Hairong Hu

Carbonyl reductase 1 as a novel target of (-)-epigallocatechin gallate against hepatocellular carcinoma.

Human carbonyl reductase 1 (CBR1) converts the antitumor drug and anthracycline daunorubicin (DNR) into the alcohol metabolite daunorubicinol (DNROL) with significantly reduced antitumor activity and cardiotoxicity, and this limits the clinical use of DNR. Inhibition of CBR1 can thus increase the efficacy and decrease the toxicity of DNR. Here we report that (−)‐epigallocatechin gallate (EGCG) from green tea is a promising inhibitor of CBR1. EGCG directly interacts with CBR1 and acts as a noncompetitive inhibitor with respect to the cofactor reduced nicotinamide adenine dinucleotide phosphate and the substrate isatin. The inhibition is dependent on the pH, and the gallate moiety of EGCG is required for activity. Molecular modeling has revealed that EGCG occupies the active site of CBR1. Furthermore, EGCG specifically enhanced the antitumor activity of DNR against hepatocellular carcinoma SMMC7721 cells expressing high levels of CBR1 and corresponding xenografts. We also demonstrated that EGCG could overcome the resistance to DNR by Hep3B cells stably expressing CBR1 but not by RNA interference of CBR1‐HepG2 cells. The level of the metabolite DNROL was negatively correlated with that of EGCG in the cell extracts. Finally, EGCG decreased the cardiotoxicity of DNR in a human carcinoma xenograft model with both SMMC7721 and Hep3B cells in mice. Conclusion: These results strongly suggest that EGCG can inhibit CBR1 activity and enhance the effectiveness and decrease the cardiotoxicity of the anticancer drug DNR. These findings also indicate that a combination of EGCG and DNR might represent a novel approach for hepatocellular carcinoma therapy or chemoprevention. (HEPATOLOGY 2010;)

A novel inhibitor of indole‐3‐glycerol phosphate synthase with activity against multidrug‐resistant Mycobacterium tuberculosis

Tuberculosis (TB) continues to be a major cause of morbidity and mortality worldwide. The increasing emergence and spread of drug‐resistant TB poses a significant threat to disease control and calls for the urgent development of new drugs. The tryptophan biosynthetic pathway plays an important role in the survival of Mycobacteriumu2003tuberculosis. Thus, indole‐3‐glycerol phosphate synthase (IGPS), as an essential enzyme in this pathway, might be a potential target for anti‐TB drug design. In this study, we deduced the structure of IGPS of M.u2003tuberculosis H37Rv by using homology modeling. On the basis of this deduced IGPS structure, screening was performed in a search for novel inhibitors, using the Maybridge database containing the structures of 60u2003000 compounds. ATB107 was identified as a potential binding molecule; it was tested, and shown to have antimycobacterial activity inu2003vitro not only against the laboratory strain M.u2003tuberculosis H37Rv, but also against clinical isolates of multidrug‐resistant TB strains. Most MDR‐TB strains tested were susceptible to 1u2003μg·mL−1 ATB107. ATB107 had little toxicity against THP‐1 macrophage cells, which are human monocytic leukemia cells. ATB107, which bound tightly to IGPS inu2003vitro, was found to be a potent competitive inhibitor of the substrate 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose‐5′‐phosphate, as shown by an increased Km value in the presence of ATB107. The results of site‐directed mutagenesis studies indicate that ATB107 might inhibit IGPS activity by reducing the binding affinity for substrate of residues Glu168 and Asn189. These results suggest that ATB107 is a novel potent inhibitor of IGPS, and that IGPS might be a potential target for the development of new anti‐TB drugs. Further evaluation of ATB107 in animal studies is warranted.

Coevolving residues of (β/α)8-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (beta/alpha)(8)-barrel proteins-the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (betaalpha)(8)-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (beta/alpha)(8)-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (beta/alpha)(8)-barrel scaffold.

A thienopyrimidine derivative induces growth inhibition and apoptosis in human cancer cell lines via inhibiting Aurora B kinase activity.

Aurora kinases play a key role in the regulation of mitosis and have been regarded as promising targets of cancer therapy. In this paper we describe a thienopyrimidine derivative (S7), a novel potent ATP-competitive hit inhibitor of Aurora B kinase screened through a HTS system, with the IC50 141.12 nM in the biochemical kinase activity assay. Human tumor cells treated with S7 showed dose-dependent inhibition of auto-phosphorylation of Aurora B on Thr232 and another widely-used marker specific for Aurora B kinase, the phosphorylation of Histone H3 (Ser 10), demonstrating endogenous Aurora B kinase activity were inhibited at cellular level. Moreover, S7 treatment induced proliferation inhibition, colony formation inhibition and apoptosis of human tumor cell lines in a dose- and time-dependent manner.

Correlated Mutation Analysis on the Catalytic Domains of Serine/Threonine Protein Kinases

Background Protein kinases (PKs) have emerged as the largest family of signaling proteins in eukaryotic cells and are involved in every aspect of cellular regulation. Great progresses have been made in understanding the mechanisms of PKs phosphorylating their substrates, but the detailed mechanisms, by which PKs ensure their substrate specificity with their structurally conserved catalytic domains, still have not been adequately understood. Correlated mutation analysis based on large sets of diverse sequence data may provide new insights into this question. Methodology/Principal Findings Statistical coupling, residue correlation and mutual information analyses along with clustering were applied to analyze the structure-based multiple sequence alignment of the catalytic domains of the Ser/Thr PK family. Two clusters of highly coupled sites were identified. Mapping these positions onto the 3D structure of PK catalytic domain showed that these two groups of positions form two physically close networks. We named these two networks as θ-shaped and γ-shaped networks, respectively. Conclusions/Significance The θ-shaped network links the active site cleft and the substrate binding regions, and might participate in PKs recognizing and interacting with their substrates. The γ-shaped network is mainly situated in one side of substrate binding regions, linking the activation loop and the substrate binding regions. It might play a role in supporting the activation loop and substrate binding regions before catalysis, and participate in product releasing after phosphoryl transfer. Our results exhibit significant correlations with experimental observations, and can be used as a guide to further experimental and theoretical studies on the mechanisms of PKs interacting with their substrates.

Characterization of a Supercluster of SnoRNA Genes from Rice Genome

Small nucleolar RNAs (snoRNAs) are mainly involved in rRNA processing and modification, including the maturation of precursor ribosomal RNAs (pre-rRNA), and methylation and pseudouridy nation of rRNA bases. These moleclules are transcripted independly, along with host gene as an intron or as polycistron, reflecting the different pathways for processing of precursor snoRNAs (pre-snoRNAs) into mature snoRNAs. Analysis ofsnoRNA gene organizations and sequences will enable us to explore their evolutions. Previously, we have briefly mentioned a surprisingly larg cluster from rice, consisting of 5 duplictions of a cluster. It provides a good opportunity to reveal the evolution machanism of the snoRNA genes. Here detailed examination were performed and the processs of snoRNA gene loss, insertion and tranfer and cluster duplication are highlighted.