Discovery of a Potent and Selective Covalent p300/CBP Inhibitor.

Aberrant gene activation driven by the histone acetyltransferases p300 and CREB binding protein (CBP) has been linked to several diseases, including cancers. Because of this, many efforts have been aimed toward the targeting of the closely related paralogues, p300 and CBP, but these endeavors have been exclusively directed toward noncovalent inhibitors. X-ray crystallography of A-485 revealed that both p300 and CBP possess a cysteine (C1450) near the active site, thus rendering covalent inhibition an attractive chemical approach. Herein we report the development of compound 2, an acrylamide-based inhibitor of p300/CBP that forms a covalent adduct with C1450. We demonstrated using mass spectrometry that compound 2 selectively targets C1450, and we also validated covalent binding using kinetics experiments and cellular washout studies. The discovery of covalent inhibitor 2 gives us a unique tool for the study of p300/CBP biology.

Discovery of spirohydantoins as selective, orally bioavailable inhibitors of p300/CBP histone acetyltransferases.

p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.

Targeting Lineage-specific MITF Pathway in Human Melanoma Cell Lines by A-485, the Selective Small-molecule Inhibitor of p300/CBP.

Metastatic melanoma is responsible for approximately 80% of deaths from skin cancer. Microphthalmia-associated transcription factor (MITF) is a melanocyte-specific transcription factor that plays an important role in the differentiation, proliferation, and survival of melanocytes as well as in melanoma oncogenesis. MITF is amplified in approximately 15% of patients with metastatic melanoma. However, no small-molecule inhibitors of MITF currently exist. MITF was shown to associate with p300/CBP, members of the KAT3 family of histone acetyltransferase. p300 and CREB-binding protein (p300/CBP) regulate a wide range of cellular events such as senescence, apoptosis, cell cycle, DNA damage response, and cellular differentiation. p300/CBP act as transcriptional coactivators for multiple proteins in cancers, including oncogenic transcription factors such as MITF. In this study, we showed that our novel p300/CBP catalytic inhibitor, A-485, induces senescence in multiple melanoma cell lines, similar to silencing expression of EP300 (encodes p300) or MITF We did not observe apoptosis and increase invasiveness upon A-485 treatment. A-485 regulates the expression of MITF and its downstream signature genes in melanoma cell lines undergoing senescence. In addition, expression and copy number of MITF is significantly higher in melanoma cell lines that undergo A-485-induced senescence than resistant cell lines. Finally, we showed that A-485 inhibits histone-H3 acetylation but did not displace p300 at promoters of MITF and its putative downstream genes. Taken together, we provide evidence that p300/CBP inhibition suppressed the melanoma-driven transcription factor, MITF, and could be further exploited as a potential therapy for treating melanoma.

Author Correction: Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.

Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours.

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.

The SUV4-20 inhibitor A-196 verifies a role for epigenetics in genomic integrity.

Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.

Pyrimidine-based tricyclic molecules as potent and orally efficacious inhibitors of wee1 kinase.

Aided by molecular modeling, compounds with a pyrimidine-based tricyclic scaffold were designed and confirmed to inhibit Wee1 kinase. Structure-activity studies identified key pharmacophores at the aminoaryl and halo-benzene regions responsible for binding affinity with sub-nM K i values. The potent inhibitors demonstrated sub-µM activities in both functional and mechanism-based cellular assays and also possessed desirable pharmacokinetic profiles. The lead molecule, 31, showed oral efficacy in potentiating the antiproliferative activity of irinotecan, a cytotoxic agent, in a NCI-H1299 mouse xenograft model.

Hit to Lead optimization of a novel class of squarate-containing polo-like kinases inhibitors.

A high throughput screening (HTS) hit, 1 (Plk1 K(i)=2.2 µM) was optimized and evaluated for the enzymatic inhibition of Plk-1 kinase. Molecular modeling suggested the importance of adding a hydrophobic aromatic amine side chain in order to improve the potency by a classic kinase H-donor-acceptor binding mode. Extensive SAR studies led to the discovery of 49 (Plk1 K(i)=5 nM; EC(50)=1.05 µM), which demonstrated moderate efficacy at 100 mpk in a MiaPaCa tumor model, with no overt toxicity.

ABT-888, an orally active poly(ADP-ribose) polymerase inhibitor that potentiates DNA-damaging agents in preclinical tumor models.

PURPOSE: To evaluate the preclinical pharmacokinetics and antitumor efficacy of a novel orally bioavailable poly(ADP-ribose) polymerase (PARP) inhibitor, ABT-888. EXPERIMENTAL DESIGN: In vitro potency was determined in a PARP-1 and PARP-2 enzyme assay. In vivo efficacy was evaluated in syngeneic and xenograft models in combination with temozolomide, platinums, cyclophosphamide, and ionizing radiation. RESULTS: ABT-888 is a potent inhibitor of both PARP-1 and PARP-2 with K(i)s of 5.2 and 2.9 nmol/L, respectively. The compound has good oral bioavailability and crosses the blood-brain barrier. ABT-888 strongly potentiated temozolomide in the B16F10 s.c. murine melanoma model. PARP inhibition dramatically increased the efficacy of temozolomide at ABT-888 doses as low as 3.1 mg/kg/d and a maximal efficacy achieved at 25 mg/kg/d. In the 9L orthotopic rat glioma model, temozolomide alone exhibited minimal efficacy, whereas ABT-888, when combined with temozolomide, significantly slowed tumor progression. In the MX-1 breast xenograft model (BRCA1 deletion and BRCA2 mutation), ABT-888 potentiated cisplatin, carboplatin, and cyclophosphamide, causing regression of established tumors, whereas with comparable doses of cytotoxic agents alone, only modest tumor inhibition was exhibited. Finally, ABT-888 potentiated radiation (2 Gy/d x 10) in an HCT-116 colon carcinoma model. In each model, ABT-888 did not display single-agent activity. CONCLUSIONS: ABT-888 is a potent inhibitor of PARP, has good oral bioavailability, can cross the blood-brain barrier, and potentiates temozolomide, platinums, cyclophosphamide, and radiation in syngeneic and xenograft tumor models. This broad spectrum of chemopotentiation and radiopotentiation makes this compound an attractive candidate for clinical evaluation.