Mechanistic and structural requirements for active site labeling of phosphoglycerate mutase by spiroepoxides

We recently reported the pharmacological screening of a natural products-inspired library of spiroepoxide probes, resulting in the discovery of an agent MJE3 that displayed anti-proliferative effects in human breast cancer cells. MJE3 was found to covalently inactivate phosphoglycerate mutase-1 (PGAM1), a glycolytic enzyme with postulated roles in cancer cell metabolism and proliferation. Considering that MJE3 is one of the first examples of a cell-permeable, small-molecule inhibitor for PGAM1, we pursued a detailed examination of its mechanism and structural requirements for covalent inactivation. MJE3 was found to label PGAM1 on lysine-100, a conserved active site residue implicated in substrate recognition. Structural features of MJE3 important for PGAM1 labeling included two key recognition elements (an indole ring and carboxylic acid), the stereochemical orientation of the spiroepoxide, and presentation of these various binding/reactive groups on a rigid cyclohexane scaffold. Modeling studies of the docked MJE3-PGAM1 complex provide a structural rationale for these stringent requirements. Overall, these studies indicate that a special combination of binding and reactive elements are united in the MJE3 structure to inactivate PGAM1. More generally, our findings provide further evidence that useful pharmacological tools can emerge from screening structurally diverse libraries of protein-reactive probes.     

Identification of bioactive molecules by adipogenesis profiling of organic compounds

An important step in the postgenomic drug discovery is the construction of high quality chemical libraries that generate bioactive molecules at high rates. Here we report a cell-based approach to composing a focused library of biologically active compounds. A collection of bioactive non-cytotoxic chemicals was identified from a divergent library through the effects on the insulin-induced adipogenesis of 3T3-L1 cells, one of the most drastic and sensitive morphological alterations in cultured mammalian cells. The resulting focused library amply contained unique compounds with a broad range of pharmacological effects, including glucose-uptake enhancement, cytokine inhibition, osteogenesis stimulation, and selective suppression of cancer cells. Adipogenesis profiling of organic compounds generates a focused chemical library for multiple biological effects that are seemingly unrelated to adipogenesis, just as genetic screens with the morphology of fly eyes identify oncogenes and neurodegenerative genes.     

Identification and validation of bioactive small molecule target through phenotypic screening

For effective bioactive small molecule discovery and development into new therapeutic drug, a systematic screening and target protein identification is required. Different from the conventional screening system, herein phenotypic screening in combination with multi-omics-based target identification and validation (MOTIV) is introduced. First, phenotypic screening provides visual effect of bioactive small molecules in the cell or organism level. It is important to know the effect on the cell or organism level since small molecules affect not only a single target but the entire cellular mechanism within a cell or organism. Secondly, MOTIV provides systemic approach to discover the target protein of bioactive small molecule. With the chemical genomics and proteomics approach of target identification methods, various target protein candidates are identified. Then network analysis and validations of these candidates result in identifying the biologically relevant target protein and cellular mechanism. Overall, the combination of phenotypic screening and MOTIV will provide an effective approach to discover new bioactive small molecules and their target protein and mechanism identification.     

Emodin-Induced Necroptosis in Prostate Cancer Cells via the Mitochondrial Fission HSP90/MLKL/PGAM Pathway

Currently, prostate cancer is one of the major malignant tumors in males. Recurrence and metastasis are the main obstacles that prevent the effective treatment of prostate cancer. In the present study, we aimed to evaluate emodin (EG) against human prostate cancer PC3 and DU145 cells. Our study showed that EG significantly decreased the cell viability of PC3 and DU145 cells and strikingly induced non-apoptotic cell death via necroptosis that was visualized through colony formation assay, Hoechst 33258 staining, and TEM analysis. Furthermore, RNA-sequencing and KEGG functional enrichment analysis revealed that the necroptosis-related pathway was activated upon EG treatment in PC3 cells. mRNA and protein expression of necroptosis markers were analyzed by qPCR and immunoblotting, which implied that EG-induced cell necroptosis via enhancing the expression of MLKL and HSP90AA1 activating PGAM pathway which is considered as a key mediator of mitochondrial fission and leading to ROS generation in PC3 and DU145 cells. Thus, our findings suggested that EG is a new small molecule agonist that induced necroptosis in prostate cancer cells via the mitochondrial fission HSP90/MLKL/PGAM pathway.     

