Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.     

Synthesis and in vitro evaluation of derivatives of the β₁-adrenergic receptor antagonist HX-CH 44

Isopropyl- and fluoroisopropyl-amino derivatives of the β(1)-adrenergic receptor antagonist 2-[4-[3-(tert-butyl-amino)-2-hydroxypropoxy]phenyl]-3-methyl-6-methoxy-4(3H)-quinazolinone ((±)HX-CH 44) were synthesized, including a concise and efficient preparation of the core, 2-(4-hydroxyphenyl)-6-methoxy-3-methylquinazolin-4(3H)-one. In vitro binding assays showed that the fluorinated analog was selective towards β(1)-adrenergic receptors over β(2)-adrenergic and 5-HT(1A) receptors. An X-ray crystallographic characterization of the fluorinated analog is also reported.     

Synthesis and in vitro evaluation of α-synuclein ligands

Accumulation of misfolded α-synuclein in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD). To identify ligands having high binding potency toward aggregated α-synuclein, we synthesized a series of phenothiazine derivatives and assessed their binding affinity to recombinant α-synuclein fibrils using a fluorescent thioflavin T competition assay. Among 16 new analogues, the in vitro data suggest that compound 11b has high affinity to α-synuclein fibrils (K(i)=32.10 ± 1.25 nM) and compounds 11d, 16a and16b have moderate affinity to α-synuclein fibrils (K(i)≈50-100 nM). Further optimization of the structure of these analogues may yield compounds with high affinity and selectivity for aggregated α-synuclein.     

Synthesis and anti-BVDV activity of novel δ-sultones in vitro: Implications for HCV therapies

In this study we report the synthesis and activity against bovine viral diarrhea virus (BVDV) of a novel series of bicycle δ-sultones containing γ-lactones. BVDV is responsible for major losses in cattle. Some of the synthesized δ-sultones showed pronounced anti-BVDV activity with EC₅₀ values of 0.12–1.0 μM and no significant cytotoxicity. Among them, the ortho bromosubstituted derivative 4f (EC₅₀ = 0.12 μM) showed better antiviral activity than other derivatives and was 10 fold more that of than positive control ribavirin (EC₅₀ = 1.3 μM). BVDV is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. The above results provided a novel candidate for the development of anti-HCV agents.     

Phenotype and growth behavior of residual β-catenin-positive hepatocytes in livers of β-catenin-deficient mice

Signaling through the Wnt/β-catenin pathway is a crucial determinant of hepatic zonal gene expression, liver development, regeneration, and tumorigenesis. Transgenic mice with hepatocyte-specific knockout of Ctnnb1 (encoding β-catenin) have proven their usefulness in elucidating these processes. We now found that a small number of hepatocytes escape the Cre-mediated gene knockout in that mouse model. The remaining β-catenin-positive hepatocytes showed approximately 25% higher cell volumes compared to the β-catenin-negative cells and exhibited a marker protein expression profile similar to that of normal perivenous hepatocytes or hepatoma cells with mutationally activated β-catenin. Surprisingly, the expression pattern was observed independent of the cell's position within the liver lobule, suggesting a malfunction of physiological periportal repression of perivenously expressed genes in β-catenin-deficient liver. Clusters of β-catenin-expressing hepatocytes lacked expression of the gap junction proteins Connexin 26 and 32. Nonetheless, β-catenin-positive hepatocytes had no striking proliferative advantage, but started to grow out on treatment with phenobarbital, a tumor-promoting agent known to facilitate the formation of mouse liver adenoma with activating mutations of Ctnnb1. Progressive re-population of Ctnnb1 knockout livers with wild-type hepatocytes was seen in aged mice with a pre-cirrhotic phenotype. In these large clusters of β-catenin-expressing hepatocytes, perivenous-specific gene expression was re-established. In summary, our data demonstrate that the zone-specificity of a hepatocyte's gene expression profile is dependent on the presence of β-catenin, and that β-catenin provides a proliferative advantage to hepatocytes when promoted with phenobarbital, or in a pre-cirrhotic environment.     

Synthesis and in vitro antikinetoplastid activity of polyamine–hydroxybenzotriazole conjugates

Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was envisioned as a potential target. Among all tested molecules, only one compound, a N³-spermidine–benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on parasites. The corresponding Boc-protected spermidine–benzotriazole was however trypanocidal against Trypanosoma brucei gambiense with an IC₅₀ value of 1 μM and was completely devoid of cytotoxicity. On the intramacrophage amastigotes of Leishmania donovani, a N²-spermidine conjugate of this series, exhibited an interesting IC₅₀ value of 3 μM associated with both low cytotoxicity against axenic Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid agents and their targets have to be deciphered.     

