Development of 3‐Phenyl‐N‐(2‐(3‐phenylureido)ethyl)‐thiophene‐2‐sulfonamide Compounds as Inhibitors of Antiapoptotic Bcl‐2 Family Proteins

Antiapoptotic Bcl‐2 family proteins, such as Bcl‐x_L, Bcl‐2, and Mcl‐1, are often overexpressed in tumor cells, which contributes to tumor cell resistance to chemotherapies and radiotherapies. Inhibitors of these proteins thus have potential applications in cancer treatment. We discovered, through structure‐based virtual screening, a lead compound with micromolar binding affinity to Mcl‐1 (inhibition constant (K_i)=3 μM ). It contains a phenyltetrazole and a hydrazinecarbothioamide moiety, and it represents a structural scaffold not observed among known Bcl‐2 inhibitors. This work presents the structural optimization of this lead compound. By following the scaffold‐hopping strategy, we have designed and synthesized a total of 82 compounds in three sets. All of the compounds were evaluated in a fluorescence‐polarization binding assay to measure their binding affinities to Bcl‐x_L, Bcl‐2, and Mcl‐1. Some of the compounds with a 3‐phenylthiophene‐2‐sulfonamide core moiety showed sub‐micromolar binding affinities to Mcl‐1 (K_i=0.3–0.4 μM ) or Bcl‐2 (K_i≈1 μM ). They also showed obvious cytotoxicity on tumor cells (IC₅₀<10 μM ). Two‐dimensional heteronuclear single quantum coherence NMR spectra of three selected compounds, that is, YCW‐E5, YCW‐E10, and YCW‐E11, indicated that they bind to the BH3‐binding groove on Bcl‐x_L in a similar mode to ABT‐737. Several apoptotic assays conducted on HL‐60 cells demonstrated that these compounds are able to induce cell apoptosis through the mitochondrial pathway. We propose that the compounds with the 3‐phenylthiophene‐2‐sulfonamide core moiety are worth further optimization as effective apoptosis inducers with an interesting selectivity towards Mcl‐1 and Bcl‐2.     

De Novo Design,Synthesis and Evaluation of Benzylpiperazine Derivatives as Highly Selective Binders of Mcl‐1

Considerable efforts have been made to the development of small‐molecule inhibitors of antiapoptotic B‐cell lymphoma 2 (Bcl‐2) family proteins (such as Bcl‐2, Bcl‐x_L, and Mcl‐1) as a new class of anticancer therapies. Unlike general inhibitors of the entire family, selective inhibitors of each member protein can hopefully reduce the adverse side effects in chemotherapy treatments of cancers overexpressing different Bcl‐2 family proteins. In this study, we designed four series of benzylpiperazine derivatives as plausible Bcl‐2 inhibitors based on the outcomes of a computational algorithm. A total of 81 compounds were synthesized, and their binding affinities to Bcl‐2, Bcl‐x_L, and Mcl‐1 measured. Encouragingly, 22 compounds exhibited binding affinities in the micromolar range (K_i<20 μM ) to at least one target protein. Moreover, some compounds were observed to be highly selective binders to Mcl‐1 with no detectable binding to Bcl‐2 or Bcl‐x_L, among which the most potent one has a K_i value of 0.18 μM for Mcl‐1. Binding modes of four selected compounds to Mcl‐1 and Bcl‐x_L were derived through molecular docking and molecular dynamics simulations. It seems that the binding affinity and selectivity of these compounds can be reasonably interpreted with these models. Our study demonstrated the possibility for obtaining selective Mcl‐1 inhibitors with relatively simple chemical scaffolds. The active compounds identified by us could be used as lead compounds for developing even more potent selective Mcl‐1 inhibitors with potential pharmaceutical applications.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

Experimental Characterization of the Binding Affinities between Proapoptotic BH3 Peptides and Antiapoptotic Bcl‐2 Proteins

