Chenglong Li | University of Florida (original) (raw)

Papers by Chenglong Li

Mol Cancer, 2010

Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibit... more Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells. Results: FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO). Conclusions: These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

Neoplasia, 2010

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling i... more Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 μM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Molecular bioSystems, Jan 29, 2015

Curcumin, a natural product, has been shown to possess notable anti-inflammatory activities and n... more Curcumin, a natural product, has been shown to possess notable anti-inflammatory activities and numerous studies have been carried out on its clinical applications. Recently, several reports mentioned that myeloid differentiation protein 2 (MD-2) may be the direct target of curcumin in the inhibition of lipopolysaccharide (LPS) signaling. However, the exact interaction between curcumin and MD-2 is still incompletely understood. In the present study, computational and experimental methods were employed to explore the underlying structural mechanism of curcumin binding to the MD-2 protein. Molecular docking and molecular dynamics (MD) simulation studies showed that curcumin could be embedded into the hydrophobic pocket of MD-2 and form stable hydrogen bonding interactions with residues R90 and Y102 of MD-2. Moreover, experimental results of curcumin binding to the MD-2(R90A/Y102A) mutant further confirmed that residues ARG-90 and TYR-102 contribute to the recognition process of curcum...

Biosensors & bioelectronics, Jan 10, 2015

We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS)... more We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS) and a fluorescence probe for multi-sulphonamide detection. Dihydropteridine pyrophosphate (DHPPP) was synthesised and acts as the first substrate for DHPS. Under optimised conditions, the half-maximal inhibitory concentrations (IC50) of the assay were less than 100ngmL(-1) for at least 29 sulphonamides and the time needed for the detection was less than 20min. More importantly, the assay revealed quite uniform affinities for all of the individual sulphonamides tested, which has never before been achieved in an antibody-based assay.

Journal of molecular graphics & modelling, 2014

Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind importan... more Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind important biochemical processes such as protein folding and molecular interaction. With the rapidly increasing power of modern computers, large-scale MD simulation experiments can be performed regularly, generating huge amounts of MD data. An important question is how to analyze and interpret such massive and complex data. One of the (many) challenges involved in analyzing MD simulation data computationally is the high-dimensionality of such data. Given a massive collection of molecular conformations, researchers typically need to rely on their expertise and prior domain knowledge in order to retrieve certain conformations of interest. It is not easy to make and test hypotheses as the data set as a whole is somewhat "invisible" due to its high dimensionality. In other words, it is hard to directly access and examine individual conformations from a sea of molecular structures, and to fu...

American journal of translational research, 2014

Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully und... more Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully understood. We demonstrate that the histone demethylases JMJD2A, JMJD2B and JMJD2C are overexpressed in colon cancer cell lines, whereas another related protein, JMJD2D, is not. Interestingly, despite their high homology, the intracellular localization of JMJD2A-C is different in colon and other cancer cells, with JMJD2A being present comparably in the cytoplasm and nucleus, JMJD2B more prevalent in the nucleus and JMJD2C strongly associated with chromatin. This suggests that each of these three proteins performs different, non-redundant functions. Moreover, we show that JMJD2C (also called KDM4C) forms complexes with β-catenin, an oncoprotein whose overexpression is crucial for the development of most colonic tumors. In addition, JMJD2C downregulation reduced both growth and clonogenic capacity of HCT-116 colon cancer cells. Further, JMJD2C was required for efficient expression of the grow...

Tetrahedron, 2014

The first one-pot copper-catalyzed highly regioselective CeN bond formation between aryl/alkyl am... more The first one-pot copper-catalyzed highly regioselective CeN bond formation between aryl/alkyl amines and sulfonamide-substituted naphthoquinones was accomplished. Facile chemoselective routes obtained diverse ring-opening 6-amino/anilino-naphthalen-dione-1-sulfonamides and ring-fused 6-amino/aniline-5H-naphth[1,8-cd]isothiazol-5-one,1,1-dioxides with great functional group tolerance. Regiochemistry was confirmed by 1D-and 2D-NMRs.

