The Pseudo-Natural Product Rhonin Targets RHOGDI.

For the discovery of novel chemical matter generally endowed with bioactivity, strategies may be particularly efficient that combine previous insight about biological relevance, e.g., natural product (NP) structure, with methods that enable efficient coverage of chemical space, such as fragment-based design. We describe the de novo combination of different 5-membered NP-derived N-heteroatom fragments to structurally unprecedented "pseudo-natural products" in an efficient complexity-generating and enantioselective one-pot synthesis sequence. The pseudo-NPs inherit characteristic elements of NP structure but occupy areas of chemical space not covered by NP-derived chemotypes, and may have novel biological targets. Investigation of the pseudo-NPs in unbiased phenotypic assays and target identification led to the discovery of the first small-molecule ligand of the RHO GDP-dissociation inhibitor 1 (RHOGDI1), termed Rhonin. Rhonin inhibits the binding of the RHOGDI1 chaperone to GDP-bound RHO GTPases and alters the subcellular localization of RHO GTPases.

Discovery of small-molecule modulator of heterotrimeric Gi-protein by integrated phenotypic profiling and chemical proteomics.

Discovery of small-molecule inducers of unique phenotypic changes combined with subsequent target identification often provides new insights into cellular functions. Here, we applied integrated profiling based on cellular morphological and proteomic changes to compound screening. We identified an indane derivative, NPD9055, which is mechanistically distinct from reference compounds with known modes of action. Employing a chemical proteomics approach, we then showed that NPD9055 binds subunits of heterotrimeric G-protein Gi. An in vitro [35S]GTPγS-binding assay revealed that NPD9055 inhibited GDP/GTP exchange on a Gαi subunit induced by a G-protein-coupled receptor agonist, but not on another G-protein from the Gαs family. In intact HeLa cells, NPD9055 induced an increase in intracellular Ca2+ levels and ERK/MAPK phosphorylation, both of which are regulated by Gßγ, following its dissociation from Gαi. Our observations suggest that NPD9055 targets Gαi and thus regulates Gßγ-dependent cellular processes, most likely by causing the dissociation of Gßγ from Gαi.

Small molecules capable of activating DNA methylation-repressed genes targeted by the p38 mitogen-activated protein kinase pathway.

Regulation of gene expression by epigenetic modifications such as DNA methylation is crucial for developmental and disease processes, including cell differentiation and cancer development. Genes repressed by DNA methylation can be derepressed by various compounds that target DNA methyltransferases, histone deacetylases, and other regulatory factors. However, some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Chemical agents that can counteract the effects of epigenetic repression that is not regulated by DNA methyltransferases or histone deacetylases therefore may be of research interest. Here, we report the results of a high-throughput screen using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation. Seven hit compounds were identified that did not directly target bulk DNA methylation or histone acetylation. Analyzing the effect of these compounds on endogenous gene expression, we discovered that three of these compounds (compounds LX-3, LX-4, and LX-5) selectively activate the p38 mitogen-activated protein kinase (MAPK) pathway and derepress a subset of endogenous genes repressed by DNA methylation. Selective agonists of the p38 pathway have been lacking, and our study now provides critical compounds for studying this pathway and p38 MAPK-targeted genes repressed by DNA methylation.

Synthesis and Evaluation of a New Type of Small Molecule Epigenetic Modulator Containing Imidazo[1,2-b][1,2,4]triazole Motif.

Epigenetic modifications such as DNA methylation is important for many cellular processes, such as cell differentiation and cell death. The disorder of epigenetic state is closely related to human diseases, especially cancers. DNA methylation is a well-characterized epigenetic modification which is related to gene silencing and is considered as a repressive epigenetic mark. DNA methylation caused gene repression can be derepressed by chemical agents. Small molecules targeting DNA methyltransferases, histone deacetylases, and other regulatory factors can activate genes silenced by DNA methylation. However, more and more studies have shown that histone deacetylation is not the only downstream event of DNA methylation. Some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Recently, through high-throughput screening using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation, we identified seven hit compounds that did not directly target bulk DNA methylation or histone acetylation. Three of them (LX-3, LX-4, LX-5) were proven to selectively activate the p38 MAPK pathway in multiple cell types. In order to identify the exact cellular targets of these compounds, we turn to work on the SAR study of LX-3 by constructing a structurally diverse chemical library based on the imidazo[1,2-b][1,2,4]triazole core structure via diversity-oriented synthesis. Our work provides a general approach to efficiently access diverse heterocyclic molecules with interesting epigenetic modulation activities.

Syntheses of [1,2,4]triazolo[1,5-a]benzazoles enabled by the transition-metal-free oxidative N-N bond formation.

A transition-metal-free oxidative N-N bond formation strategy was developed to generate various structurally interesting [1,2,4]triazolo[1,5-a]benzazoles efficiently. The mechanism of the key oxidative N-N bond formation was investigated by using an intramolecular competition reaction. Notably, the first single crystal structure was also obtained to confirm the structure of 2-aryl[1,2,4]triazolo[1,5-a]benzimidazole.

