VEGF inhibition increases expression of HIF-regulated angiogenic genes by the RPE limiting the response of wet AMD eyes to aflibercept.

Neovascular age-related macular degeneration (nvAMD) is the leading cause of severe vision loss in the elderly in the developed world. While the introduction of therapies targeting vascular endothelial growth factor (VEGF) has provided the first opportunity to significantly improve vision in patients with nvAMD, many patients respond inadequately to current anti-VEGF therapies. It was recently demonstrated that expression of a second angiogenic mediator, angiopoietin-like 4 (ANGPTL4), synergizes with VEGF to promote choroidal neovascularization (CNV) in mice and correlates with reduced response to anti-VEGF therapy in patients with nvAMD.

Pharmacologic HIF stabilization activates costimulatory receptor expression to increase antitumor efficacy of adoptive T cell therapy.

Adoptive cell transfer (ACT) is a therapeutic strategy to augment antitumor immunity. Here, we report that ex vivo treatment of mouse CD8 T cells with dimethyloxalylglycine (DMOG), a stabilizer of hypoxia-inducible factors (HIFs), induced HIF binding to the genes encoding the costimulatory receptors CD81, GITR, OX40, and 4-1BB, leading to increased expression. DMOG treatment increased T cell killing of melanoma cells, which was further augmented by agonist antibodies targeting each costimulatory receptor.

Targeting hypoxia-inducible factors with 32-134D safely and effectively treats diabetic eye disease in mice.

Many patients with diabetic eye disease respond inadequately to anti-VEGF therapies, implicating additional vasoactive mediators in its pathogenesis. We demonstrate that levels of angiogenic proteins regulated by HIF-1 and -2 remain elevated in the eyes of people with diabetes despite treatment with anti-VEGF therapy. Conversely, by inhibiting HIFs, we normalized the expression of multiple vasoactive mediators in mouse models of diabetic eye disease.

NMR as a readout to monitor and restore the integrity of complex chemoenzymatic reactions.

The non-invasive nature of NMR offers a means to monitor biochemical reactions in situ at the atomic level. We harness this advantage to monitor a complex chemoenzymatic reaction that sequentially modifies reagents and loads the product on a nonribosomal peptide synthetase carrier protein. We present a protocol including a pulse sequence that permits to assess both the integrity of reagents and the completion of each step in the reaction, thus alleviating otherwise time-consuming and costly approaches to debug and repeat inefficient reactions.

HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy.

Hepatocellular carcinoma (HCC) is a major cause of cancer mortality worldwide and available therapies, including immunotherapies, are ineffective for many patients. HCC is characterized by intratumoral hypoxia, and increased expression of hypoxia-inducible factor 1α (HIF-1α) in diagnostic biopsies is associated with patient mortality. Here we report the development of 32-134D, a low-molecular-weight compound that effectively inhibits gene expression mediated by HIF-1 and HIF-2 in HCC cells, and blocks human and mouse HCC tumor growth.

CREB Promotes Beta Cell Gene Expression by Targeting Its Coactivators to Tissue-Specific Enhancers.

CREB mediates effects of cyclic AMP on cellular gene expression. Ubiquitous CREB target genes are induced following recruitment of CREB and its coactivators to promoter proximal binding sites. We found that CREB stimulates the expression of pancreatic beta cell-specific genes by targeting CBP/p300 to promoter-distal enhancer regions.

Combination Targeting of the Bromodomain and Acetyltransferase Active Site of p300/CBP.

p300 and CBP are highly related histone acetyltransferase (HAT) enzymes that regulate gene expression, and their dysregulation has been linked to cancer and other diseases. p300/CBP is composed of a number of domains including a HAT domain, which is inhibited by the small molecule A-485, and an acetyl-lysine binding bromodomain, which was recently found to be selectively antagonized by the small molecule I-CBP112. Here we show that the combination of I-CBP112 and A-485 can synergize to inhibit prostate cancer cell proliferation.

Hydrazide Mimics for Protein Lysine Acylation To Assess Nucleosome Dynamics and Deubiquitinase Action.

A range of acyl-lysine (acyl-Lys) modifications on histones and other proteins have been mapped over the past decade but for most, their functional and structural significance remains poorly characterized. One limitation in the study of acyl-Lys containing proteins is the challenge of producing them or their mimics in site-specifically modified forms. We describe a cysteine alkylation-based method to install hydrazide mimics of acyl-Lys post-translational modifications (PTMs) on proteins.

Enzyme-catalyzed expressed protein ligation.

