Galactosyl Human Serum Albumin-NMP1 Conjugate: A Near Infrared (NIR)-Activatable Fluorescence Imaging Agent to Detect Peritoneal Ovarian Cancer Metastases

Patient survival depends on the completeness of resection of peritoneal ovarian cancer metastases (POCM), and therefore, it is important to develop methods to enhance detection. Previous probe designs based on activatable galactosyl human serum albumin (hGSA)-fluorophore pairs, which target lectin receptors expressed on POCM, have used only visible range dyes conjugated to hGSA. However, imaging probes emitting fluorescence in the NIR range are advantageous because NIR photons have deeper in vivo tissue penetration and result in lower background autofluorescence than those emitting in the visible range. A NIR-activatable hGSA fluorophore was synthesized using a bacteriochlorin-based dye, NMP1. NMP1 has two unique absorption peaks, one in the green range and the other in the NIR range, but emits at a NIR peak of 780 nm. NMP1, thus, has two different Stokes shifts that have the potential to allow imaging of POCM both at the peritoneal surface and just below it. hGSA was conjugated with 2 NMP1 molecules to create a self-quenching complex (hGSA-NMP1). The activation ratio of hGSA-NMP1 was measured by the fluorescence intensity before and after exposure to 10% SDS. The activation ratio of hGSA-NMP1 was ～100-fold in vitro. Flow cytometry, fluorescence microscopy, and in vivo spectral fluorescence imaging were carried out to compare hGSA-NMP1 with hGSA-IR800 and hGSA-ICG (two always-on control agents with similar emission to NMP1) in terms of comparative fluorescence signal and the ability to detect POCM in mice models. The sensitivity and specificity of hGSA-NMP1 for POCM implant detection were determined by colocalizing NMP1 emission spectra with red fluorescent protein (RFP) expressed constitutively in SHIN3 tumor implants at different depths below the peritoneal surface. In vitro, SHIN3 cells were easily detectable after 3 h of incubation with hGSA-NMP1. In vivo submillimeter POCM foci were clearly detectable with spectral fluorescence imaging using hGSA-NMP1. Among 555 peritoneal lesions, hGSA-NMP, using NIR and green excitation light, respectively, detect 75% of all lesions and 91% of lesions ～0.8 mm or greater in diameter. Few false positives were encountered. Nodules located at a depth below the small bowel surface were only depicted with hGSA-NMP1. We conclude that hGSA-NMP1 is useful in imaging peritoneal ovarian cancer metastases, located both superficially and deep in the abdominal cavity.     

3D-QSAR based pharmacophore modeling and virtual screening for identification of novel pteridine reductase inhibitors

Pteridine reductase is a promising target for development of novel therapeutic agents against Trypanosomatid parasites. A 3D-QSAR pharmacophore hypothesis has been generated for a series of L. major pteridine reductase inhibitors using Catalyst/HypoGen algorithm for identification of the chemical features that are responsible for the inhibitory activity. Four pharmacophore features, namely: two H-bond donors (D), one Hydrophobic aromatic (H) and one Ring aromatic (R) have been identified as key features involved in inhibitor-PTR1 interaction. These features are able to predict the activity of external test set of pteridine reductase inhibitors with a correlation coefficient (r) of 0.80. Based on the analysis of the best hypotheses, some potent Pteridine reductase inhibitors were screened out and predicted with anti-PTR1 activity. It turned out that the newly identified inhibitory molecules are at least 300 fold more potent than the current crop of existing inhibitors. Overall the current SAR study is an effort for elucidating quantitative structure-activity relationship for the PTR1 inhibitors. The results from the combined 3D-QSAR modeling and molecular docking approach have led to the prediction of new potent inhibitory scaffolds.     

