Plumbagin Suppresses Pulmonary Fibrosis via Inhibition of p300 Histone Acetyltransferase Activity.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing interstitial lung disease with a poor prognosis similar to that of malignancy. The causes of IPF are not clearly known, and there is no effective therapy to date. In this study, the natural compound plumbagin, which was isolated from Plumbago rosea root extract, was screened for p300 inhibitory activity. Plumbagin specifically inhibited the activity of p300 toward histone acetyltransferases. Plumbagin treatment significantly suppressed transforming growth factor-ß-induced profibrotic target-gene expression and proliferation of fibroblast cell lines. Moreover, plumbagin significantly inhibited bleomycin-induced pulmonary fibrosis in mice. Taken together, these data demonstrate the inhibitory effects of plumbagin on lung fibrosis and its promise as a therapeutic agent for IPF.

Preclinical Safety Assessment of a Highly Selective and Potent Dual Small-Molecule Inhibitor of CBP/P300 in Rats and Dogs.

Cyclic adenosine monophosphate-response element (CREB)-binding protein (CBP) and EP300E1A-binding protein (p300) are members of the bromodomain and extraterminal motif (BET) family. These highly homologous proteins have a key role in modulating transcription, including altering the status of chromatin or through interactions with or posttranslational modifications of transcription factors. As CBP and p300 have known roles for stimulating c-Myc oncogenic activity, a small-molecule inhibitor, GNE-781, was developed to selectively and potently inhibit the CBP/p300 bromodomains (BRDs). Genetic models have been challenging to develop due to embryonic lethality arising from germline homozygous mutations in either CBP or P300. Hence, the purpose of this study was to characterize the role of dual inhibition of these proteins in adult rats and dogs. Repeat dose toxicity studies were conducted, and toxicologic and pathologic end points were assessed. GNE-781 was generally tolerated; however, marked effects on thrombopoiesis occurred in both species. Evidence of inhibition of erythroid, granulocytic, and lymphoid cell differentiation was also present, as well as deleterious changes in gastrointestinal and reproductive tissues. These findings are consistent with many preclinical (and clinical) effects reported with BET inhibitors targeting BRD proteins; thus, the current study findings indicate a likely important role for CBP/p300 in stem cell differentiation.

Inhibition of p300/CBP-Associated Factor Attenuates Renal Tubulointerstitial Fibrosis through Modulation of NF-kB and Nrf2.

p300/CBP-associated factor (PCAF), a histone acetyltransferase, is involved in many cellular processes such as differentiation, proliferation, apoptosis, and reaction to cell damage by modulating the activities of several genes and proteins through the acetylation of either the histones or transcription factors. Here, we examined a pathogenic role of PCAF and its potential as a novel therapeutic target in the progression of renal tubulointerstitial fibrosis induced by non-diabetic unilateral ureteral obstruction (UUO) in male C57BL/6 mice. Administration of garcinol, a PCAF inhibitor, reversed a UUO-induced increase in the renal expression of total PCAF and histone 3 lysine 9 acetylation and reduced positive areas of trichrome and α-smooth muscle actin and collagen content. Treatment with garcinol also decreased mRNA levels of transforming growth factor-ß, matrix metalloproteinase (MMP)-2, MMP-9, and fibronectin. Furthermore, garcinol suppressed nuclear factor-κB (NF-κB) and pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6, whereas it preserved the nuclear expression of nuclear factor erythroid-derived 2-like factor 2 (Nrf2) and levels of Nrf2-dependent antioxidants including heme oxygense-1, catalase, superoxide dismutase 1, and NAD(P)H:quinone oxidoreductase 1. These results suggest that the inhibition of inordinately enhanced PCAF could mitigate renal fibrosis by redressing aberrant balance between inflammatory signaling and antioxidant response through the modulation of NF-κB and Nrf2.

Curcumin and its demethoxy derivatives possess p300 HAT inhibitory activity and suppress hypertrophic responses in cardiomyocytes.

The natural compound, curcumin (CUR), possesses several pharmacological properties, including p300-specific histone acetyltransferase (HAT) inhibitory activity. In our previous study, we demonstrated that CUR could prevent the development of cardiac hypertrophy by inhibiting p300-HAT activity. Other major curcuminoids isolated from Curcuma longa including demethoxycurcumin (DMC) and bisdemethoxycurcumin (BDMC) are structural analogs of CUR. In present study, we first confirmed the effect of these three curcuminoid analogs on p300-HAT activity and cardiomyocyte hypertrophy. Our results showed that DMC and BDMC inhibited p300-HAT activity and cardiomyocyte hypertrophy to almost the same extent as CUR. As the three compounds have structural differences in methoxy groups at the 3-position of their phenol rings, our results suggest that these methoxy groups are not involved in the inhibitory effects on p300-HAT activity and cardiac hypertrophy. These findings provide useful insights into the structure-activity relationship and biological activity of curcuminoids for p300-HAT activity and cardiomyocyte hypertrophy.