Expressed protein ligation is a valuable method for protein semisynthesis that involves the reaction of recombinant protein C-terminal thioesters with N-terminal cysteine (N-Cys)-containing peptides, but the requirement of a Cys residue at the ligation junction can limit the utility of this method. Here we employ subtiligase variants to efficiently ligate Cys-free peptides to protein thioesters. Using this method, we have more accurately determined the effect of C-terminal phosphorylation on the tumor suppressor protein PTEN.

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs.

Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features.

Architectural organization of the metabolic regulatory enzyme ghrelin O-acyltransferase.

Ghrelin O-acyltransferase (GOAT) is a polytopic integral membrane protein required for activation of ghrelin, a secreted metabolism-regulating peptide hormone. Although GOAT is a potential therapeutic target for the treatment of obesity and diabetes and plays a key role in other physiologic processes, little is known about its structure or mechanism. GOAT is a member of the membrane-bound O-acyltransferase (MBOAT) family, a group of polytopic integral membrane proteins involved in lipid-biosynthetic and lipid-signaling reactions from prokaryotes to humans.

Ghrelin O-acyltransferase assays and inhibition.

Ghrelin O-acyltransferase (GOAT) is responsible for catalyzing the attachment of the eight-carbon fatty acid octanoyl to the Ser3 side chain of the peptide ghrelin to generate the active form of this metabolic hormone. As such, GOAT is viewed as a potential therapeutic target for the treatment of obesity and diabetes mellitus. Here, we review recent progress in the development of cell and in vitro assays to measure GOAT action and the identification of several synthetic GOAT inhibitors.

Glucose and weight control in mice with a designed ghrelin O-acyltransferase inhibitor.

Ghrelin is a gastric peptide hormone that stimulates weight gain in vertebrates. The biological activities of ghrelin require octanoylation of the peptide on Ser(3), an unusual posttranslational modification that is catalyzed by the enzyme ghrelin O-acyltransferase (GOAT). Here, we describe the design, synthesis, and characterization of GO-CoA-Tat, a peptide-based bisubstrate analog that antagonizes GOAT.

Site-specific introduction of an acetyl-lysine mimic into peptides and proteins by cysteine alkylation.

Protein acetylation on Lys residues is recognized as a significant post-translational modification in cells, but it is often difficult to discern the direct structural and functional effects of individual acetylation events. Here we describe a new tool, methylthiocarbonyl-aziridine, to install acetyl-Lys mimics site-specifically into peptides and proteins by alkylation of Cys residues. We demonstrate that the resultant thiocarbamate modification can be recognized by the Brdt bromodomain and site-specific antiacetyl-Lys antibodies, is resistant to histone deacetylase cleavage, and can confer activation of the histone acetyltransferase Rtt109 by simulating autoacetylation.

Electrophilic tuning of the chemoprotective natural product sulforaphane.

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)butane], a naturally occurring isothiocyanate derived from cruciferous vegetables, is a highly potent inducer of phase 2 cytoprotective enzymes and can protect against electrophiles including carcinogens, oxidative stress, and inflammation. The mechanism of action of sulforaphane is believed to involve modifications of critical cysteine residues of Keap1, which lead to stabilization of Nrf2 to activate the antioxidant response element of phase 2 enzymes. However, the dithiocarbamate functional group formed by a reversible reaction between isothiocyanate of sulforaphane and sulfhydryl nucleophiles of Keap1 is kinetically labile, and such modification in intact cells has not yet been demonstrated.

The structural basis of protein acetylation by the p300/CBP transcriptional coactivator.

The transcriptional coactivator p300/CBP (CREBBP) is a histone acetyltransferase (HAT) that regulates gene expression by acetylating histones and other transcription factors. Dysregulation of p300/CBP HAT activity contributes to various diseases including cancer. Sequence alignments, enzymology experiments and inhibitor studies on p300/CBP have led to contradictory results about its catalytic mechanism and its structural relation to the Gcn5/PCAF and MYST HATs.

Enzymatic and cellular study of a serotonin N-acetyltransferase phosphopantetheine-based prodrug.

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) regulates the daily rhythm in the production of melatonin and is therefore an attractive target for pharmacologic modulation of the synthesis of this hormone. Previously prepared bisubstrate analogs show potent inhibition of AANAT but have unfavorable pharmacokinetic properties due to the presence of phosphate groups which prevents transfer across the plasma membrane. Here, we examine a bis-pivaloyloxymethylene (POM)-tryptamine-phosphopantetheine prodrug (2) and its biotransformations in vitro by homogenates and pineal cells.

Efficient synthesis of phosphorylated prodrugs with bis(POM)-phosphoryl chloride.

An efficient method for the synthesis of phosphorylated prodrugs is described. The preparation of various bis-pivaloyloxymethyl (POM) phosphate triesters was accomplished in moderate to good yields with the use of bis(POM) phosphoryl chloride under mild conditions.