Small-Molecule Quinolinol Inhibitor Identified Provides Protection against BoNT/A in Mice

Botulinum neurotoxins (BoNTs), etiological agents of the life threatening neuroparalytic disease botulism, are the most toxic substances currently known. The potential for the use as bioweapon makes the development of small-molecule inhibitor against these deadly toxins is a top priority. Currently, there are no approved pharmacological treatments for BoNT intoxication. Although an effective vaccine/immunotherapy is available for immuno-prophylaxis but this cannot reverse the effects of toxin inside neurons. A small-molecule pharmacological intervention, especially one that would be effective against the light chain protease, would be highly desirable. Similarity search was carried out from ChemBridge and NSC libraries to the hit (7-(phenyl(8-quinolinylamino)methyl)-8-quinolinol; NSC 84096) to mine its analogs. Several hits obtained were screened for in silico inhibition using AutoDock 4.1 and 19 new molecules selected based on binding energy and Ki. Among these, eleven quinolinol derivatives potently inhibited in vitro endopeptidase activity of botulinum neurotoxin type A light chain (rBoNT/A-LC) on synaptosomes isolated from rat brain which simulate the in vivo system. Five of these inhibitor molecules exhibited IC₅₀ values ranging from 3.0 nM to 10.0 µM. NSC 84087 is the most potent inhibitor reported so far, found to be a promising lead for therapeutic development, as it exhibits no toxicity, and is able to protect animals from pre and post challenge of botulinum neurotoxin type A (BoNT/A).     

Axonal and dendritic synaptotagmin isoforms revealed by a pHluorin-syt functional screen

The synaptotagmins (syts) are a family of molecules that regulate membrane fusion. There are 17 mammalian syt isoforms, most of which are expressed in the brain. However, little is known regarding the subcellular location and function of the majority of these syts in neurons, largely due to a lack of isoform-specific antibodies. Here we generated pHluorin-syt constructs harboring a luminal domain pH sensor, which reports localization, pH of organelles to which syts are targeted, and the kinetics and sites of exocytosis and endocytosis. Of interest, only syt-1 and 2 are targeted to synaptic vesicles, whereas other isoforms selectively recycle in dendrites (syt-3 and 11), axons (syt-5, 7, 10, and 17), or both axons and dendrites (syt-4, 6, 9, and 12), where they undergo exocytosis and endocytosis with distinctive kinetics. Hence most syt isoforms localize to distinct secretory organelles in both axons and dendrites and may regulate neuropeptide/neurotrophin release to modulate neuronal function.     

Identification of novel hrp-regulated genes through functional genomic analysis of the Pseudomonas syringae pv. tomato DC3000 genome

Pseudomonas syringae pv. tomato (Pst) strain DC3000 infects the model plants Arabidopsis thaliana and tomato, causing disease symptoms characterized by necrotic lesions surrounded by chlorosis. One mechanism used by Pst DC3000 to infect host plants is the type III protein secretion system, which is thought to deliver multiple effector proteins to the plant cell. The exact number of type III effectors in Pst DC3000 or any other plant pathogenic bacterium is not known. All known type III effector genes of P. syringae are regulated by HrpS, an NtrC family protein, and the HrpL alternative sigma factor, which presumably binds to a conserved cis element (called the "hrp box") in the promoters of type III secretion-associated genes. In this study, we designed a search motif based on the promoter sequences conserved in 12 published hrp operons and putative effector genes in Pst DC3000. Seventy-three predicted genes were retrieved from the January 2001 release of the Pst DC3000 genome sequence, which had 95% genome coverage. The expression of the 73 genes was analysed by microarray and Northern blotting, revealing 24 genes/operons (including eight novel genes), the expression of which was consistently higher in hrp-inducing minimal medium than in nutrient-rich Luria-Bertani broth. Expression of all eight genes was dependent on the hrpS gene. Most were also dependent on the hrpL gene, but at least one was dependent on the hrpS gene, but not on the hrpL gene. An AvrRpt2-based type III translocation assay provides evidence that some of the hrpS-regulated novel genes encode putative effector proteins.     

Evaluation of Mycobacterium smegmatis as a possible surrogate screen for selecting molecules active against multi-drug resistant Mycobacterium tuberculosis

New and better drugs are needed for tuberculosis (TB), particularly for the multi-drug resistant (MDR) disease. However, the highly infectious nature of MDR Mycobacterium tuberculosis restricts its use for large scale screening of probable drug candidates. We have evaluated the potential of a screen based on a 'fast grower' mycobacterium to shortlist compounds which could be active against MDR M. tuberculosis. Sensitivity profiles of M. smegmatis, M. phlei and M. fortuitum as well as MDR clinical isolates of M. tuberculosis were determined against anti-TB drugs isoniazid and rifampicin. Among the three fast growers, M. smegmatis was found to display a profile similar to MDR M. tuberculosis. Subsequently we evaluated the performance of M. smegmatis as a 'surrogate' screen for 120 compounds which were synthesized for anti-TB activity. Fifty of these molecules were active against M. tuberculosis H(37)Rv at a minimum inhibitory concentration (MIC) cutoff of 相似文献   