The Bcl‐2 family proteins are key regulators of the intrinsic apoptotic pathway and are among the validated targets for developing anticancer drugs. Protein–protein interactions between the pro‐ and antiapoptotic members of this family determine mitochondrial outer‐membrane permeabilization. Elucidating such protein–protein interactions in a quantitative way is helpful for network pharmacology studies on the Bcl‐2 family, which, in turn, will provide valuable guidance for developing new anticancer therapies. In this study, the binding affinities of the BH3 peptides derived from eight proapoptotic BH3‐only proteins (i.e., Bid, Bim, Puma, Noxa, Bad, Bmf, Bik, Hrk) against five well‐studied antiapoptotic proteins (i.e., Bcl‐x_L, Bcl‐2, Mcl‐1, Bcl‐w, Bfl‐1) in the Bcl‐2 family have been measured. Three different types of binding assay (i.e., surface plasmon resonance, fluorescence polarization, and homogeneous time‐resolved fluorescence) were employed for cross‐validation. The results confirmed that each proapoptotic BH3 peptide exhibited a distinct binding profile against the five antiapoptotic proteins. The binding data obtained herein serve as a fresh update or correction to existing knowledge. It is expected that such binding data will be helpful for building more accurate mathematical network models for depicting the complex protein–protein interactions within the Bcl‐2 family.     

Structure‐Guided Rational Design of α/β‐Peptide Foldamers with High Affinity for BCL‐2 Family Prosurvival Proteins

We have used computational methods to improve the affinity of a foldamer ligand for its target protein. The effort began with a previously reported α/β‐peptide based on the BH3 domain of the proapoptotic protein Puma; this foldamer binds tightly to Bcl‐x_L but weakly to Mcl‐1. The crystal structure of the Puma‐derived α/β‐peptide complexed to Bcl‐x_L was used as the basis for computational design of variants intended to display improved binding to Mcl‐1. Molecular modelling suggested modification of three α residues of the original α/β backbone. Individually, each substitution caused only a modest (4‐ to 15‐fold) gain in affinity; however, together the three substitutions led to a 250‐fold increase in binding to Mcl‐1. These modifications had very little effect on affinity for Bcl‐x_L. Crystal structures of a number of the new α/β‐peptides bound to either Mcl‐1 or Bcl‐x_L validated the selection of each substitution. Overall, our findings demonstrate that structure‐guided rational design can be used to improve affinity and alter partner selectivity of peptidic ligands with unnatural backbones that bind to specific protein partners.     

Two‐Face,Two‐Turn α‐Helix Mimetics Based on a Cross‐Acridine Scaffold: Analogues of the Bim BH3 Domain

The design of a cross‐acridine scaffold mimicking the i, i+3, i+5, and i+7 residues distributed over a two‐face, two‐turn α‐helix is described. Docking studies and 2D ¹H,¹⁵N HSQC NMR spectroscopy provide compelling evidence that compound 3 d accurately reproduces the arrangement of four hotspots in the Bim BH3 peptide to permit binding to the Mcl‐1 and Bcl‐2 proteins (K_i 0.079 and 0.056 μM , respectively). Furthermore, the hotspot mutation could also be mimicked by individual or multiple deletions of side chains on the scaffold.     

Isolation of a Small‐Molecule Inhibitor of the Antiapoptotic Protein Bcl‐xL from a DNA‐Encoded Chemical Library

Bcl‐xL is an antiapoptotic member of the Bcl‐2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl‐xL from a DNA‐encoded chemical library using affinity‐capture selections and massively parallel high‐throughput sequencing of >30 000 sequence tags of library members. The most potent binder identified, compound 19 / 93 [(R)‐3‐(amido indomethacin)‐4‐(naphthalen‐1‐yl)butanoic acid], bound to Bcl‐xL with a dissociation constant (K_d) of 930 nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function.     

Synthetic Inhibitors of Extended Helix–Protein Interactions Based on a Biphenyl 4,4′‐Dicarboxamide Scaffold

Turn Bak : We present rationally designed scaffolds that mimic the spatial projection of the i, i+4, i+7, and i+11 residues of an α‐helix. A library of biphenyl derivatives was shown by competition fluorescence polarization and ITC to mimic Bak and disrupt the Bak/Bcl‐x_L protein–protein interaction. ¹⁵N HSQC experiments confirmed that the surface of Bcl‐x_L normally occupied by Bak was the target area of our new synthetic inhibitors.