Discovery of Novel 15-Lipoxygenase Activators To Shift the Human Arachidonic Acid Metabolic Network toward Inflammation Resolution.

For disease network intervention, up-regulating enzyme activities is equally as important as down-regulating activities. However, the design of enzyme activators presents a challenging route for drug discovery. Previous studies have suggested that activating 15-lipoxygenase (15-LOX) is a promising strategy to intervene the arachidonic acid (AA) metabolite network and control inflammation. To prove this concept, we used a computational approach to discover a previously unknown allosteric site on 15-LOX. Both allosteric inhibitors and novel activators were discovered using this site. The influence of activating 15-LOX on the AA metabolite network was then investigated experimentally. The activator was found to increase levels of 15-LOX products and reduce production of pro-inflammatory mediators in human whole blood assays. These results demonstrate the promising therapeutic value of enzyme activators and aid in further development of activators of other proteins.

Benzo[d]isothiazole 1,1-dioxide derivatives as dual functional inhibitors of 5-lipoxygenase and microsomal prostaglandin E(2) synthase-1.

A series of 6-nitro-3-(m-tolylamino) benzo[d]isothiazole 1,1-dioxide analogues were synthesized and evaluated for their inhibition activity against 5-lipoxygenase (5-LOX) and microsomal prostaglandin E2 synthase (mPGES-1). These compounds can inhibit both enzymes with IC50 values ranging from 0.15 to 23.6µM. One of the most potential compounds, 3g, inhibits 5-LOX and mPGES-1 with IC50 values of 0.6µM, 2.1µM, respectively.

Development of 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors.

Human 5-lipoxygenase (5-LOX) is a well-validated target for anti-inflammatory therapy. Development of novel 5-LOX inhibitors with higher activities is highly demanded. In previous study, we have built a model for the active conformation of human 5-LOX, and identified naphthalen-1-yl 3,5-dinitrobenzoate (JMC-4) as a 5-LOX inhibitor by virtual screening. In the present work, 3,5-dinitrobenzoate-based 5-lipoxygenase inhibitors were developed. Twenty aryl 3,5-dinitrobenzoates, N-aryl 3,5-dinitrobenzamides and analogues were designed and synthesized. Several of them were found with significantly increased activities according to cell-free assay and human whole blood assay. The structure-activity relationship study may provide useful insights for designing effective 5-LOX inhibitors.

De novo design of multitarget ligands with an iterative fragment-growing strategy.

The discovery of multitarget drugs has recently attracted much attention. Most of the reported multitarget ligands have been serendipitous discoveries. Although a few methods have been developed for rational multitarget drug discovery, there is a lack of elegant methods for de novo multitarget drug design and optimization, especially for multiple targets with large differences in their binding sites. In this paper, we report the first de novo multitarget ligand design method, with an iterative fragment-growing strategy. Using this method, dual-target inhibitors for COX-2 and LTA4H were designed, with the most potent one inhibiting PGE2 and LTB4 production in the human whole blood assay with IC50 values of 7.0 and 7.1 µM, respectively. Our strategy is generally applicable in rational and efficient multitarget drug design, especially for the design of highly integrated inhibitors for proteins with dissimilar binding pockets.

Structure and novel functional mechanism of Drosophila SNF in sex-lethal splicing.

Sans-fille (SNF) is the Drosophila homologue of mammalian general splicing factors U1A and U2B'', and it is essential in Drosophila sex determination. We found that, besides its ability to bind U1 snRNA, SNF can also bind polyuridine RNA tracts flanking the male-specific exon of the master switch gene Sex-lethal (Sxl) pre-mRNA specifically, similar to Sex-lethal protein (SXL). The polyuridine RNA binding enables SNF directly inhibit Sxl exon 3 splicing, as the dominant negative mutant SNF(1621) binds U1 snRNA but not polyuridine RNA. Unlike U1A, both RNA recognition motifs (RRMs) of SNF can recognize polyuridine RNA tracts independently, even though SNF and U1A share very high sequence identity and overall structure similarity. As SNF RRM1 tends to self-associate on the opposite side of the RNA binding surface, it is possible for SNF to bridge the formation of super-complexes between two introns flanking Sxl exon 3 or between a intron and U1 snRNP, which serves the molecular basis for SNF to directly regulate Sxl splicing. Taken together, a new functional model for SNF in Drosophila sex determination is proposed. The key of the new model is that SXL and SNF function similarly in promoting Sxl male-specific exon skipping with SNF being an auxiliary or backup to SXL, and it is the combined dose of SXL and SNF governs Drosophila sex determination.

[Controlling arachidonic acid metabolic network: from single- to multi-target inhibitors of key enzymes].

Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.