Prostaglandin E(2) production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E(2) production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE(2) production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC(50) of 1.0 microM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE(2) production by activated macrophages.     

Prostaglandin E2 production and induction of prostaglandin endoperoxide synthase-2 is inhibited in a murine macrophage-like cell line,RAW 264.7, by Mallotus japonicus phloroglucinol derivatives

An aqueous acetone extract obtained from the pericarps of Mallotus japonicus (MJE) was observed to inhibit prostaglandin (PG) E₂ production in a lipopolysaccharide (LPS)-activated murine macrophage-like cell line, RAW 264.7. Six phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against PGE₂ production. Among these phloroglucinol derivatives, isomallotochromanol showed the strongest inhibitory activity, with an IC₅₀ of 1.0 μM. MJE and its phloroglucinol derivatives did not effect the enzyme activity of either prostaglandin endoperoxide synthase (PGHS)-1 or PGHS-2. However, induction of PGHS-2 in LPS-activated macrophages was inhibited by MJE and its phloroglucinol derivatives, whereas the level of PGHS-1 protein was not affected. Moreover, RT-PCR analysis showed that MJE and its phloroglucinol derivatives significantly suppressed PGHS-2 mRNA expression. Therefore, the observed inhibition of PGHS-2 induction by MJE and its phloroglucinol derivatives was likely due to a suppression of PGHS-2 mRNA expression. These results suggest that MJE and its phloroglucinol derivatives have the pharmacological ability to suppress PGE₂ production by activated macrophages.     

Recent advances in target identification of bioactive natural products

Natural products are a tremendous source of tool discovery for basic science and drug discovery for clinical uses. In contrast to the large number of compounds isolated from nature, however, the number of compounds whose target molecules have been identified so far is fairly limited. Elucidation of the mechanism of how bioactive small molecules act in cells to induce biological activity (mode of action) is an attractive but challenging field of basic biology. At the same time, this is the major bottleneck for drug development of compounds identified in cell-based and phenotype-based screening. Although researchers’ experience and inspiration have been crucial for successful target identification, recent advancements in genomics, proteomics, and chemical genomics have made this challenging task possible in a systematic fashion.     

Application of encoded library technology (ELT) to a protein–protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists

The inhibition of protein–protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.     

Analysis of multiple compound–protein interactions reveals novel bioactive molecules

The discovery of novel bioactive molecules advances our systems‐level understanding of biological processes and is crucial for innovation in drug development. For this purpose, the emerging field of chemical genomics is currently focused on accumulating large assay data sets describing compound–protein interactions (CPIs). Although new target proteins for known drugs have recently been identified through mining of CPI databases, using these resources to identify novel ligands remains unexplored. Herein, we demonstrate that machine learning of multiple CPIs can not only assess drug polypharmacology but can also efficiently identify novel bioactive scaffold‐hopping compounds. Through a machine‐learning technique that uses multiple CPIs, we have successfully identified novel lead compounds for two pharmaceutically important protein families, G‐protein‐coupled receptors and protein kinases. These novel compounds were not identified by existing computational ligand‐screening methods in comparative studies. The results of this study indicate that data derived from chemical genomics can be highly useful for exploring chemical space, and this systems biology perspective could accelerate drug discovery processes.     

Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2

Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage–induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.     

Inhibitory effects of phloroglucinol derivatives from Mallotus japonicus on nitric oxide production by a murine macrophage-like cell line, RAW 264.7, activated by lipopolysaccharide and interferon-gamma.

