C646

C646, a pyrazolone-containing small molecule, is a cell-permeable histone acetyltransferase (HAT) inhibitor that competitively and selectively inhibits the HAT activity of p300, a transcriptional co-activator involved in a variety of gene regulatory pathways and protein acetylation events, with the inhibition constant Ki value of 400 nM and the half maximal inhibition concentration IC50 value of 1.6 μM [1].

C646 binds to p300 as the Z-isomer forming hydrogen bonds between Thr1411, tYR1467, Trp1466 and Arg1410 on the side chains of p300 and oxygen atoms of C646 [1].

C646 is also capable of inhibiting a variety of p300 HAT mutants, including T1411A, Y1467F, W1466F and R1410A with IC50 values of 3.4 μM, 7 μM, 5 μM and 2.5 μM respectively [1].

Reference

References:
[1] Bowers EM, Yan G, Mukherjee C, Orry A, Wang L, Holbert MA, Crump NT, Hazzalin CA, Liszczak G, Yuan H, Larocca C, Saldanha SA, Abagyan R, Sun Y, Meyers DJ, Marmorstein R, Mahadevan LC, Alani RM, Cole PA. Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem Biol. 2010 May 28;17(5):471-82. doi: 10.1016/j.chembiol.2010.03.006.

C646, a selective small molecule inhibitor of histone acetyltransferase p300, radiosensitizes lung cancer cells by enhancing mitotic catastrophe.[Pubmed:24746574]

Radiother Oncol. 2014 May;111(2):222-7.

BACKGROUND AND PURPOSE: Chromatin remodeling through histone modifications, including acetylation, plays an important role in the appropriate response to DNA damage induced by ionizing radiation (IR). Here we investigated the radiosensitizing effect of C646, a selective small molecule inhibitor of p300 histone acetyltransferase, and explored the underlying mechanisms. MATERIALS AND METHODS: A549, H157 and H460 human non-small cell lung carcinoma (NSCLC) cells, and HFL-III human lung fibroblasts were assessed by clonogenic survival assay. Apoptosis and necrosis were assessed by annexin V staining. Senescence was assessed by Senescence-associated beta-galactosidase staining. Mitotic catastrophe was assessed by evaluating nuclear morphology with DAPI staining. Cell cycle profiles were analyzed by flow cytometry. Protein expression was analyzed by immunoblotting. RESULTS: C646 sensitized A549, H460 and H157 cells to IR with a dose enhancement ratio at 10% surviving fraction of 1.4, 1.2 and 1.2, respectively. C646 did not radiosensitize HFL-III cells. In A549 cells, but not in HFL-III cells, C646 (i) enhanced mitotic catastrophe but not apoptosis, necrosis, or senescence after IR; (ii) increased the hyperploid cell population after IR; and (iii) suppressed the phosphorylation of CHK1 after IR. CONCLUSIONS: C646 radiosensitizes NSCLC cells by enhancing mitotic catastrophe through the abrogation of G2 checkpoint maintenance.

C646, a Novel p300/CREB-Binding Protein-Specific Inhibitor of Histone Acetyltransferase, Attenuates Influenza A Virus Infection.[Pubmed:26711748]

Antimicrob Agents Chemother. 2015 Dec 28;60(3):1902-6.

New strategies to develop novel broad-spectrum antiviral drugs against influenza virus infections are needed due to the emergence of antigenic variants and drug-resistant viruses. Here, we evaluated C646, a novel p300/CREB-binding protein-specific inhibitor of histone acetyltransferase (HAT), as an anti-influenza virus agent in vitro and in vivo and explored how C646 affects the viral life cycle and host response. Our studies highlight the value of targeting HAT activity for anti-influenza drug development.

Histone acetyltransferase inhibitor C646 reverses epithelial to mesenchymal transition of human peritoneal mesothelial cells via blocking TGF-beta1/Smad3 signaling pathway in vitro.[Pubmed:26045780]

Int J Clin Exp Pathol. 2015 Mar 1;8(3):2746-54. eCollection 2015.