     

Discovery of a Small‐Molecule pBcl‐2 Inhibitor that Overcomes pBcl‐2‐Mediated Resistance to Apoptosis

Although the role of Bcl‐2 phosphorylation is still under debate, it has been identified in a resistance mechanism to BH3 mimetics, for example ABT‐737 and S1 . We identified an S1 analogue, S1‐16 , as a small‐molecule inhibitor of pBcl‐2. S1‐16 efficiently kills EEE‐Bcl‐2 (a T69E, S70E, and S87E mutant mimicking phosphorylation)‐expressing HL‐60 cells and high endogenously expressing pBcl‐2 cells, by disrupting EEE‐Bcl‐2 or native pBcl‐2 interactions with Bax and Bak, followed by apoptosis. In vitro binding assays showed that S1‐16 binds to the BH3 binding groove of EEE‐Bcl‐2 (K_d=0.38 μM by ITC; IC₅₀=0.16 μM by ELISA), as well as nonphosphorylated Bcl‐2 (npBcl‐2; K_d=0.38 μM ; IC₅₀=0.12 μM ). However, ABT‐737 and S1 had much weaker affinities to EEE‐Bcl‐2 (IC₅₀=1.43 and >10 μM , respectively), compared with npBcl‐2 (IC₅₀=0.011 and 0.74 μM , respectively). The allosteric effect on BH3 binding groove by Bcl‐2 phosphorylation in the loop region was illustrated for the first time.     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

Tailor‐Made Designer Helical Peptides that Induce Mitochondrion‐Mediated Cell Death without Necrosis

Managing protein–protein interactions is essential for resolving unknown biological events at the molecular level and developing drugs. We have designed and synthesized a side‐chain‐crosslinked helical peptides based on the binding domain of a pro‐apoptotic protein (Bad) that induces programed cell death. The peptide showed high helical content and bound to its target, Bcl‐X_L, more strongly than its non‐crosslinked counterparts. When HeLa cells were incubated with the crosslinked peptide, the peptide entered the cytosol across the plasma membrane. The peptide formed a stable complex with Bcl‐X_L localized at the outer mitochondrial membrane, and this binding event caused the release of cytochrome c from the intermembrane space of mitochondria into the cytosol. This activated the caspase cascade: 70 % of HeLa cells died by the apoptosis pathway (without evidence of necrosis).     

Separation and concentration of bilberry impact aroma compound from dilute model solution by pervaporation

BACKGROUND: In the present work, the flavor compounds of natural juice of bilberries were qualitatively analyzed by GC‐MS, leading to the identification of trans‐Hex‐2‐en‐1‐ol as one of the major ‘impact aroma’ compounds of this fruit. The pervaporation of trans‐Hex‐2‐en‐1‐ol from diluted aqueous solutions was studied using commercial polydimethylsiloxane (PDMS) capillary membranes. The influences of solvent composition (water/ethanol mixtures), initial concentration of the aroma compound, flow rate and temperature of the feed were studied. RESULTS: High selectivity of the PDMS membrane towards the aroma compound was obtained, leading to enrichment factors in the range 100 < β < 200. Mass transfer resistance was found to be located in the pervaporation membrane. Experimental data showed a linear dependency of the permeation flux of trans‐Hex‐2‐en‐1‐ol on the differences in partial pressures of the compound across the pervaporation membrane. The permeability coefficient of the PDMS membrane to the transport of trans‐Hex‐2‐en‐1‐ol was calculated as P_{m, Hex}(50 °C) = 7.62 × 10^?11 mol m^?1 s^?1 Pa^?1. CONCLUSION: The membrane used was found to be very selective toward trans‐Hex‐2‐en‐1‐ol. A model based on the solution‐diffusion mechanism was applied. The mass transfer parameters needed for the design of a pervaporation process for aroma compound recovery were obtained. Copyright © 2008 Society of Chemical Industry     

Effect of feed composition on PHB production from methanol by HCDC of Methylobacterium extorquens (DSMZ 1340)

