Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: INDISPENSABILITY OF THE STILBENOID CORE*

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential.     

A role for lymphotoxin in primary Sjogren's disease

The etiology of salivary gland injury in primary Sj?gren's disease is not well understood. We have previously described a mouse model of Sj?gren's disease, IL-14α transgenic (IL14αTG) mice, which reproduces many of the features of the human disease. We now demonstrate a critical role for lymphotoxin α (LTA) in the pathogenesis of Sj?gren's disease in IL14αTG mice. IL14αTG mice express LTA mRNA in their salivary glands and spleen and produce soluble LTA protein in their salivary secretions. When IL14αTG mice were crossed with LTA(-/-) mice, the IL14αTG.LTA(-/-) mice retained normal salivary gland secretions and did not develop either lymphocytic infiltration of their salivary glands or secondary lymphomas. However, both IL14αTG and IL14αTG.LTA(-/-) mice produced similar amounts of IFN-α and had similar deposition of autoantibodies in their salivary glands. Both IL14α and IL14α/LTA(-/-) mice had similar B cell responses to T-dependent and T-independent Ags, L-selectin expression, and expression of RelA, RelB, and NF-κB2 in their spleens. These studies suggest that LTA plays a critical role in the local rather than systemic inflammatory process of Sj?gren's disease. Furthermore, local production of soluble LTA in the salivary glands of IL14αTG mice is necessary for the development of overt Sj?gren's disease. Autoantibody deposition alone is not sufficient to produce salivary gland dysfunction. We also demonstrate that LTA is increased in the salivary gland secretions and sera of patients with Sj?gren's disease, further strengthening the biological relevance of the IL14αTG model to understanding the pathogenesis of human disease.     

A combined approach to data mining of textual and structured data to identify cancer-related targets

Background  

We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas.     

Synthesis and anticancer activity of certain mononuclear Ru (II) complexes

Bis(1,10-phenanthroline/2,2'-bipyridine) ruthenium(II)complexes containing TCP, TTZ OPBI, and BTSC ligands (where, TCP = 1-thiocarbamoyl-3,5-diphenyl-2-pyrazoline, TTZ = 2-(3,5-diphenyl-4,5-dihydropyrazol-1-yl)-4-phenylthiazole, OPBI = 2-hydroxyphenyl benzimidazole and BTSC = benzoin thiosemicarbazone) have been prepared and characterized. The spectral data suggested that the ligands were coordinated with the metal through nitrogen, sulfur and oxygen atoms. The target complexes were tested in vivo for anticancer activity against transplantable murine tumor cell line, Ehrlich's Ascitic Carcinoma (EAC). All these complexes increased the life span of the EAC-bearing mice, decreased their tumor volume and viable ascitic cell count as well as improved Hb, RBC and WBC counts. These results suggest that the Ru(II) complexes exhibit significant antitumor activity in EAC-bearing mice. It was also observed that the ruthenium complexes protected red blood cells from 2,2'-azo-bis(2-methylpropionamidine) dihydrochloride (AAPH)- induced hemolysis. The inhibitory effect was dose-dependent at a concentration of 20-120 microg/ml.     

Spatial distribution of tissue level properties in a human femoral cortical bone

The mechanical properties of cortical bone are determined by a combination bone tissue composition, and structure at several hierarchical length scales. In this study the spatial distribution of tissue level properties within a human femoral shaft has been investigated. Cylindrically shaped samples (diameter: 4.4mm, N=56) were prepared from cortical regions along the entire length (20-85% of the total femur length), and around the periphery (anterior, medial, posterior and lateral quadrants). The samples were analyzed using scanning acoustic microscopy (SAM) at 50MHz and synchrotron radiation micro computed tomography (SRμCT). For all samples the average cortical porosity (Ct.Po), tissue elastic coefficients (c(ij)) and the average tissue degree of mineralization (DMB) were determined. The smallest coefficient of variation was observed for DMB (1.8%), followed by BV/TV (5.4%), c(ij) (8.2-45.5%), and Ct.Po (47.5%). Different variations with respect to the anatomical position were found for DMB, Ct.Po and c(ij). These data address the anatomical variations in anisotropic elastic properties and link them to tissue mineralization and porosity, which are important input parameters for numerical multi-scale bone models.     