Structure, 2002

Most TNFRs activate intracellular signaling pathways, including the NF-B and c-JUN N-terminal kin... more Most TNFRs activate intracellular signaling pathways, including the NF-B and c-JUN N-terminal kinase (JNK) 1 Cancer Research Center The Burnham Institute [7-9]. With the identification of proteins that bind directly to La Jolla, California 92037 2 Johnsson Comprehensive Cancer Center the cytoplasmic domains of TNFRs called TNFR-associated factors (TRAFs) [10], an understanding of TNF

Molecular Cancer, 2010

Background: Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an ... more Background: Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells. Results: FLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ. Conclusion: Our findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling.

Journal of Pharmacology and Experimental Therapeutics, 2009

Hypermethylation of CpG islands of tumor suppressor genes resulting in their silencing has been p... more Hypermethylation of CpG islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents with a more favorable toxicity profile is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys 38 of p65 to inhibit NF-κB activation and exhibit anti-tumor activity in human malignancies. In this paper, we report that parthenolide: 1) inhibits DNMT1 with an IC 50 of 3.5 µM possibly through alkylation of the proximal thiolate of C1226 of the catalytic domain by its γmethylene lactone and 2) down-regulates DNMT1 expression possibly associated with its SubG1 cell cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in-vitro and in-vivo global DNA hypomethylation. Additionally, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in-vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitors discovery and development from natural structural-diversified sesquiterpene lactones.

Journal of Molecular Recognition, 2002

Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling path... more Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling pathways that activate the NF-kappaB transcription factor. The resulting signals elicit immune responses and have important implications for disorders such as autoimmunity or allergic reactions. TNF-receptor-associated factors (TRAFs) bind to the cytoplasmic portion of TNFRs as well as downstream regulators and thus are co-inducers of the signal transduction. TRAF3 binds to diverse receptors and regulators by accomodating a conserved motif that is embedded in completely different structural frameworks. Thus, the protein-protein contact region on TRAF3 represents a binding interface that is structurally and functionally adaptive. In this report, three 'hot spots' at the TRAF3 protein-interaction interface are defined that provide the principal contact regions for different binding partners. The side-chains of residues at these 'hot spots' are flexible and undergo movements on binding the different partners. These side chain rearrangements provide a structural adaptability that promotes interaction with a variety of distinct proteins. It is proposed that similar adaptive 'hot spots' are also present on the binding surfaces of TRAF1, TRAF2 and TRAF5.

Journal of Chemical Information and Modeling, 2012

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand ... more Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein-ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (15; K(d) = 0.37 μM), one moderately active (12; K(d) = 3.2 μM), and one weak but very selective (9; K(d) = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general.

Journal of Biological Chemistry, 2011

Cancer Research, 2010

The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is freq... more The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colon...

Cancer Prevention Research, 2011

Growing evidence shows an association between chronic liver inflammation and hepatocellular carci... more Growing evidence shows an association between chronic liver inflammation and hepatocellular carcinoma (HCC) development. STAT3, which is associated with inflammation and cellular transformation, is constitutively activated in human HCC tissues but not in normal human liver tissues. Although interleukin-6 (IL-6) is elevated in the serum of patients with HCC, it is not fully understood whether STAT3 constitutive activation is positively correlated with autocrine IL-6 secreted by HCC cells. Here, we reported that in HCC cells, the elevated levels of both IL-6 and IL-6 receptor (IL-6R, gp80), not IL-6 alone, correlated with STAT3 activation. We also explored whether the anticancer effects of celecoxib, an anti-inflammatory drug, may be due to the inhibition of the IL-6/STAT3 pathway in HCC cells. Our results showed that celecoxib decreased STAT3 phosphorylation by reducing Janus-activated kinase (JAK2) phosphorylation and caused apoptosis in HCC cells. Celecoxib could also block exogeno...

Blood, 2012

T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia ... more T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The i...

Biochemistry, 2003

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic inte... more Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

Biochemical and Biophysical Research Communications, 2011

Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequent... more Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existence of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH(+)/CD133(+)). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower IC50 in colon cancer stem-like cells. In summary, our results indicate that STAT3 is a novel therapeutic target in colon cancer stem-like cells and inhibition of STAT3 in cancer stem-like cells may offer a potential treatment for colorectal cancer.