BACKGROUND: Poly‐β‐hydroxybutyrate (PHB), produced by several species of bacteria, has attracted great attention as a biodegradable and biocompatible compound with similar properties to polypropylene. Unfortunately, its use is currently limited due to high production costs. One of the most common methods for overcoming this constraint is the use of inexpensive substrates, like methanol, in high cell density cultivations (HCDC). RESULTS: Fermentation was carried out with optimized feed composition (639 g l^?1 methanol, 4 g l^?1 MgSO₄.7H₂O, 41 mL L^?1 trace elements, 5.6 g L^?1 NaH₂PO₄.H₂O and 24.3 g L^?1 K₂HPO₄) and a feeding strategy based on the detection of substrate limitation by dissolved oxygen (DO). After 35 h, at which dry cell weight (DCW) reached a value of 70 g L^?1, PHB production was stimulated, and biomass and PHB productivities of 2.8 and 0.98 g L^?1 h^?1 were obtained, respectively. These results surpassed those reported in the literature for PHB production from methanol by Methylobacterium species. CONCLUSION: The proposed feed composition and feeding strategy for PHB production from methanol by Methylobacterium extorquens 1340 in fed‐batch cultivation resulted in high biomass and PHB productivity. They can be implemented for recombinant bioproducts (proteins) produced by M. extorquens due to the lack of PHB accumulation in the growth phase. Copyright © 2009 Society of Chemical Industry     

Synthesis of cationic nickel(II) compounds for butadiene polymerization

A series of cationic nickel(II) compounds with various counter ions ([BF₄]^–, [CF₃SO₃]^–) and N‐bearing ligands (1,10‐phenanthroline, benzimidazole and terpyridine) were synthesized. The simple compound, Ni(Phen)₂Cl₂, was prepared as a reference compound. All title compounds were characterized by elemental analysis, and infrared spectra analyses and compounds, some specific ones, were further examined by X‐ray crystallographic analysis. Upon treatment with ethylaluminum sesquichloride, these nickel(II) compounds exhibited high activities (up to 88.5%/4 h) for butadiene polymerization and afforded liquid polybutadiene (M_w < 30,000) with a high cis‐content (up to 92.1%). Various polymerization conditions were investigated in detail, and it was found that less steric hindrance of the ligands enhanced the catalytic activities of the nickel(II) compounds. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40511.     

Treatment of paper and pulp wastewater and removal of odorous compounds by a Fenton‐like process at the pilot scale

A Fenton‐like process, involving oxidation and coagulation, was evaluated for the removal of odorous compounds and treatment of a pulp and paper wastewater. The main parameters that govern the complex reactive system [pH and Fe(III) and hydrogen peroxide concentrations] were studied. Concentrations of Fe(III) between 100 and 1000 mg L^?1 and of H₂O₂ between 0 and 2000 mg L^?1 were chosen. The main mechanism for color removal was coagulation. The maximum COD, color and aromatic compound removals were 75, 98 and 95%, respectively, under optimal operating conditions ([Fe(III)] = 400 mg L^?1; [H₂O₂] = 500–1000 mg L^?1; pH = 2.5; followed by coagulation at pH 5.0). The biodegradability of the wastewater treated increased from 0.4 to 0.7 under optimal conditions and no residual hydrogen peroxide was found after treatment. However, partially or non‐oxidized compounds present in the treated wastewater presented higher acute toxicity to Artemia salina than the untreated wastewater. Based on the optimum conditions, pilot‐scale experiments were conducted and revealed a high efficiency in relation to the mineralization of organic compounds. Terpenes [(1S)‐α‐pinene, β‐pinene, (1R)‐α‐pinene and limonene] were identified in the wastewater and were completely eliminated by the Fenton‐like treatment. Copyright © 2006 Society of Chemical Industry     

Effect of Li2O on the defect polarization in CuO‐added (K0.9Na0.1)NbO3 piezoelectric ceramics

CuO‐added (Li_xK_0.9?xNa_0.1)NbO₃ [C(L_xK_0.9?xN_0.1)N] ceramics with 0.0≤x≤0.05 were well‐sintered at 960°C for 6 hours. The lattice parameters of the specimens decreased with the addition of Li₂O. Defect polarization (P_D) formed between Cu²⁺ions and oxygen vacancies. Double polarization vs electric field (P‐E) hysteresis and sprout‐shaped strain vs electric field (S‐E) curves were observed in these specimens with a large strain of 0.16% at 7.0 kV/mm, possibly owing to the presence of P_D. When the P‐E curve was measured at temperatures higher than 75°C, the C(K_0.9N_0.1)N ceramic exhibited a normal P‐E hysteresis curve, whereas the C(L_0.04K_0.86N_0.1)N ceramic maintained the double P‐E hysteresis curve up to 125°C, indicating that Li₂O increased the thermal stability of P_D. The latter specimen also showed the sprout shaped S‐E curve with a strain of 0.15% at 7.0 kV/mm after 10⁴ cycles of a high electric field of 7.0 kV/mm.     

