Hongbo Shen

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 Mycobacterium tuberculosis. 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. tuberculosis 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 60 000 compounds. ATB107 was identified as a potential binding molecule; it was tested, and shown to have antimycobacterial activity in vitro not only against the laboratory strain M. tuberculosis H37Rv, but also against clinical isolates of multidrug‐resistant TB strains. Most MDR‐TB strains tested were susceptible to 1 μg·mL−1 ATB107. ATB107 had little toxicity against THP‐1 macrophage cells, which are human monocytic leukemia cells. ATB107, which bound tightly to IGPS in vitro, 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.

Recombinant Mycobacterium bovis BCG expressing chimaeric protein of Ag85B and ESAT-6 enhances immunostimulatory activity of human macrophages.

Recombinant BCG strain that secretes the chimaeric protein of Ag85B and ESAT-6 has been demonstrated to augment Th1 immune response in C57BL/6 mice. In this paper, we studied the immunostimulatory activity of the recombinant BCG strains in vitro and found out that rBCG-A(N)-E-A(C) activated THP-1 cells and induced higher expression levels of CD86, CD80, CD40 and HLA-DR, especially increased the ratio of CD86/CD80. Likewise, rBCG-A(N)-E-A(C) infection was able to stimulate an increase in TNF-alpha production of macrophages. Moreover, rBCG-A(N)-E-A(C) up-regulated the expression of EFHD2, ACTB and ACTG1 in the macrophages and improved the ability of antigen presentation and the CD8(+) T-cells immune response. Taken together, this rBCG-A(N)-E-A(C) strain enhanced the immunostimulatory activity of human macrophages and could be a potential vaccine against Mycobacterium tuberculosis.

Novel recombinant BCG coexpressing Ag85B, ESAT-6 and mouse TNF-α induces significantly enhanced cellular immune and antibody responses in C57BL/6 mice

The recent emergence of multidrug‐resistant and extensively drug‐resistant strains of Mtb and the epidemic of TB in populations co‐infected with human immunodeficiency virus demonstrate that TB remains a leading infectious disease. Moreover, the failure of BCG to protect against this disease indicates that new vaccines against TB are urgently needed. Experimental evidence has revealed that TNF plays a major role in host defense against Mtb in both active and latent phases of infection. Release of TNF, which would induce mycobacteria‐mediated macrophage apoptosis and thus reduce the spread of mycobacteria, is one of the most important and early responses of macrophages challenged with Mtb. In order to identify the usefulness of TNF in improving the effectiveness of TB vaccine, in the current study a novel rBCG strain expressing the fusion gene of Ag85B‐Esat6‐TNF‐α in BCG Danish strain was constructed, and its ability to induce an immune response in C57BL/6 mice evaluated. It was found that immunization with strains of rBCG‐Ag85B‐Esat6‐TNF‐α can induce a stronger immune response than does immunization with rBCG‐Ag85B‐Esat6 or parental BCG. The results indicate that rBCG‐Ag85B‐Esat6‐TNF‐α is a promising candidate for further study.

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.

Evolution of hepatitis B virus polymerase mutations in a patient with HBeAg-positive chronic hepatitis B virus treated with sequential monotherapy and add-on nucleoside/nucleotide analogues