Peritoneal fibrosis resulting from long-term peritoneal dialysis is a major cause of failure of peritoneal ultrafiltration function and main reason of dropout from peritoneal dialysis. Epithelial-mesenchymal transition (EMT) of peritoneal mesochelial cells (HPMCs) is a major contributor of peritoneal fibrosis. Recently, the association between histone acetylation and kinds of fibrosis including liver, lung and kidney fibrosis is well established. Thus, in this study we tried to profile whether histone acetylation is also operates EMT process in HPMCs and what's the regulatory mechanism. We established an EMT model of HPMCs through high glucose treatment. And hyperacetylation of H3 histone was found using western blot in EMT model. After treated with C646, a histone acetyltransferase (HAT) inhibitor, high glucose-induced EMT in HPMCs was counteracted. To further understand the molecular mechanism of C646 rescues high glucose-induced EMT, CHIP-qPCRwas used to examine the modulation of histone H3 acetylation at promoters of series signaling target genes. We found that the H3 acetylation level at TGF-beta1 gene promoter was down-regulation by C646 treatment. Moreover, we also found that TGF-beta1/Smad3 signaling was blocked. Hence, our results suggest that histone H3 acetylation activated TGF-beta1/Smad3 signaling during EMT of HPMCs, and C646 can rescue the mesenchymal phenotype transition. These findings may provide a novel pathogenic mechanism and therapeutic target for peritoneal fibrosis.

The histone acetyltransferase p300 inhibitor C646 reduces pro-inflammatory gene expression and inhibits histone deacetylases.[Pubmed:26718586]

Biochem Pharmacol. 2016 Feb 15;102:130-140.

Lysine acetylations are reversible posttranslational modifications of histone and non-histone proteins that play important regulatory roles in signal transduction cascades and gene expression. Lysine acetylations are regulated by histone acetyltransferases as writers and histone deacetylases as erasers. Because of their role in signal transduction cascades, these enzymes are important players in inflammation. Therefore, histone acetyltransferase inhibitors could reduce inflammatory responses. Among the few histone acetyltransferase inhibitors described, C646 is one of the most potent (Ki of 0.4muM for histone acetyltransferase p300). C646 was described to affect the NF-kappaB pathway; an important pathway in inflammatory responses, which is regulated by acetylation. This pathway has been implicated in asthma and COPD. Therefore, we hypothesized that via regulation of the NF-kappaB signaling pathway, C646 can inhibit pro-inflammatory gene expression, and have potential for the treatment of inflammatory lung diseases. In line with this, we demonstrate here that C646 reduces pro-inflammatory gene expression in RAW264.7 murine macrophages and murine precision-cut lung slices. To unravel its effects on cellular substrates we applied mass spectrometry and found, counterintuitively, a slight increase in acetylation of histone H3. Based on this finding, and structural features of C646, we presumed inhibitory activity of C646 on histone deacetylases, and indeed found inhibition of histone deacetylases from 7muM and higher concentrations. This indicates that C646 has potential for further development towards applications in the treatment of inflammation, however, its newly discovered lack of selectivity at higher concentrations needs to be taken into account.

Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor.[Pubmed:20534345]

Chem Biol. 2010 May 28;17(5):471-82.

The histone acetyltransferase (HAT) p300/CBP is a transcriptional coactivator implicated in many gene regulatory pathways and protein acetylation events. Although p300 inhibitors have been reported, a potent, selective, and readily available active-site-directed small molecule inhibitor is not yet known. Here we use a structure-based, in silico screening approach to identify a commercially available pyrazolone-containing small molecule p300 HAT inhibitor, C646. C646 is a competitive p300 inhibitor with a K(i) of 400 nM and is selective versus other acetyltransferases. Studies on site-directed p300 HAT mutants and synthetic modifications of C646 confirm the importance of predicted interactions in conferring potency. Inhibition of histone acetylation and cell growth by C646 in cells validate its utility as a pharmacologic probe and suggest that p300/CBP HAT is a worthy anticancer target.

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.[Pubmed:20201064]

Stem Cells. 2010 Apr;28(4):713-20.

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.

HAT trick: p300, small molecule, inhibitor.[Pubmed:20534339]

Chem Biol. 2010 May 28;17(5):417-8.

p300 is an acetyltransferase that targets histone and nonhistone proteins for lysine acetylation. Based on a p300 structure, Bowers et al. (2010) have conducted an in silico screen for active site small molecule inhibitors of p300. We describe this important discovery and its potential therapeutic applications.

C646 is a selective and competitive histone acetyltransferase p300 inhibitor with Ki of 400 nM, and is less potent for other acetyltransferases.

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