Mol Cancer, 2010

Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibit... more Background: We characterized the biologic effects of a novel small molecule STAT3 pathway inhibitor that is derived from the natural product curcumin. We hypothesized this lead compound would specifically inhibit the STAT3 signaling pathway to induce apoptosis in melanoma cells. Results: FLLL32 specifically reduced STAT3 phosphorylation at Tyr705 (pSTAT3) and induced apoptosis at micromolar amounts in human melanoma cell lines and primary melanoma cultures as determined by annexin V/propidium iodide staining and immunoblot analysis. FLLL32 treatment reduced expression of STAT3-target genes, induced caspase-dependent apoptosis, and reduced mitochondrial membrane potential. FLLL32 displayed specificity for STAT3 over other homologous STAT proteins. In contrast to other STAT3 pathway inhibitors (WP1066, JSI-124, Stattic), FLLL32 did not abrogate IFN-γ-induced pSTAT1 or downstream STAT1-mediated gene expression as determined by Real Time PCR. In addition, FLLL32 did not adversely affect the function or viability of immune cells from normal donors. In peripheral blood mononuclear cells (PBMCs), FLLL32 inhibited IL-6-induced pSTAT3 but did not reduce signaling in response to immunostimulatory cytokines (IFN-γ, IL 2). Treatment of PBMCs or natural killer (NK) cells with FLLL32 also did not decrease viability or granzyme b and IFN-γ production when cultured with K562 targets as compared to vehicle (DMSO). Conclusions: These data suggest that FLLL32 represents a lead compound that could serve as a platform for further optimization to develop improved STAT3 specific inhibitors for melanoma therapy.

Neoplasia, 2010

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling i... more Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 μM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.

Molecular bioSystems, Jan 29, 2015

Curcumin, a natural product, has been shown to possess notable anti-inflammatory activities and n... more Curcumin, a natural product, has been shown to possess notable anti-inflammatory activities and numerous studies have been carried out on its clinical applications. Recently, several reports mentioned that myeloid differentiation protein 2 (MD-2) may be the direct target of curcumin in the inhibition of lipopolysaccharide (LPS) signaling. However, the exact interaction between curcumin and MD-2 is still incompletely understood. In the present study, computational and experimental methods were employed to explore the underlying structural mechanism of curcumin binding to the MD-2 protein. Molecular docking and molecular dynamics (MD) simulation studies showed that curcumin could be embedded into the hydrophobic pocket of MD-2 and form stable hydrogen bonding interactions with residues R90 and Y102 of MD-2. Moreover, experimental results of curcumin binding to the MD-2(R90A/Y102A) mutant further confirmed that residues ARG-90 and TYR-102 contribute to the recognition process of curcum...

Biosensors & bioelectronics, Jan 10, 2015

We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS)... more We describe a fluorescence polarisation assay based on the use of dihydropteroate synthase (DHPS) and a fluorescence probe for multi-sulphonamide detection. Dihydropteridine pyrophosphate (DHPPP) was synthesised and acts as the first substrate for DHPS. Under optimised conditions, the half-maximal inhibitory concentrations (IC50) of the assay were less than 100ngmL(-1) for at least 29 sulphonamides and the time needed for the detection was less than 20min. More importantly, the assay revealed quite uniform affinities for all of the individual sulphonamides tested, which has never before been achieved in an antibody-based assay.

Journal of molecular graphics & modelling, 2014

Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind importan... more Molecular dynamics (MD) simulation is a crucial tool for understanding principles behind important biochemical processes such as protein folding and molecular interaction. With the rapidly increasing power of modern computers, large-scale MD simulation experiments can be performed regularly, generating huge amounts of MD data. An important question is how to analyze and interpret such massive and complex data. One of the (many) challenges involved in analyzing MD simulation data computationally is the high-dimensionality of such data. Given a massive collection of molecular conformations, researchers typically need to rely on their expertise and prior domain knowledge in order to retrieve certain conformations of interest. It is not easy to make and test hypotheses as the data set as a whole is somewhat "invisible" due to its high dimensionality. In other words, it is hard to directly access and examine individual conformations from a sea of molecular structures, and to fu...