Discovery of 1,8-acridinedione derivatives as novel GCN5 inhibitors via high throughput screening.

The general control nonrepressed protein 5 (GCN5) plays a crucial role in many biological processes. Dysregulation of GCN5 has been closely related to various human diseases, especially cancers. Hence, the exploitation of small molecules targeting GCN5 is essential for drug design and academic research. Based on the amplified luminescent proximity homogeneous assay screen methodology, we performed high throughput screening and discovered a novel GCN5 inhibitor DC_G16 with 1,8-acridinedione scaffold. Structure optimization led to the identification of a highly potent inhibitor, namely DC_G16-11 with the half-maximal inhibitory concentration (IC50) value of 6.8 µM. The binding between DC_G16-11 and GCN5 was demonstrated by NMR and SPR with a KD of 4.2 µM. It could also inhibit proliferation and induce cell cycle arrest and apoptosis in cancer cells while it presented minimal effects on normal cells. Herein, DC_G16-11 could be applied as a validated chemical probe for GCN5-related biological function research and presented great potential for clinical disease treatment.

Therapeutic effect of Cryptotanshinone on experimental rheumatoid arthritis through downregulating p300 mediated-STAT3 acetylation.

BACKGROUND AND PURPOSE: The balance between T helper 17 (Th17) cells and regulatory T (Treg) cells, plays a critical role in rheumatoid arthritis (RA). The differentiation of Th17 cells requires the activation of STAT3, which determines the balance of Th17/Treg. Here, we investigated the therapeutic effect of Cryptotanshinone (CTS) on collagen induced mouse arthritis and explored the underlying mechanisms. EXPERIMENTAL APPROACH: Arthritis was induced in DBA/1 mice with bovine collagen type II and complete Freund's adjuvant. CTS was given at 20mgkg-1d-1 or 60mgkg-1d-1 by gavage for 6weeks. The immuno-inflammation and joint destruction were evaluated and the balance of Th17/Treg was determined. STAT3 acetylation and phosphorylation were detected by western blotting, and the involvement of p300 was investigated by siRNA and plasmid overexpression. KEY RESULTS: CTS at a dose of 60mgkg-1d-1 ameliorated the inflammation and joint destruction in CIA mice. It improved Th17/Treg imbalance, and inhibited both acetylation and phosphorylation of STAT3. CTS reduced p300 expression and its binding to STAT3, but increased phosphorylated AMPK. Knockdown of p300 mimicked the inhibitory effect of CTS on STAT3 acetylation and phosphorylation, which could be partially rescued by overexpression of p300-WT, but not p300-dominant negative (DN) construct. CONCLUSION AND IMPLICATIONS: Our study suggested that the anti-arthritis effects of CTS were attained through suppression of p300-mediated STAT3 acetylation. Our data suggest that CTS might be a potential immune modulator for RA treatment.

MAP30 inhibits autophagy through enhancing acetyltransferase p300 and induces apoptosis in acute myeloid leukemia cells.

Momordica anti-human immunodeficiency virus protein of 30 kDa (MAP30) has been shown to exhibit potent antitumor activities against several solid tumors. In the present investigation we demonstrated that MAP30 significantly inhibited the proliferation of acute myeloid leukemia (AML) HL-60 and THP-1 cell lines and patient AML cells through autophagy inhibition and apoptosis induction. Intriguingly, MAP30-induced cell death and apoptosis were partially rescued in combination with an autophagy activator rapamycin, and aggravated in combination with an autophagy inhibitor bafilomycin A1 in HL-60 cells, suggesting that autophagy is a pro-survival signal and its inhibition contributes to the induction of apoptosis in MAP30­induced cell death. Further mechanism analysis demonstrated that MAP30 enhanced p300, and C646, a selective inhibitor of p300, markedly promoted autophagy and partially rescued the MAP30-induced cell death in HL-60 cells and patient AML cells. Collectively, our findings suggest that apoptosis and autophagy act cooperatively to elicit MAP30-induced cell death and MAP30 may be a potential antitumor drug candidate against AML.

CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses.