Role of Direct Repeat and Stem-Loop Motifs in mtDNA Deletions: Cause or Coincidence?

Deletion mutations within mitochondrial DNA (mtDNA) have been implicated in degenerative and aging related conditions, such as sarcopenia and neuro-degeneration. While the precise molecular mechanism of deletion formation in mtDNA is still not completely understood, genome motifs such as direct repeat (DR) and stem-loop (SL) have been observed in the neighborhood of deletion breakpoints and thus have been postulated to take part in mutagenesis. In this study, we have analyzed the mitochondrial genomes from four different mammals: human, rhesus monkey, mouse and rat, and compared them to randomly generated sequences to further elucidate the role of direct repeat and stem-loop motifs in aging associated mtDNA deletions. Our analysis revealed that in the four species, DR and SL structures are abundant and that their distributions in mtDNA are not statistically different from randomized sequences. However, the average distance between the reported age associated mtDNA breakpoints and their respective nearest DR motifs is significantly shorter than what is expected of random chance in human (p<10(-4)) and rhesus monkey (p = 0.0034), but not in mouse (p = 0.0719) and rat (p = 0.0437), indicating the existence of species specific difference in the relationship between DR motifs and deletion breakpoints. In addition, the frequencies of large DRs (>10 bp) tend to decrease with increasing lifespan among the four mammals studied here, further suggesting an evolutionary selection against stable mtDNA misalignments associated with long DRs in long-living animals. In contrast to the results on DR, the probability of finding SL motifs near a deletion breakpoint does not differ from random in any of the four mtDNA sequences considered. Taken together, the findings in this study give support for the importance of stable mtDNA misalignments, aided by long DRs, as a major mechanism of deletion formation in long-living, but not in short-living mammals.     

Surface loop dynamics in adeno-associated virus capsid assembly

The HI loop is a prominent domain on the adeno-associated virus (AAV) capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring fivefold VP. Despite the highly conserved nature of the residues at the fivefold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion, and substitution mutants. A mutant lacking the HI loop was unable to assemble particles, while a substitution mutant (10 glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5, and AAV8 yielded (i) particles with titers and infectivity identical to those of AAV2 (AAV2 HI1 and HI8), (ii) particles with a decreased virus titer (1 log) but normal infectivity (HI4), and (iii) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we replaced the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino-acid peptide substitutions unless they spanned a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer but a substantial decrease (1 log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the fivefold axis. Western blot analysis confirmed inefficient incorporation of VP1, as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging, all of which implies a potential role for this unique surface domain in viral infectivity.     

Structural and mutational characterization of -carnitine binding to human carnitine acetyltransferase

We report the crystal structure of a binary complex of human peroxisomal carnitine acetyltransferase and the substrate l-carnitine, refined to a resolution of 1.8 Angstrom with an R(factor) value of 18.9% (R(free)=22.3%). L-carnitine binds to a preformed pocket in the active site tunnel of carnitine acetyltransferase aligned with His(322). The quaternary nitrogen of carnitine forms a pi-cation interaction with Phe(545), while Arg(497) forms an electrostatic interaction with the negatively charged carboxylate group. An extensive hydrogen bond network also occurs between the carboxylate group and Tyr(431), Thr(444), and a bound water molecule. Site-directed mutagenesis and kinetic characterization reveals that Tyr(431), Thr(444), Arg(497), and Phe(545) are essential for high affinity binding of L-carnitine.     

Inhibition of carbonic anhydrase II by thioxolone: a mechanistic and structural study

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791 . Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.     

STUDIES ON THE TISSUE AND SUBCELLULAR DISTRIBUTION OF β-N-OXALYL-l-α, β-DIAMINOPROPIONIC ACID, THE LATHYRUS SATIVUS NEUROTOXIN

Abstract— β- N -Oxalyl- l -α, β-diaminopropionic acid (ODAP), the Lathyrus sativus neurotoxin can be detected in significant concentrations in the synaptosomal fractions isolated from young rat brain and adult monkey spinal cord, when these animals manifest neurological symptoms after ODAP administration. However, isolated synaptosomes fail to exhibit any transport system for ODAP uptake. ODAP administered in vivo appears to get localized in a population of synaptosomes which exhibit a high affinity uptake system for glutamate.