American journal of translational research, 2014

Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully und... more Colon tumors are a major cause of cancer death, yet their molecular intricacies are not fully understood. We demonstrate that the histone demethylases JMJD2A, JMJD2B and JMJD2C are overexpressed in colon cancer cell lines, whereas another related protein, JMJD2D, is not. Interestingly, despite their high homology, the intracellular localization of JMJD2A-C is different in colon and other cancer cells, with JMJD2A being present comparably in the cytoplasm and nucleus, JMJD2B more prevalent in the nucleus and JMJD2C strongly associated with chromatin. This suggests that each of these three proteins performs different, non-redundant functions. Moreover, we show that JMJD2C (also called KDM4C) forms complexes with β-catenin, an oncoprotein whose overexpression is crucial for the development of most colonic tumors. In addition, JMJD2C downregulation reduced both growth and clonogenic capacity of HCT-116 colon cancer cells. Further, JMJD2C was required for efficient expression of the grow...

Tetrahedron, 2014

The first one-pot copper-catalyzed highly regioselective CeN bond formation between aryl/alkyl am... more The first one-pot copper-catalyzed highly regioselective CeN bond formation between aryl/alkyl amines and sulfonamide-substituted naphthoquinones was accomplished. Facile chemoselective routes obtained diverse ring-opening 6-amino/anilino-naphthalen-dione-1-sulfonamides and ring-fused 6-amino/aniline-5H-naphth[1,8-cd]isothiazol-5-one,1,1-dioxides with great functional group tolerance. Regiochemistry was confirmed by 1D-and 2D-NMRs.

Structure, 2002

Most TNFRs activate intracellular signaling pathways, including the NF-B and c-JUN N-terminal kin... more Most TNFRs activate intracellular signaling pathways, including the NF-B and c-JUN N-terminal kinase (JNK) 1 Cancer Research Center The Burnham Institute [7-9]. With the identification of proteins that bind directly to La Jolla, California 92037 2 Johnsson Comprehensive Cancer Center the cytoplasmic domains of TNFRs called TNFR-associated factors (TRAFs) [10], an understanding of TNF

Molecular Cancer, 2010

Background: Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an ... more Background: Targeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells. Results: FLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ. Conclusion: Our findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling.

Journal of Pharmacology and Experimental Therapeutics, 2009

Hypermethylation of CpG islands of tumor suppressor genes resulting in their silencing has been p... more Hypermethylation of CpG islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents with a more favorable toxicity profile is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys 38 of p65 to inhibit NF-κB activation and exhibit anti-tumor activity in human malignancies. In this paper, we report that parthenolide: 1) inhibits DNMT1 with an IC 50 of 3.5 µM possibly through alkylation of the proximal thiolate of C1226 of the catalytic domain by its γmethylene lactone and 2) down-regulates DNMT1 expression possibly associated with its SubG1 cell cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in-vitro and in-vivo global DNA hypomethylation. Additionally, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in-vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitors discovery and development from natural structural-diversified sesquiterpene lactones.

Journal of Molecular Recognition, 2002

Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling path... more Tumor necrosis factor (TNF) signaling is controlled by receptors and intracellular signaling pathways that activate the NF-kappaB transcription factor. The resulting signals elicit immune responses and have important implications for disorders such as autoimmunity or allergic reactions. TNF-receptor-associated factors (TRAFs) bind to the cytoplasmic portion of TNFRs as well as downstream regulators and thus are co-inducers of the signal transduction. TRAF3 binds to diverse receptors and regulators by accomodating a conserved motif that is embedded in completely different structural frameworks. Thus, the protein-protein contact region on TRAF3 represents a binding interface that is structurally and functionally adaptive. In this report, three 'hot spots' at the TRAF3 protein-interaction interface are defined that provide the principal contact regions for different binding partners. The side-chains of residues at these 'hot spots' are flexible and undergo movements on binding the different partners. These side chain rearrangements provide a structural adaptability that promotes interaction with a variety of distinct proteins. It is proposed that similar adaptive 'hot spots' are also present on the binding surfaces of TRAF1, TRAF2 and TRAF5.