Th17 responses are critical to a variety of human autoimmune diseases, and therapeutic targeting with monoclonal antibodies against IL-17 and IL-23 has shown considerable promise. Here, we report data to support selective bromodomain blockade of the transcriptional coactivators CBP (CREB binding protein) and p300 as an alternative approach to inhibit human Th17 responses. We show that CBP30 has marked molecular specificity for the bromodomains of CBP and p300, compared with 43 other bromodomains. In unbiased cellular testing on a diverse panel of cultured primary human cells, CBP30 reduced immune cell production of IL-17A and other proinflammatory cytokines. CBP30 also inhibited IL-17A secretion by Th17 cells from healthy donors and patients with ankylosing spondylitis and psoriatic arthritis. Transcriptional profiling of human T cells after CBP30 treatment showed a much more restricted effect on gene expression than that observed with the pan-BET (bromo and extraterminal domain protein family) bromodomain inhibitor JQ1. This selective targeting of the CBP/p300 bromodomain by CBP30 will potentially lead to fewer side effects than with the broadly acting epigenetic inhibitors currently in clinical trials.

Assessment of dual inhibition property of newly discovered inhibitors against PCAF and GCN5 through in silico screening, molecular dynamics simulation and DFT approach.

p300/CBP-associated factor (PCAF) is one among the histone acetyltransferase (HAT) family enzymes. It is involved in the regulation of transcription by modifying the chromatin structure indirectly through the acetylation of histones. It has been emerged as a promising drug target for various types of cancer. A four-point pharmacophore with two hydrogen bond acceptor, one aromatic ring and one hydrophobic feature, was generated for six highly active isothiazolone derivatives as PCAF inhibitors in order to elucidate their anticancer activity. The generated pharmacophore was used for screening three different databases such as Maybridge, Life Chemicals and Chembridge databases. The screened compounds were further filtered through docking studies. Then the compounds were further carried for ADME prediction. The best three compounds BTB09406, F1418-0051 and F1880-1727 were docked to GCN5 to explore the dual inhibitory properties. The conformational stability of the protein-ligand complexes were analyzed through molecular dynamics simulation. Three best compounds were finally went through electronic structure analysis using density functional theory (DFT) at B3LYP/6-31**G level to understand their molecular reactivity. The results obtained from this study exploit that the three best compounds (BTB09406, F1418-0051 and F1880-1727) were found to have more potent and dual inhibitory properties.

Pharmacophore-based virtual screening and biological evaluation of small molecule inhibitors for protein arginine methylation.

Protein arginine methyltransferases (PRMTs) are proved to play vital roles in chromatin remodeling, RNA metabolism, and signal transduction. Aberrant regulation of PRMT activity is associated with various pathological states such as cancer and cardiovascular disorders. Development and application of small molecule PRMT inhibitors will provide new avenues for therapeutic discovery. The combination of pharmacophore-based virtual screening methods with radioactive methylation assays provided six hits identified as inhibitors against the predominant arginine methyltransferase PRMT1 within micromolar potency. Two potent compounds, A9 and A36, exhibited the inhibitory effect by directly targeting substrate H4 other than PRMT1 and displayed even higher inhibition activity than the well-known PRMT inhibitors AMI-1. A9 significantly inhibits proliferation of castrate-resistant prostate cancer cells. Together, A9 may be a potential inhibitor against advanced hormone-independent cancers, and the work will provide clues for the future development of specific compounds that block the interaction of PRMTs with their targets.

A novel drug delivery system of oral curcumin markedly improves efficacy of treatment for heart failure after myocardial infarction in rats.

Curcumin is an inhibitor of p300 histone acetyltransferase activity, which is associated with the deterioration of heart failure. We reported that native curcumin, at a dosage of 50 mg/kg, prevented deterioration of the systolic function in rat models of heart failure. To achieve more efficient oral pharmacological therapy against heart failure by curcumin, we have developed a novel drug delivery system (DDS) which markedly increases plasma curcumin levels. At the dosage of 0.5 mg/kg, DDS curcumin but not native curcumin restored left ventricular fractional shortening in post-myocardial infarction rats. Thus, our DDS strategy will be applicable to the clinical setting in humans.

Mutant hypoxia inducible factor-1α improves angiogenesis and tissue perfusion in ischemic rabbit skeletal muscle.