An aqueous acetone extract of the pericarps of Mallotus japonicus (MJE) inhibited nitric oxide (NO) production by a murine macrophage-like cell line, RAW 264.7, which was activated by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma). Seven phloroglucinol derivatives isolated from MJE exhibited inhibitory activity against NO production. Among these phloroglucinol derivatives, isomallotochromanol exhibited strong inhibitory activity toward NO production, exhibiting an IC(50) of 10.7 microM. MJE and the phloroglucinol derivatives significantly reduced both the induction of inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. NO production by macrophages preactivated with LPS and IFN-gamma for 16 h was also inhibited by MJE and the phloroglucinol derivatives. Furthermore, MJE and the derivatives directly affected the conversion of L-[(14)C]arginine to L-[(14)C]citrulline by the cell extract. These results suggest that MJE and the phloroglucinol derivatives have the pharmacological ability to suppress NO production by activated macrophages. They inhibited NO production by two mechanisms: reduction of iNOS protein induction and inhibition of enzyme activity.     

Xanthone derivatives as phosphoglycerate mutase 1 inhibitors: Design,synthesis, and biological evaluation

Phosphoglycerate mutase 1 (PGAM1) is a glycolytic enzyme that dynamically converts 3-phosphoglycerate (3PG) to 2-phosphoglycerate (2PG), which was upregulated to coordinate glycolysis, pentose phosphate pathway (PPP) and serine biosynthesis to promote cancer cell proliferation and tumor growth in a variety of cancers. However, only a few inhibitors of PGAM1 have been reported with poor molecular or cellular efficacy. In this paper, a series of xanthone derivatives were discovered as novel PGAM1 inhibitors through scaffold hopping and sulfonamide reversal strategy based on the lead compound PGMI-004A. Most xanthone derivatives showed higher potency against PGAM1 than PGMI-004A and exhibited moderate anti-proliferation activity on different cancer cell lines.     

Targeting Mcl-1 for Radiosensitization of Pancreatic Cancers

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-X_L, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.     

Sphingosine inhibits nitric oxide synthase from cerebellar granule cells differentiated in vitro.

The effects of different bioactive sphingoid molecules on NOS activity of differentiated cerebellar granule cells were investigated by measuring the conversion of [3H]arginine to [3H]citrulline. Cytosolic Ca2+-dependent NOS activity was strongly inhibited in a dose-dependent manner by sphingosine in concentrations of 1-40 microM. This inhibition seems to be peculiar to sphingosine in that ceramide, N-acetylsphingosine, sphingosine-1P, sphinganine and tetradecylamine have no effect on the cytosolic enzyme at the considered concentrations, suggesting that it is the bulk of the sphingosine hydrophilic portion that is critical for cytosolic NOS inhibition. This inhibition of cytosolic NOS is not reversed by increasing the arginine concentration, so a competitive mechanism can be excluded. Instead, increasing the concentrations of calmodulin led to loss of sphingosine inhibition, suggesting that sphingosine interferes with the calmodulin-dependent activation of the enzyme by a competitive mechanism. Sphingosine and related compounds had no effect on the particulate Ca2+-independent NOS activity. The data obtained suggest that sphingosine could be involved in the regulation of NO production in neurons.     

Cyclosporine A and bromocriptine attenuate cell death mediated by intracellular calcium mobilization

To identify the novel inhibitors of endoplasmic reticulum stress-induced cell death, we performed a high throughput assay with a chemical library containing a total of 3280 bioactive small molecules. Cyclosporine A and bromocriptine were identified as potent inhibitors of thapsigargiin-induced cell death (cut-off at 4σ standard score) . However, U74389G, the potent inhibitor of lipid peroxidation had lower activity in inhibiting cell death. The inhibition effect of cyclosporine A and bromocriptine was specific for only thapsigargin-induced cell death. The mechanism of inhibition by these compounds was identified as modification of the expression of glucose regulated protein-78 (GRP-78/Bip) and inhibition of phosphorylation of p38 mitogen activated protein kinase (MAPK). However, these compounds did not inhibit the same events triggered by tunicamycin, which was in agreement with the cell survival data. We suggest that the induction of protective unfolded protein response by these compounds confers resistance to cell death. In summary, we identified compounds that may provide insights on cell death mechanisms stimulated by ER stress. [BMB Reports 2012; 45(8): 482-487].