BACKGROUND Nucleoside/nucleotide analogues are a fundamental tool for the treatment of chronic hepatitis B virus (HBV). Sequential anti-HBV treatment might lead to the selection of mutations. OBJECTIVE This report aimed to analyze the genetic evolution of the reverse-transcriptase (RT) gene of viral quasispecies in a patient with hepatitis B e antigen (HBeAg)-positive chronic HBV who received, sequentially, lamivudine (LAM), adefovir dipivoxil (ADV), and ADV + telbivudine (LDT) combination treatment over a total of 108 weeks. METHODS A 20-year-old Chinese man presented to Huashan Hospital, Fudan University, Shanghai, Peoples Republic of China, with hepatitis B surface antigen-positive and HBeAg-positive chronic HBV and was sequentially treated with LAM 100 mg/d for 18weeks,ADV 10mg/d for 68weeks, and ADV 10mg/d + LDT 600 mg/d combination treatment for 22 weeks. Compliance was monitored every 4 weeks using a pill count. For genotypic analysis, the RT region of the polymerase gene from the serum of this patient was amplified, cloned, and sequenced. Fifty clones with HBV insert were selected for sequencing at weeks 0 (baseline), 18, 22, 60, 70, 86, and 108. RESULTS The rtM204V/L LAM-resistance mutation was detected in 4.4% (2/45) of clones prior to LAM treatment. At week 18 during LAM treatment, the rtM204I mutation became predominant, being present in 79.5% (35/44) of clones. The rtM204I mutation was associated with compensatory mutations (rtL180M and rtT184L). A total of 9.1% (4/44) of the clones harbored the rtL180M + rtT184L + rtM204I mutations. Two new mutations, rtL229V and rtV191I, were detected in 75.0% (33/44) and 11.4% (5/44) of clones, respectively. At week 22 during ADV treatment, LAM-resistance mutations (rtL180M, rtT184L, rtM204I, rtV191I, and rtL229V) were not detected. At week 86 during ADV therapy, the rtN236T ADV-resistance mutation was detected in 58.8% (20/34) of clones. A total of 20.6% (7/34) of the clones harbored the rtK212T + rtM250L mutation, and rtA181V was found in 2.9% (1/34) of the clones. At week 108, after the patient had been receiving ADV + LDT combination therapy for 22 weeks, rtS202G and rtI269T had emerged, representing 28.9% (13/45) and 8.9% (4/45), respectively, of the viral population during ADV + LDT combination treatment. We also detected several polymorphic sites,including rtF221Y, rtS223A, rtI224V, rtN238H, rtL267Q, and rtQ271M, during the sequential treatment. After 22 weeks of combination treatment, HBV DNA count was decreased to less than the lower limit of quantitation (<200 copies/mL). CONCLUSIONS This report identified HBV mutations that escaped the antiviral pressure of LAM, ADV, and ADV + LDT in this patient and provided insight into the process of mutation selection through genotypic analysis during antiviral treatment. Mutations selected under sequential treatments of LAM, ADV, and ADV + LDT can lead to a series of compensatory mutations, which partially restore the level of viral replication. ADV administered in combination with LDT appeared to be effective in this selected case with clinical or virologic resistance to sequential treatment with LAM and ADV.

High-resolution melting facilitates mutation screening of rpsL gene associated with streptomycin resistance in Mycobacterium tuberculosis

Drug resistance remains a serious threat to tuberculosis control worldwide. As one of the important first-line antitubercular agents, resistance to streptomycin (SM) and its derivatives has increased in recent years and has become one of the characteristics of extensively drug-resistant tuberculosis (XDR-TB). A novel rapid assay to screen for rpsL gene mutations associated with SM resistance in Mycobacterium tuberculosis (M. tuberculosis), was developed using high-resolution melting (HRM) analysis. The HRM results of 134 SM-resistant clinical isolates and 20 SM-susceptible clinical isolates of M. tuberculosis for rpsL gene mutations were perfectly matched with those of DNA sequencing. SM resistance was highly associated with rpsL mutations in M. tuberculosis. HRM technique represented an inexpensive, highly sensitive and high-throughput method to facilitate the screening of large numbers of clinical samples for epidemiological studies of drug-resistance of M. tuberculosis, especially in developing countries.

