N-terminal acetylation is an abundant modification influencing protein functions. Since ≈80% of mammalian cytosolic proteins are N-terminally acetylated, this potentially represents an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions, suggesting it may be a druggable target. We report the development of chemical probes targeting the N-terminal acetylation-dependent interaction between an E2 conjugating enzyme (UBE2M, aka UBC12) and DCN1 (aka DCUN1D1), a subunit of a multiprotein E3 ligase for the ubiquitin-like protein NEDD8. The inhibitors are highly selective with respect to other protein acetyl amide binding sites, inhibit NEDD8 ligation in vitro and in cells, and suppress the anchorage-independent growth of a cell line harboring DCN1 amplification. Overall, the data demonstrate that N-terminal acetyl-dependent protein interactions are druggable targets, and provide insights into targeting multiprotein E2–E3 ligases.

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.

SUMMARY Pantothenate kinase (PanK) catalyzes the rate-controlling step in coenzyme A (CoA) biosynthesis. PanK3 is stringently regulated by acetyl-CoA and uses an ordered kinetic mechanism with ATP as the leading substrate. Biochemical analysis of site-directed mutants indicates that pantothenate binds in a tunnel adjacent to the active site that is occupied by the pantothenate moiety of the acetyl-CoA regulator in the PanK3•acetyl-CoA binary complex. A high-throughput screen for PanK3 inhibitors and activators was applied to a bioactive compound library. Thiazolidinediones, sulfonylureas and steroids were inhibitors, and fatty acyl-amides and tamoxifen were activators. The PanK3 activators and inhibitors either stimulated or repressed CoA biosynthesis in HepG2/C3A cells. The flexible allosteric acetyl-CoA regulatory domain of PanK3 also binds the substrates, pantothenate and pantetheine, and small molecule inhibitors and activators to modulate PanK3 activity.

ATP‐binding cassette sub‐family G member 2 (ABCG2) is a homodimeric ATP‐binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno‐ and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π‐π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π‐π interaction “clamps” the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π‐π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.

Here we describe three new small-molecule activators of BMP signaling found by high throughput screening of a library of ~600,000 small molecules. Using a cell-based luciferase assay in the BMP4-responsive human cervical carcinoma clonal cell line, C33A-2D2, we identified three compounds with similar chemotypes that each ventralize zebrafish embryos and stimulate increased expression of the BMP target genes, bmp2b and szl. Because these compounds ventralize zebrafish embryos, we have termed them “ventromorphins.” As expected for BMP pathway activators, they induce the differentiation of C2C12 myoblasts to osteoblasts. Affymetrix RNA analysis confirmed the differentiation results and showed that ventromorphins treatment elicits a response similar to BMP4 treatment. Unlike Isoliquiritigenin (SJ000286237), a flavone that maximally activates the pathway after 24 hours of treatment, all three ventromorphins induced SMAD1/5/8 phosphorylation within 30 minutes of treatment and achieved peak activity within 1 hour, indicating that their responses are consistent with directly activated BMP signaling.

Pantothenate kinase (PanK) is a regulatory enzyme that controls coenzyme A (CoA) biosynthesis. The association of PanK with neurodegeneration and diabetes suggests that chemical modifiers of PanK activity may be useful therapeutics. We performed a high throughput screen of >520000 compounds from the St. Jude compound library and identified new potent PanK inhibitors and activators with chemically tractable scaffolds. The HTS identified PanK inhibitors exemplified by the detailed characterization of a tricyclic compound (7) and a preliminary SAR. Biophysical studies reveal that the PanK inhibitor acts by binding to the ATP–enzyme complex.

Introduction The constitutive androstane receptor (CAR) induces drug-metabolizing enzymes for xenobiotic metabolism. Areas covered This review covers recent advances in elucidating the biological functions of CAR and its modulation by a growing number of agonists and inhibitors. Expert opinion Extrapolation of animal CAR function to that of humans should be carefully scrutinized, particularly when rodents are used in evaluating the metabolic profile and carcinogenic properties of clinical drugs and environmental chemicals. Continuous efforts are needed to discover novel CAR inhibitors, with extensive understanding of their inhibitory mechanism, species selectivity, and discriminating power against other xenobiotic sensors.

The pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) are ligandactivated nuclear receptors (NRs) that are notorious for their role in drug metabolism, causing unintended drug-drug interactions and decreasing drug efficacy. They control the xenobiotic detoxification system by regulating the expression of an array of drug-metabolizing enzymes and transporters that excrete exogenous chemicals and maintain homeostasis of endogenous metabolites. Much effort has been invested in recognizing potential drugs for clinical use that can activate PXR and CAR to enhance the expression of their target genes, and in identifying PXR and CAR inhibitors that can be used as co-therapeutics to prevent adverse effects. Here, we present current technologies and assays used in the quest to characterize PXR and CAR modulators, which range from biochemical to cell-based and animal models.