Generation and exploration of new classes of antitubercular agents: The optimization of oxazolines, oxazoles, thiazolines, thiazoles to imidazo[1,2-a]pyridines and isomeric 5,6-fused scaffolds

Tuberculosis (TB) is a devastating disease resulting in a death every 20s. Thus, new drugs are urgently needed. Herein we report ten classes of compounds-oxazoline, oxazole, thiazoline, thiazole, pyrazole, pyridine, isoxazole, imidazo[1,2-a]pyridine, imidazo[1,2-a]pyrimidine and imidazo[1,2-c]pyrimidine-which have good (micromolar) to excellent (sub-micromolar) antitubercular potency. The 5,6-fused heteroaromatic compounds were the most potent with MIC's as low as <0.195 μM (9 and 11). Overall, the imidazo[1,2-a]pyridine class was determined to be most promising, with potency similar to isoniazid and PA-824 against replicating Mtb H(37)Rv, clinically relevant drug sensitive, multi- and extensively resistant Mtb strains as well as having good in vitro metabolic stability.     

Solid-phase synthesis of 2'-hydroxychalcones. Effects on cell growth inhibition, cell cycle and apoptosis of human tumor cell lines

Thirty-one 2'-hydroxychalcones were prepared via solid-phase synthesis by base-catalyzed aldol condensation of substituted 2'-hydroxyacetophenones and benzaldehydes. Chalcones were tested for their growth inhibitory activity in three human tumor cell lines (MCF-7, NCI-H460 and A375-C5) using the SRB assay. Results revealed that several of the tested compounds caused a pronounced dose-dependent growth inhibitory effect on the tumor cell lines studied in the low micromolar range. To gain further insight on the cellular mechanism of action of this class of compounds, studies of their effect on cell cycle profile as well as on induction of cellular apoptosis were also carried out. Generally, the tested chalcones interfered with the cell cycle profile and increased the percentage of apoptotic MCF-7 cells. The results here presented may help to identify new chalcone-like structures with optimized cell growth inhibitory activity which may be further tested as potential antitumor agents.     

Sarcosin (Krp1) in skeletal muscle differentiation: gene expression profiling and knockdown experiments

SARCOSIN, also named Krp1, has been identified as a protein exclusively expressed in striated muscle tissue. Here we report on the role of SARCOSIN in skeletal muscle development and differentiation. We demonstrate, by means of whole-mount in situ hybridization, that Sarcosin mRNA is expressed in the myotome part of the mature somites in mouse embryos from embryonic day 9.5 onwards. Sarcosin is not expressed in the developing heart at these embryonic stages, and in adult tissues the mRNA expression levels are five times lower in the heart than in skeletal muscle. SARCOSIN protein partially co-localizes with the M-band protein myomesin and between and below laterally fusing myofibrils in adult skeletal muscle tissue. RNA interference mediated knock-down of SARCOSIN in the C2C12 myoblast cell line appeared to be stimulatory in the early phase of differentiation, but inhibitory at a later phase of differentiation.     

Solution structures of the first and fourth TSR domains of F-spondin

F-spondin is a protein mainly associated with neuronal development. It attaches to the extracellular matrix and acts in the axon guidance of the developing nervous system. F-spondin consists of eight domains, six of which are TSR domains. The TSR domain family binds a wide range of targets. Here we present the NMR solution structures of TSR1 and TSR4. TSR domains have an unusual fold that is characterized by a long, nonglobular shape, consisting of two beta-strands and one irregular extended strand. Three disulfide bridges and stack of alternating tryptophan and arginine side-chains stabilize the structure. TSR1 and TSR4 structures are similar to each other and to the previously determined TSR domain X-ray structures from another protein, TSP, although TSR4 exhibits a mobile loop not seen in other structures.     

Comprehensive repertoire and phylogenetic analysis of the G protein-coupled receptors in human and mouse

Understanding differences in the repertoire of orthologous gene pairs is vital for interpretation of pharmacological and physiological experiments if conclusions are conveyed between species. Here we present a comprehensive dataset for G protein-coupled receptors (GPCRs) in both human and mouse with a phylogenetic road map. We performed systematic searches applying several search tools such as BLAST, BLAT, and Hidden Markov models and searches in literature data. We aimed to gather a full-length version of each human or mouse GPCR in only one copy referring to a single chromosomal position. Moreover, we performed detailed phylogenetic analysis of the transmembrane regions of the receptors to establish accurate orthologous pairs. The results show the identity of 495 mouse and 400 human functional nonolfactory GPCRs. Overall, 329 of the receptors are found in one-to-one orthologous pairs, while 119 mouse and 31 human receptors originate from species-specific expansions or deletions. The average percentage similarity of the orthologue pairs is 85%, while it varies between the main GRAFS families from an average of 59 to 94%. The orthologous pairs for the lipid-binding GPCRs had the lowest levels of conservation, while the biogenic amines had highest levels of conservation. Moreover, we searched for expressed sequence tags (ESTs) and identified more than 17,000 ESTs matching GPCRs in mouse and human, providing information about their expression patterns. On the whole, this is the most comprehensive study of the gene repertoire that codes for human and mouse GPCRs. The datasets are available for downloading.     