Synthesis and in vitro stability of amino acid prodrugs of 6-β-naltrexol for microneedle-enhanced transdermal delivery

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a–f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid bioconversion to NTXOL in human plasma (t_1/2 = 2.2 ± 0.1 h).     

Synthesis and in vitro cytotoxicity evaluation of β-carboline-combretastatin carboxamides as apoptosis inducing agents: DNA intercalation and topoisomerase-II inhibition

To explore a new set of cytotoxic agents, β-carboline-combretastatin carboxamide conjugates were designed, synthesized and evaluated for their in vitro cytotoxicity potential, DNA binding affinity and Topoisomerase-II (topo-II) inhibition activity. Among the designed hybrids, 10v and 10af have shown significant cytotoxic effect against A549 (lung cancer) cell line having IC₅₀ value 1.01 µM and 1.17 µM respectively. Further, it was speculated that treatment with compound 10v may induce apoptosis among A549 cells, which was supported by Hoechst staining, DCFDA, Annexin V-FITC and morphological assays. Flow cytometric analysis revealed that the hybrid 10v arrests A549 cells in G2/M phase of cell cycle in a dose dependent manner. Amongst the active hybrids, most potent hybrid 10v was tested for DNA topo-II inhibition activity. Moreover, to further support the biological activity and to infer the mode of interaction between ligands and DNA, spectroscopy and molecular docking studies were carried out. The docking and spectroscopy results showed that the ligands exhibited an intercalative mode of binding with DNA and could efficiently bind to DNA and form topo-II ternary complex. Based on these experiments, the hybrids 10v and 10af were identified as proficient new scaffolds which need to be developed as hit molecules for therapeutic interest.     

Synthesis and in vitro α-chymotrypsin inhibitory activity of 6-chlorobenzimidazole derivatives

A library of benzimidazole derivatives 1–20 were synthesized, and studied for their α-chymotrypsin (α-CT) inhibitory activity in vitro. Kinetics and molecular docking studies were performed to identify the type of inhibition. Compound 1 was found to be a good inhibitor of α-chymotrypsin enzyme (IC₅₀ = 14.8 ± 0.1 μM, K_i = 16.4 μM), when compared with standard chymostatin (IC₅₀ = 5.7 ± 0.13 μM). Compounds 2–8, 15, 17, and 18 showed significant inhibitory activities. All the inhibitors were found to be competitive inhibitors, except compound 17, which was a mixed type inhibitor. The substituents (R) in para and ortho positions of phenyl ring B, apparently played a key role in the inhibitory potential of the series. Compounds 1–20 were also studied for their cytotoxicity profile by using 3T3 mouse fibroblast cells and compounds 3, 5, 6, 8, 12–14, 16, 17, 19, and 20 were found to be cytotoxic. Molecular docking was performed on the most active members of the series in comparison to the standard compound, chymostatin, to identify the most likely binding modes. The compounds reported here can serve as templates for further studies for new inhibitors of α-chymotrypsin and other chymotrypsin-like serine proteases enzymes.

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β-Puromycin selection of modified ribosomes for in vitro incorporation of β-amino acids

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against β-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated β-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing β-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of β-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence β-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. β-Puromycin, a putative mimetic for β-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of β-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to β-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of β-alanyl-tRNA(CUA).     

N⁶-Alkyladenosines: Synthesis and evaluation of in vitro anticancer activity

A series of adenosine analogues differently substituted in N⁶-position were synthesized to continue our studies on the relationships between structure and biological activity of iPA. The structures of the compounds were confirmed by standard studies of ¹H NMR, MS and elemental analysis. These molecules were then evaluated for their anti-proliferative activity on bladder cancer cells. We found that some of these compounds possess anti-proliferative activity but have no effect on cell invasion and metalloprotease activity.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Porogen Effect on Structural and Physical Properties of β-TCP Scaffolds for Bone Tissue Regeneration

Scaffolds for bone tissue applications have been an outstanding alternative to repair and regenerate bone tissue defects caused by traumas or illness. There are many methods available to fabricate porous scaffold such as solvent casting, gas bubble, phase separation, electrospinning, particle-leaching, among others. The particle-leaching technique has shown advantages in bone tissue regeneration applications, the main benefit of this technique is related to the porogen particle size and the porogen content in the manufacture of scaffolds. Tricalcium phosphate is one calcium phosphate that presented appropriated characteristic to be used for bone tissue engineering due to the chemical properties similar to the human bones. Scaffolds of tricalcium phosphate β phase were made using sugar particles. The porogen was varied in amounts of 50, 60 and 70 wt.% of two commercial sugars with the remainder of the composition made up of tricalcium phosphate powders. The pore sizes in all the scaffolds were in the range of 90 to 600 μm with an irregular pore morphology and the porosity was in the range of 63 to 77%.     

