). All samples were run in quadruplicate.

LuCap-77 CR xenograft efficacy studies.[1]
The LuCap-77 CR prostate PDX model was used. Donor tumors were dissociated and injected as a brie (1:2) into the right flank of 16 week old male C.B.-17 SCID mice on day 0 in a volume of 0.2 ml. Tumors were size matched on day 26 post-inoculation with a mean tumor volume of 211 ± 3 (SEM) mm3 with dosing beginning on day 28. No mice on study were excluded from the analysis. Mice were randomized into 1 treatment groups using Studylog software based on tumor volume. Tumor volume was calculated twice weekly. Measurements of the length (L) and width (W) of the tumor were taken via electronic caliper and the volume was calculated according to the following equation: V = L x W2/2 using Study Director version 3.1. Partial blinding was used. A different technician formulated and dosed compounds while the main investigator randomized and measured tumor volumes during the study. Tumor growth inhibition was calculated according to the following equation: TGI% = (mean tumor volume of the control group – mean tumor volume of the treated group) / mean tumor volume of the control group x 100.

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LuCap-77 CR xenograft PD studies [1]
LuCap-77 CR xenograft tumors were established in SCID mice and animals were dosed with A-485 as described above in “LuCap-77 CR xenograft growth studies” for 7 days. Three hours post the final dose, tumors were harvested and snap frozen on dry ice. For RNA isolation, tumors were homogenized in lysis solution from the 96 well RNA spin kit using a Precellys 24 homogenizer and further processed according to manufacturer’s instructions. For western blotting, tumors were homogenized in ice cold lysis buffer (as described in “Western blotting”) and centrifuged at 20,000 g for 20 min. The supernatant was then processed for western blotting as described above.

[1]. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature. 2017 Oct 5;550(7674):128-132.

The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.[1]

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In summary, we have overcome the long-standing challenge of developing a drug-like HAT inhibitor by identifying a first-in-class highly potent, selective, cell and in vivo active p300/CBP catalytic inhibitor, A-485. A similar approach also can be applied more broadly to develop inhibitors of other HATs. Furthermore, they underscore the value of therapeutically targeting the HAT activity of p300/CBP, providing a major advance in the road to evaluating the clinical utility of HAT inhibitors for multiple human diseases.[1]

C25H24F4N4O5

536.4755

536.168

C, 55.97; H, 4.51; F, 14.17; N, 10.44; O, 14.91

1889279-16-6

1889279-16-6

118958122

White to off-white solid powder

3.3

2

9

6

38

941

2

C[C@@H](C(F)(F)F)N(CC1=CC=C(C=C1)F)C(=O)CN2C(=O)[C@]3(CCC4=C3C=CC(=C4)NC(=O)NC)OC2=O

VRVJKILQRBSEAG-LFPIHBKWSA-N

InChI=1S/C25H24F4N4O5/c1-14(25(27,28)29)32(12-15-3-5-17(26)6-4-15)20(34)13-33-21(35)24(38-23(33)37)10-9-16-11-18(7-8-19(16)24)31-22(36)30-2/h3-8,11,14H,9-10,12-13H2,1-2H3,(H2,30,31,36)/t14-,24+/m0/s1

N-(4-fluorobenzyl)-2-((R)-5-(3-methylureido)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidin]-3'-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)acetamide

A-485; A 485; N-(4-Fluorobenzyl)-2-((R)-5-(3-methylureido)-2',4'-dioxo-2,3-dihydrospiro[indene-1,5'-oxazolidin]-3'-yl)-N-((S)-1,1,1-trifluoropropan-2-yl)acetamide; (1R)-N-[(4-Fluorophenyl)methyl]-2,3-dihydro-5-[[(methylamino)carbonyl]amino]-2',4'-dioxo-N-[(1S)-2,2,2-trifluoro-1-methylethyl]spiro[1H-indene-1,5'-oxazolidine]-3'-acetamide; CHEMBL4282264; A485.

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)

DMSO : ~125 mg/mL (~233.00 mM)

Solubility in Formulation 1: 11 mg/mL (20.50 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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: ≥ 2.08 mg/mL (3.88 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 20.8 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 3: ≥ 2.08 mg/mL (3.88 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 20.8 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 4: ≥ 2.08 mg/mL (3.88 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 20.8 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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