Journal of Chemical Information and Modeling, 2012

Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand ... more Kinase targets have been demonstrated to undergo major conformational reorganization upon ligand binding. Such protein conformational plasticity remains a significant challenge in structure-based virtual screening methodology and may be approximated by screening against an ensemble of diverse protein conformations. Maternal embryonic leucine zipper kinase (MELK), a member of serine-threonine kinase family, has been recently found to be involved in the tumerogenic state of glioblastoma, breast, ovarian, and colon cancers. We therefore modeled several conformers of MELK utilizing the available chemogenomic and crystallographic data of homologous kinases. We carried out docking pose prediction and virtual screening enrichment studies with these conformers. The performances of the ensembles were evaluated by their ability to reproduce known inhibitor bioactive conformations and to efficiently recover known active compounds early in the virtual screen when seeded with decoy sets. A few of the individual MELK conformers performed satisfactorily in reproducing the native protein-ligand pharmacophoric interactions up to 50% of the cases. By selecting an ensemble of a few representative conformational states, most of the known inhibitor binding poses could be rationalized. For example, a four conformer ensemble is able to recover 95% of the studied actives, especially with imperfect scoring function(s). The virtual screening enrichment varied considerably among different MELK conformers. Enrichment appears to improve by selection of a proper protein conformation. For example, several holo and unliganded active conformations are better to accommodate diverse chemotypes than ATP-bound conformer. These results prove that using an ensemble of diverse conformations could give a better performance. Applying this approach, we were able to screen a commercially available library of half a million compounds against three conformers to discover three novel inhibitors of MELK, one from each template. Among the three compounds validated via experimental enzyme inhibition assays, one is relatively potent (15; K(d) = 0.37 μM), one moderately active (12; K(d) = 3.2 μM), and one weak but very selective (9; K(d) = 18 μM). These novel hits may be utilized to assist in the development of small molecule therapeutic agents useful in diseases caused by deregulated MELK, and perhaps more importantly, the approach demonstrates the advantages of choosing an appropriate ensemble of a few conformers in pursuing compound potency, selectivity, and novel chemotypes over using single target conformation for structure-based drug design in general.

Journal of Biological Chemistry, 2011

Cancer Research, 2010

The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is freq... more The constitutive activation of signal transducer and activator of transcription 3 (STAT3) is frequently detected in most types of human cancer where it plays important roles in survival, drug resistance, angiogenesis, and other functions. Targeting constitutive STAT3 signaling is thus an attractive therapeutic approach for these cancers. We have recently developed novel small-molecule STAT3 inhibitors, known as FLLL31 and FLLL32, which are derived from curcumin (the primary bioactive compound of turmeric). These compounds are designed to bind selectively to Janus kinase 2 and the STAT3 Src homology-2 domain, which serve crucial roles in STAT3 dimerization and signal transduction. Here we show that FLLL31 and FLLL32 are effective inhibitors of STAT3 phosphorylation, DNA-binding activity, and transactivation in vitro, leading to the impediment of multiple oncogenic processes and the induction of apoptosis in pancreatic and breast cancer cell lines. FLLL31 and FLLL32 also inhibit colon...

Cancer Prevention Research, 2011

Growing evidence shows an association between chronic liver inflammation and hepatocellular carci... more Growing evidence shows an association between chronic liver inflammation and hepatocellular carcinoma (HCC) development. STAT3, which is associated with inflammation and cellular transformation, is constitutively activated in human HCC tissues but not in normal human liver tissues. Although interleukin-6 (IL-6) is elevated in the serum of patients with HCC, it is not fully understood whether STAT3 constitutive activation is positively correlated with autocrine IL-6 secreted by HCC cells. Here, we reported that in HCC cells, the elevated levels of both IL-6 and IL-6 receptor (IL-6R, gp80), not IL-6 alone, correlated with STAT3 activation. We also explored whether the anticancer effects of celecoxib, an anti-inflammatory drug, may be due to the inhibition of the IL-6/STAT3 pathway in HCC cells. Our results showed that celecoxib decreased STAT3 phosphorylation by reducing Janus-activated kinase (JAK2) phosphorylation and caused apoptosis in HCC cells. Celecoxib could also block exogeno...