A histidine in the β-CASP domain of Artemis is critical for its full in vitro and in vivo functions

Artemis is a key factor of the nonhomologous end-joining (NHEJ) pathway, which is critical for DNA double-strand break (DSB) repair in eukaryotic cells. It belongs to the β-CASP family of nucleases, forming a distinct group within the metallo-β-lactamase superfamily. Proteins of this group are specific for nucleic acids and contain an original domain, the β-CASP domain, which serves as a cap covering the active site displayed by the metallo-β-lactamase domain.Here, we have identified in the highly divergent sequences of the β-CASP domains from DNA-specific nucleases two conserved residues (Artemis E213 and H254), which are not present in RNA-specific enzymes, and shown that H254 plays a key role in the Artemis function, as it is critical for its full activity in vitro. Moreover, inherited mutation of H254 results in radiosensitive severe combined immune deficiency (RS-SCID) in humans. This residue might play a key role in specificity towards DNA, if not directly in zinc binding.     

Synthesis and in vitro evaluation of vanillin derivatives as multi-target therapeutics for the treatment of Alzheimer’s disease

A number of novel naphthalimido and phthalimido vanillin derivatives were synthesised, and evaluated as antioxidants and cholinesterase inhibitors in vitro. Antioxidant activity was assessed using DPPH, FRAP, and ORAC assays. All compounds demonstrated enhanced activity compared to the parent compound, vanillin. They also exhibited BuChE selectivity in Ellman’s assay. A lead compound, 2a (2-(3-(bis(4-hydroxy-3-methoxybenzyl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione), was identified and displayed strong antioxidant activity (IC₅₀ of 16.67 µM in the DPPH assay, a 25-fold increase in activity compared to vanillin in the FRAP assay, and 9.43 TE in the ORAC assay). Furthermore, 2a exhibited potent BuChE selectivity, with an IC₅₀ of 0.27 µM which was around 53-fold greater than the corresponding AChE inhibitory activity. Molecular modelling studies showed that molecules with bulkier groups, as in 2a, exhibited better BuChE selectivity. This work provides a promising basis for the development of multi-target hybrid compounds based on vanillin as potential AD therapeutics.     

2,5-Disubstituted thiadiazoles as potent β-glucuronidase inhibitors; Synthesis,in vitro and in silico studies

Twenty-five thiadiazole derivatives 1–25 were synthesized from methyl 4-methoxybenzoate via hydrazide and thio-hydrazide intermediates, and evaluated for their potential against β-glucuronidase enzyme. Most of the compounds including 1 (IC₅₀ = 26.05 ± 0.60 μM), 2 (IC₅₀ = 42.53 ± 0.80 μM), 4 (IC₅₀ = 38.74 ± 0.70 μM), 5 (IC₅₀ = 9.30 ± 0.29 μM), 6 (IC₅₀ = 6.74 ± 0.26 μM), 7 (IC₅₀ = 18.40 ± 0.66 μM), and 15 (IC₅₀ = 18.10 ± 0.53 μM) exhibited superior activity potential than the standard d-saccharic acid-1,4-lactone (IC₅₀ = 48.4 ± 1.25 μM). Molecular docking studies were conducted to correlate the in vitro results and to identify possible mode of interaction with enzyme active site.     

Synthesis, docking and in vitro antimalarial evaluation of bifunctional hybrids derived from β-lactams and 7-chloroquinoline using click chemistry

1,2,3-Triazole tethered β-lactam and 7-chloroquinoline bifunctional hybrids were synthesized and evaluated as potential antimalarial agents. Activity against cultured Plasmodium falciparum was dependent on the N-substituent of the β-lactam ring as well as the presence of bis-triazole at the C-3 position. The observed activity profiles were further substantiated by docking studies via inhibition of P. falciparum dihydrofolate reductase (PfDHFR), a potential target for the development of new anti-malarials.     

Synthesis of β-functionalized temoporfin derivatives for an application in photodynamic therapy

The synthesis of novel β-functionalized derivatives of the clinically used photosensitizer Temoporfin has been achieved by nucleophilic addition reactions to a corresponding diketo chlorin. The β-substituted dihydroxychlorin products exhibit a strong absorption in the red spectral region, a high singlet oxygen quantum yield, and were found to be highly effective in in vitro assays against HT-29 tumor cells.