Chemo-enzymatic synthesis of poly-<Emphasis Type="Italic">N</Emphasis>-acetyllactosamine (poly-LacNAc) structures and their characterization for CGL2-galectin-mediated binding of ECM glycoproteins to biomaterial surfaces

Poly-N-acetyllactosamine (poly-LacNAc) structures have been identified as important ligands for galectin-mediated cell adhesion to extra-cellular matrix (ECM) proteins. We here present the biofunctionalization of surfaces with poly-LacNAc structures and subsequent binding of ECM glycoproteins. First, we synthesized β-GlcNAc glycosides carrying a linker for controlled coupling onto chemically functionalized surfaces. Then we produced poly-LacNAc structures with defined lengths using human β1,4-galactosyltransferase-1 and β1,3-N-acetylglucosaminyltransferase from Helicobacter pylori. These compounds were also used for kinetic characterization of glycosyltransferases and lectin binding assays. A mixture of poly-LacNAc-structures covalently coupled to functionalized microtiter plates were identified for best binding to our model galectin His₆CGL2. We further demonstrate for the first time that these poly-LacNAc surfaces are suitable for further galectin-mediated binding of the ECM glycoproteins laminin and fibronectin. This new technology should facilitate cell adhesion to biofunctionalized surfaces by imitating the natural ECM microenvironment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Inhibition of cancer cell growth by ruthenium(II) cyclopentadienyl derivative complexes with heteroaromatic ligands

Inhibition of the growth of LoVo human colon adenocarcinoma and MiaPaCa pancreatic cancer cell lines by two new organometallic ruthenium(II) complexes of general formula [Ru(η⁵-C₅H₅)(PP) L][CF₃SO₃], where PP is 1,2-bis(diphenylphosphino)ethane and L is 1,3,5-triazine (Tzn) 1 or PP is 2x triphenylphosphine and L is pyridazine (Pyd) 2 has been investigated. Crystal structures of compounds 1 and 2 were determined by X-ray diffraction studies. Atomic force microscopy (AFM) images suggest different mechanisms of interaction with the plasmid pBR322 DNA; while the mode of binding of compound 1 could be intercalation between base pairs of DNA, compound 2 might be involved in a covalent bond formation with N from the purine base.     

Variation in the Distribution of Trace Elements in Renal Cell Carcinoma

The development of cancer is a complex, multistage process during which a normal cell undergoes genetic changes that result in phenotypic alterations and in the acquisition of the ability to invade other sites. Inductively coupled plasma optical emission spectroscopy was used to estimate the contents of Al, Ca, Cd, Cr, Cu, Fe, K, Mg, Mn, Na, P, Pb, and Zn in healthy kidney and renal cell carcinoma (RCC), and significant differences were found for all elements. Along with the progression of the malignant disease, a progressive decrease of Cd and K was observed. In fact, for Cd, the concentration in stage T4 was 263.9 times lower than in stage T1, and for K, the concentration in stage T4 was 1.73 times lower than in stage T1. Progressive accumulation was detected for P, Pb, and Zn in stage T4. For P, the concentration in stage T4 was 11.1 times higher than in stage T1; for Pb, the concentration in stage T4 was 232.7 times higher than in T1; and for Zn, the concentration in T4 was 8.452 times higher than in T1. This study highlights the marked differences in the concentrations of selected trace metals in different malignant tumor stages. These findings indicate that some trace metals may play important roles in the pathogenesis of RCC.     

Islet neogenesis-associated protein pentadecapeptide (INGAP-PP): Mechanisms involved in its effect upon β-cell mass and function

The effect of islet neogenesis-associated protein pentadecapeptide (INGAP-PP) administration to normal male hamsters upon serum glucose and triglyceride levels, β-cell mass and function was studied. INGAP-PP (500 μg) or saline was injected twice daily during 10 days. Both groups showed comparable body weight, serum glucose and triglyceride levels. INGAP-PP treated animals had significantly higher HOMA-IR and HOMA-β and their islets released more insulin in response to glucose; they had lower islet DNA content, significantly increased number of islets/unit area, β-cell replication rate and mass, cells co-expressing Pdx-1/INGAP and islets in contact with ducts, and decreased β-cell apoptosis rate. The percentage of cells expressing Pdx-1 alone or together with INGAP or insulin increased significantly in ducts. These animals also showed a significantly higher concentration of Pdx-1 and Ngn-3 mRNA and a lower number of INGAP-positive cells. In conclusion, INGAP-PP promoted a controlled and functionally active increase of β-cell mass; our data demonstrate for the first time the mechanism responsible for such changes; that Ngn-3 would be involved in INGAP-PP-induced neogenesis; and the existence of a negative feedback loop with endogenous INGAP-producing cells. Accordingly, INGAP-PP could be used to induce these effects in people with or at risk of developing diabetes.