Blood, 2012

T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia ... more T-cell leukemia/lymphoma 1 (TCL1) is an oncogene overexpressed in T-cell prolymphocytic leukemia and in B-cell malignancies including B-cell chronic lymphocytic leukemia and lymphomas. To date, only a limited number of Tcl1-interacting proteins that regulate its oncogenic function have been identified. Prior studies used a proteomic approach to identify a novel interaction between Tcl1 with Ataxia Telangiectasia Mutated. The association of Tcl1 and Ataxia Telangiectasia Mutated leads to activation of the NF-κB pathway. Here, we demonstrate that Tcl1 also interacts with heat shock protein (Hsp) 70. The Tcl1-Hsp70 complex was validated by coimmunoprecipitation experiments. In addition, we report that Hsp70, a protein that plays a critical role in the folding and maturation of several oncogenic proteins, associates with Tcl1 protein and stabilizes its expression. The inhibition of the ATPase activity of Hsp70 results in ubiquitination and proteasome-dependent degradation of Tcl1. The i...

Biochemistry, 2003

Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic inte... more Glycinamide ribonucleotide transformylase (GAR Tfase) has been the target of anti-neoplastic intervention for almost two decades. Here, we use a structure-based approach to design a novel folate analogue, 10-(trifluoroacetyl)-5,10-dideazaacyclic-5,6,7,8-tetrahydrofolic acid (10-CF(3)CO-DDACTHF, 1), which specifically inhibits recombinant human GAR Tfase (K(i) = 15 nM), but is inactive (K(i) > 100 microM) against other folate-dependent enzymes that have been examined. Moreover, compound 1 is a potent inhibitor of tumor cell proliferation (IC(50) = 16 nM, CCRF-CEM), which represents a 10-fold improvement over Lometrexol, a GAR Tfase inhibitor that has been in clinical trials. Thus, this folate analogue 1 is among the most potent and selective inhibitors known toward GAR Tfase. Contributing to its efficacious activity, compound 1 is effectively transported into the cell by the reduced folate carrier and intracellularly sequestered by polyglutamation. The crystal structure of human GAR Tfase with folate analogue 1 at 1.98 A resolution represents the first structure of any GAR Tfase to be determined with a cofactor or cofactor analogue without the presence of substrate. The folate-binding loop of residues 141-146, which is highly flexible in both Escherichia coli and unliganded human GAR Tfase structures, becomes highly ordered upon binding 1 in the folate-binding site. Computational docking of the natural cofactor into this and other apo or complexed structures provides a rational basis for modeling how the natural cofactor 10-formyltetrahydrofolic acid interacts with GAR Tfase, and suggests that this folate analogue-bound conformation represents the best template to date for inhibitor design.

Biochemical and Biophysical Research Communications, 2011

Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequent... more Persistent activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in colon cancer. Increasing evidence suggests the existence of a small population of colon cancer stem or cancer-initiating cells may be responsible for tumor initiation, metastasis, and resistance to chemotherapy and radiation. Whether STAT3 plays a role in colon cancer-initiating cells and the effect of STAT3 inhibition is still unknown. Flow cytometry was used to isolate colon cancer stem-like cells from three independent human colon cancer cell lines characterized by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulation (ALDH(+)/CD133(+)). The effects of STAT3 inhibition in colon cancer stem-like cells were examined. The phosphorylated or activated form of STAT3 was expressed in colon cancer stem-like cells and was reduced by a STAT3-selective small molecular inhibitor, FLLL32. FLLL32 also inhibited the expression of potential STAT3 downstream target genes in colon cancer stem-like cells including survivin, Bcl-XL, as well as Notch-1, -3, and -4, which may be involved in stem cell function. Furthermore, FLLL32 inhibited cell viability and tumorsphere formation as well as induced cleaved caspase-3 in colon cancer stem-like cells. FLLL32 is more potent than curcumin as evidenced with lower IC50 in colon cancer stem-like cells. In summary, our results indicate that STAT3 is a novel therapeutic target in colon cancer stem-like cells and inhibition of STAT3 in cancer stem-like cells may offer a potential treatment for colorectal cancer.