Synthesis and characterization of 3,4‐dihydroxyphenylacetic acid imprinted polymers

Eight molecularly imprinted polymers (MIP1–MIP8) were synthesized with different functional monomers and porogens using 3,4‐dihydroxyphenylacetic acid (DOPAC) as a template. Thermal, radical bulk polymerization was employed in the presence of ethylene glycol dimethacrylate as a cross‐linker. A computational analysis indicated that complexes with four molecules of 4‐vinylpyridine, 1‐vinylimidazole and acrylonitrile had high positive enthalpies of formation. The polymers synthesized with these monomers showed an imprinting factor below 1. Polymer MIP8 synthesized with allylamine as the functional monomer, with the highest energy of interaction with DOPAC, was characterized by the highest imprinting factor equal to 1.91. Examination of the binding ability of DOPAC and a group of structurally related compounds showed that the strong interactions between amine groups in the polymer and carboxylic groups in the analyte governed the recognition mechanism. The Langmuir adsorption model and the pseudo‐second‐order mechanism properly evaluated the MIP8 and non‐imprinted polymer 8 adsorption characteristics. Scatchard analysis revealed that MIP8 had two classes of heterogeneous binding sites with K_d(1) = 0.12 µmol L^?1 and K_d(2) = 1.46 µmol L^?1. Finally, the potential application of MIP8 for separation of DOPAC was demonstrated. Copyright © 2011 Society of Chemical Industry     

A phase diagram of Ba1‐xCaxTiO3 (x = 0‐0.30) piezoceramics by Raman spectroscopy

Piezoelectric ceramics are widely used in sensors, actuators, and ultrasonic transducers due to their ability to achieve efficient conversion between electric and mechanical energy. There is a current drive toward lead‐free material systems capable of achieving comparable piezoelectric performance to environmentally hazardous, but commonly used, lead‐based materials such as lead zirconate titanate. This work constructs an accurate phase diagram for barium calcium titanate (Ba_1‐xCa_xTiO₃ or BCTO). The substitution of Ca²⁺ on the Ba‐site of BCTO is of much interest due to electric property improvements compared to BaTiO₃. In this work, Ba_1‐xCa_xTiO₃ (x = 0‐0.30) samples have been made by solid‐state methods and, following initial characterization by X‐ray diffraction (XRD), evaluation of changes in the Raman spectra has allowed accurate determination of the phase transition temperatures and the construction of a phase diagram.     

Improved cultivation conditions for polysaccharide production by H. influenzae type b

Haemophilus influenzae b (Hib), an encapsulated Gram‐negative cocco‐bacillus, is one of the most common agents of meningitis worldwide. The capsular polysaccharide conjugated to a carrier protein is the antigen of the vaccine against Hib. An optimized cultivation process that could lead to an increase in the polysaccharide production would be of great interest for mass vaccination programs. The aim of this work was to evaluate different culture conditions in attempt to improve the capsular polysaccharide yield. Hib was cultivated in a bioreactor with modified soy‐peptone and yeast‐extract (MP) medium and optimal hemin and nicotinamide adenine dinucleotide (NAD) concentration in the culture medium was established at 30 mg L^?1 and 15 mg L^?1, respectively. The batch experiments were carried out as follows: (a) overlay aeration without pH control; (b) air‐sparged with dissolved oxygen tension (DOT) controlled at 10 and 30% air saturation, with and without pH control. The cultures with air‐sparged aeration, without pH control, showed values for the specific production (SP_p/x) of 180–190 mg PRP g^?1 dry cell weight (DCW) and overall polysaccharide productivity of 22–29 mg L^?1 h^?1, accounting for an increase of ca 47% over the polysaccharide production with overlay aeration. Batch cultivations with air sparged aeration led to an improvement in the poly(ribosylribitol phosphate) (PRP) production for both conditions (DOT at 10 and 30% air saturation) investigated upon pH control, achieving up to 980 PRP mg L^?1. The SP_p/x and overall polysaccharide productivity were 280–300 mg PRP g^?1 DCW and 45–41 mg L^?1h^?1, respectively. The best production of capsular polysaccharide was obtained in the modified MP‐medium, with 30 mg L^?1 hemin and 15 mg L^?1 NAD, upon sparged aeration and pH control. Copyright © 2005 Society of Chemical Industry