Purification and characterization of anthranilate synthase component I (TrpE) from Mycobacterium tuberculosis H37Rv

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis is the main reason why tuberculosis (TB) continues to be a major health problem worldwide. It is urgent to discover novel anti-mycobacterial agents based on new drug targets for the treatment of TB, especially MDR-TB. Tryptophan biosynthetic pathway, which is essential for the survival of M. tuberculosis and meanwhile absent in mammals, provides potential anti-TB drug targets. One of the promising drug targets in this pathway is anthranilate synthase component I (TrpE), whose role is to catalyze the conversion of chorismate to anthranilate using ammonia as amino source. In order to get a deep understanding of TrpE, a study on purification and characteristic identification of TrpE is required. In this work, the putative trpE gene of M. tuberculosis H37Rv was expressed as a fusion protein with a 6x His-tag on the N-terminal (His-TrpE) in Escherichia coli. The recombinant TrpE protein was successfully purified and then its enzymatic characteristics were analyzed. The native TrpE without His-tag was obtained by removal of the N-terminal fusion partner of His-TrpE using enterokinase. It was found that N-terminal fusion partner had little influence on TrpE catalytic activity. In addition, the key residues related to enzyme catalytic activity and that involved in l-tryptophan inhibition were predicted in the structure of M. tuberculosis H37Rv TrpE. These results would be beneficial to the designing of novel anti-TB drugs with high potency and selectivity.

Altered protein expression patterns of Mycobacterium tuberculosis induced by ATB107

ATB107 is a potent inhibitor of indole-3-glycerol phosphate synthase (IGPS). It can effectively inhibit the growth of clinical isolates of drug-resistant Mycobacterium tuberculosis strains as well as M. tuberculosis H37Rv. To investigate the mechanism of ATB107 action in M. tuberculosis, two-dimensional gel electrophoresis coupled with MALDI-TOF-MS analysis (2-DE-MS) was performed to illustrate alterations in the protein expression profile in response to ATB107. Results show that ATB107 affected tryptophan biosynthesis by decreasing the expression of protein encoded by Rv3246c, the transcriptional regulatory protein of MtrA belonging to the MtrA-MtrB two-component regulatory system, in both drug-sensitive and drug-resistant virulent strains. ATB107 might present a stress condition similar to isoniazid (INH) or ethionamide for M. tuberculosis since the altered expression in response to ATB107 of some genes, such as Rv3140, Rv2243, and Rv2428, is consistent with INH or ethionamide treatment. After incubation with ATB107, the expression of 2 proteins encoded by Rv0685 and Rv2624c was down-regulated while that of protein encoded by Rv3140 was up-regulated in all M. tuberculosis strains used in this study. This may be the common response to tryptophan absence; however, relations to ATB107 are unknown and further evaluation is warranted.

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.

Recombinant BCG coexpressing Ag85B, ESAT-6 and Rv3620c elicits specific Th1 immune responses in C57BL/6 mice

Tuberculosis (TB) remains to be an enormous global health problem. The inconsistent protection efficacy of Bacille Calmette-Guérin (BCG) calls for new vaccines for TB. One choice to improve the efficacy of BCG vaccine is recombinant BCG (rBCG). Experimental evidences have revealed that Ag85B, ESAT-6 and Rv3620c are important immunodominant antigens of Mycobacterium tuberculosis. In this study, we have constructed a novel rBCG expressing fusion protein Ag85B-ESAT6-Rv3620c and evaluated the immunogenicity of this rBCG in C57BL/6 mice. Results show that there is a strong TB-specific CD4(+) and CD8(+) T lymphocytes proliferation in mice immunized with this rBCG vaccine. A single dose immunization of rBCG could induce a significantly strong Th1 immune response characterized by an increasing ratio of antigen-specific IgG2b/IgG1 as well as a high expression level of Th1 cytokines such as IFN-γ, TNF-α and IL-2. This conclusion was confirmed by a decreased secretion of Th2 cytokine IL-10. Moreover, this rBCG induced a strong humoral response in mice with an increasing antigen-specific IgG titer. Therefore, we concluded that this rBCG could significantly increase both Th1 type cellular immune response and antigen-specific humoral response compared with BCG. The above observations demonstrated that rBCG::Ag85B-ESAT6-Rv3620c is a potential candidate vaccine against M. tuberculosis for further study.