Hypoxia-inducible factor-1α (HIF-1α) is one of the most potent angiogenic growth factors. It regulates genes involved in angiogenesis, but is inactivated rapidly by normoxia. Ad-HIF-1α-Trip was constructed by transforming Pro402, Pro564, and Asn803 in HIF-1α to alanine in order to delay degradation and create a constitutive transcriptional activator. In this study, we investigated whether Ad-HIF-1α-Trip could induce functional mature angiogenesis and the possible mechanisms involved. We found that Ad-HIF-1α-Trip increased the expression of multiple angiogenic genes in cultured HMVEC-Ls, including VEGF, PLGF, PAI-1, and PDGF. In a rabbit model of acute hind limb ischemia, Ad-HIF-1α-Trip improved tissue perfusion and collateral vessels, as measured by contrast-enhanced ultrasound (CEU), CT angiography, and vascular casting. Ad-HIF-1α-Trip also produced more histologically identifiable capillaries, which were verified by immunostaining, compared with controls. Interestingly, inhibition of CBP/p300 by curcumin prevented HIF-1α from inducing the expression of several angiogenic genes. The present study suggests that Ad-HIF-1α-Trip can induce mature angiogenesis and improve tissue perfusion in ischemic rabbit skeletal muscle. CBP/p300, which interacts with the transactivation domains of HIF-1α, is important for HIF-1α-induced transcription of angiogenic genes.

Novel heart failure therapy targeting transcriptional pathway in cardiomyocytes by a natural compound, curcumin.

Hypertensive heart disease and post-myocardial-infarction heart failure (HF) are leading causes of cardiovascular mortality in industrialized countries. To date, pharmacological agents that block cell surface receptors for neurohormonal factors have been used, but despite such conventional therapy, HF is increasing in incidence worldwide. During the development and deterioration process of HF, cardiomyocytes undergo maladaptive hypertrophy, which markedly influences their gene expression. Regulation of histone acetylation by histone acetyltransferase (eg, p300) and histone deacetylase plays an important role in this process. Increasing evidence suggests that the excessive acetylation of cardiomyocyte nuclei is a hallmark of maladaptive cardiomyocyte hypertrophy. Curcumin inhibits p300-mediated nuclear acetylation, suggesting its usefulness in HF treatment. Clinical application of this natural compound, which is inexpensive and safe, should be established in the near future.

A dual-mode fluorescence strategy for screening HAT modulators.

Histone acetyltransferases (HATs) are an important class of epigenetic enzymes involved in chromatin restructuring and transcriptional regulation. We describe in this paper a novel approach for the identification and characterization of HAT inhibitors using both fluorescence resonance energy transfer (FRET) and fluorescence polarization. Expressed protein ligation (EPL) was used to label HATs PCAF and p300 with Dabcyl (Dab) as FRET acceptors. Methoxycoumarin (Mca) is conjugated to HAT substrate analogues to function as fluorescent donors, namely, H3CoA20Mca for interacting with PCAF and LysCoAMca for p300. When a ligand-protein interaction occurs, the fluorescent intensity of the donor fluorophore decreases due to FRET quenching by the Dab acceptor. Meanwhile, the formation of ligand-protein complexes causes reduction of the molecular mobility of the donor fluorophore, resulting in increased fluorescence anisotropy. Thus, dual modes of fluorescence measurement, FRET and anisotropy, are integrated in the same assay system. In particular, we demonstrated that both FRET and anisotropy measurements can be used to effectively detect and characterize HAT inhibitors. The developed strategy should be useful in the search of new anticancer drugs that target the substrate interfaces of the HAT targets, as well as find values in mechanistic study of HATs.

Anti-histone acetyltransferase activity from allspice extracts inhibits androgen receptor-dependent prostate cancer cell growth.

Histone acetylation depends on the activity of two enzyme families, histone acetyltransferase (HAT) and deacetylase (HDAC). In this study, we screened various plant extracts to find potent HAT inhibitors. Hot water extracts of allspice inhibited HAT activity, especially p300 and CBP (40% at 100 microg/ml). The mRNA levels of two androgen receptor (AR) regulated genes, PSA and TSC22, decreased with allspice treatment (100 microg/ml). Importantly, in IP western analysis, AR acetylation was dramatically decreased by allspice treatment.Furthermore, chromatin immunoprecipitation indicated that the acetylation of histone H3 in the PSA and B2M promoter regions was also repressed. Finally, allspice treatment reduced the growth of human prostate cancer cells, LNCaP (50% growth inhibition at 200 microg/ml). Taken together, our data indicate that the potent HAT inhibitory activity of allspice reduced AR and histone acetylation and led to decreased transcription of AR target genes, resulting in inhibition of prostate cancer cell growth.