The C terminus of apolipoprotein A-V modulates lipid-binding activity

Human apolipoprotein A-V (apoA-V) is a potent modulator of plasma triacylglycerol (TG) levels. To probe different regions of this 343-amino-acid protein, four single Trp apoA-V variants were prepared. The variant with a Trp at position 325, distal to the tetraproline sequence at residues 293-296, displayed an 11-nm blue shift in wavelength of maximum fluorescence emission upon lipid association. To evaluate the structural and functional role of this C-terminal segment, a truncated apoA-V comprising amino acids 1-292 was generated. Far UV circular dichroism spectra of full-length apoA-V and apoA-V-(1-292) were similar, with approximately 50% alpha-helix content. In guanidine HCl denaturation experiments, both full-length and truncated apoA-V yielded biphasic profiles consistent with the presence of two structural domains. The denaturation profile of the lower stability component (but not the higher stability component) was affected by truncation. Truncated apoA-V displayed an attenuated ability to solubilize l-alpha-dimyristoylphosphatidylcholine phospholipid vesicles compared with full-length apoA-V, whereas a peptide corresponding to the deleted C-terminal segment displayed markedly enhanced kinetics. The data support the concept that the C-terminal region is not required for apoA-V to adopt a folded protein structure, yet functions to modulate apoA-V lipid-binding activity; therefore, this concept may be relevant to the mechanism whereby apoA-V influences plasma TG levels.     

Social dominance regulates androgen and estrogen receptor gene expression

In Astatotilapia burtoni, dominant males have higher levels of sex steroid hormones than subordinate males. Because of the complex regulatory interactions between steroid hormones and receptors, we asked whether dominance is also associated with variation in sex steroid receptor gene expression. Using quantitative PCR, we compared the expression of specific subtypes of androgen (AR) and estrogen (ER) receptor genes between dominant and subordinated males in 3 divisions of the brain, the pituitary, and the testes. We measured mRNA levels of AR-alpha, AR-beta, ER-alpha, ER-betaa, and ER-betab, gonadotropin-releasing hormone 1 (GnRH1), and GnRH receptor 1 (GnRH-R1) relative to 18S rRNA. In the anterior part of the brain, we found that dominant males had higher mRNA expression of AR-alpha, AR-beta, ER-betaa, and ER-betab, but not ER-alpha, compared to subordinate males. This effect of dominance was reflected in a positive correlation between testes size and AR-alpha, AR-beta, ER-betaa, and ER-betab in the anterior brain. In addition, mRNA levels of all ARs and ERs in the anterior brain were positively correlated with mRNA level of GnRH1. In the middle and posterior portions of the brain, as well as the testes, steroid receptor mRNA levels were similar among dominants and subordinates. In the pituitary, ER-alpha mRNA level was positively correlated with testes size and AR-alpha mRNA was positively correlated with GnRH-R1 mRNA level. These data suggest that dominant male brains could be more sensitive to sex steroids, which may contribute to the increased complexity of the behavioral repertoires of dominant males.     

Copper conjugates of nimesulide Schiff bases targeting VEGF, COX and Bcl-2 in pancreatic cancer cells

Copper conjugates of Schiff base derivatives of nimesulide (1), a well-known cyclooxygenase-2 (COX-2) inhibitor, were synthesized, structurally characterized and evaluated for their COX selectivity indices and cytotoxicities on pancreatic tumor, BxPC-3 (COX-2 positive) and MiaPaCa (COX-2 negative) cell lines. Copper conjugates exhibit distorted square planar geometries as revealed by the single crystal X-ray structure determination of Cu(L1)(2) and show significant growth inhibition in both cell lines (IC50 values 3-26 microM for COX-2 positive and 5-9 microM for COX-2 negative cell line) than the parent nimesulide (35 microM for COX-2 positive and >100 microM for COX-2 negative cell line). The mechanistic pathway for the biological activity involves inhibition of vascular endothelial growth factor (VEGF) and COX inhibition, as well as down regulation of antiapoptotic Bcl-2 and Bcl-(XL) proteins.