In Vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis*.

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Construction of Charring-Functional Polyheptanazine towards Improvements in Flame Retardants of Polyurethane.

Nitrogen-containing flame retardants have been extensively applied due to their low toxicity and smoke-suppression properties; however, their poor charring ability restricts their applications. Herein, a representative nitrogen-containing flame retardant, polyheptanazine, was investigated. Two novel, cost-effective phosphorus-doped polyheptazine (PCN) and cobalt-anchored PCN (Co@PCN) flame retardants were synthesized via a thermal condensation method. The X-ray photoelectron spectroscopy (XPS) results indicated effective doping of P into triazine. Then, flame-retardant particles were introduced into thermoplastic polyurethane (TPU) using a melt-blending approach. The introduction of 3 wt% PCN and Co@PCN could remarkably suppress peak heat release rate (pHRR) (48.5% and 40.0%), peak smoke production rate (pSPR) (25.5% and 21.8%), and increasing residues (10.18 wt%→17.04 wt% and 14.08 wt%). Improvements in charring stability and flame retardancy were ascribed to the formation of P-N bonds and P=N bonds in triazine rings, which promoted the retention of P in the condensed phase, which produced additional high-quality residues.

Bioapplications of small molecule Aza-BODIPY: from rational structural design to in vivo investigations.

Boron-dipyrromethene (BODIPY) belongs to a family of organoboron compounds, commercialized as fluorescent dyes by Invitrogen™. As BODIPY derivatives, Aza-boron-dipyrromethene (Aza-BODIPY) dyes display superior spectral performances, such as red-shifted spectra and high molar extinction coefficients, and are considered to be extremely attractive organic materials for various bioapplications. Therefore, scientists from different disciplinary backgrounds would benefit from a review that provides a timely summary and outlook regarding Aza-BODIPY dyes. In this review, we report on the latest advances of Aza-BODIPY dyes, along with the empirical design guidelines and photophysical property manipulation of these dyes. In addition, we will discuss the biological applications of Aza-BODIPY dyes in probing various biological activities, as well as in fluorescence bioimaging/detection, newly-emerging photoacoustic bioimaging/detection, and phototherapy together with future challenges and implications in this field. We aim at providing an insightful design guideline and a clear overview of Aza-BODIPY dyes, which might entice new ideas and directions.

The azatryptophan-based fluorescent platform for in vitro rapid screening of inhibitors disrupting IKKß-NEMO interaction.

The nuclear factor-κB (NF-κB) plays an important role in inflammatory and immune responses. Aberrant NF-κB signaling is implicated in multiple disorders, including cancer. Targeting the regulatory scaffold subunit IκB kinase γ (IKKγ/NEMO) as therapeutic interventions could be promising due to its specific involvement in canonical NF-κB activation without interfering with non-canonical signaling. In this study, the use of unnatural amino acid substituted IKKß with unique photophysical activity to sense water environment changes upon interaction with NEMO provides a powerful in vitro screening platform that would greatly facilitate the identification of compounds having the potential to disrupt IKKß-NEMO interaction, and thus specifically modulate the canonical NF-κB pathway. We then utilized a competitive binding platform to screen the binding ability of a number of potential molecules being synthesized. Our results suggest that a lead compound (-)-PDC-099 is a potent agent with ascertained potency to disrupt IKKß-NEMO complex for modulating NF-κB canonical pathway.

A Self-Assembled Biocompatible Nanoplatform for Multimodal MR/Fluorescence Imaging Assisted Photothermal Therapy and Prognosis Analysis.

The need for better imaging assisted cancer therapy calls for new biocompatible agents with excellent imaging and therapeutic capabilities. This study successfully fabricates albumin-cooperated human serum albumin (HSA)-GGD-ICG nanoparticles (NPs), which are comprised of a magnetic resonance (MR) contrast agent, glycyrrhetinic-acid-modified gadolinium (III)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (GGD), and a fluorescence (FL) dye, indocyanine green (ICG), for multimodal MR/FL imaging assisted cancer therapy. These HSA-GGD-ICG NPs with excellent biocompatibility are stable under physiological conditions, and exhibit enhanced T1 contrast capability and improved fluorescence imaging capacity. In vitro experiments reveal an apparent effect of the NPs in killing tumor cells under low laser irradiation, due to the enhanced photothermal conversion efficiency (≈85.1%). Importantly, multimodal MR/FL imaging clearly shows the in vivo behaviors and the efficiency of tumor accumulation of HSA-GGD-ICG NPs, as confirmed by a pharmacokinetic study. With the guidance of multimodal imaging, photothermal therapy is subsequently conducted, which demonstrates again high photothermal conversion capability for eliminating tumors without relapse. Notably, real-time monitoring of tumor ablation for prognosis and therapy evaluation is also achieved by MR imaging. This strategy of constructing nanoplatforms through albumin-mediated methods is both convenient and efficient, which would enlighten the design of multimodal imaging assisted cancer therapy for potential clinical translation.

Discovery of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a potent nucleoside inhibitor of respiratory syncytial virus with excellent selectivity over mitochondrial RNA and DNA polymerases.

Novel 4'-substituted ß-d-2'-deoxy-2'-α-fluoro (2'd2'F) nucleoside inhibitors of respiratory syncytial virus (RSV) are reported. The introduction of 4'-substitution onto 2'd2'F nucleoside analogs resulted in compounds demonstrating potent cell based RSV inhibition, improved inhibition of the RSV polymerase by the nucleoside triphosphate metabolites, and enhanced selectivity over incorporation by mitochondrial RNA and DNA polymerases. Selectivity over the mitochondrial polymerases was found to be extremely sensitive to the specific 4'-substitution and not readily predictable. Combining the most potent and selective 4'-groups from N-nucleoside analogs onto a 2'd2'F C-nucleoside analog resulted in the identification of ß-D-2'-deoxy-2'-α-fluoro-4'-α-cyano-5-aza-7,9-dideaza adenosine as a promising nucleoside lead for RSV.

Discovery of 7-azaindole derivatives as potent Orai inhibitors showing efficacy in a preclinical model of asthma.

Synthesis and SAR of a series of 7-azaindoles as Orai channel inhibitors showing good potency inhibiting IL-2 production in Jurkat cells is described. Compound 14d displaying best pharmacokinetic properties was further characterized in a model of allergen induced asthma showing inhibition in the number of eosinophils in BALF. High lipophilicity remains as one of the main challenges for this class of compounds.

Ranking the Binding Energies of p53 Mutant Activators and Their ADMET Properties.

The guardian of the genome, p53, is the most mutated protein found in all cancer cells. Restoration of wild-type activity to mutant p53 offers promise to eradicate cancer cells using novel pharmacological agents. Several molecules have already been found to activate mutant p53. While the exact mechanism of action of these compounds has not been fully understood, a transiently open pocket has been identified in some mutants. In our study, we docked twelve known activators to p53 into the open pocket to further understand their mechanism of action and rank the best binders. In addition, we predicted the absorption, distribution, metabolism, excretion and toxicity properties of these compounds to assess their pharmaceutical usefulness. Our studies showed that alkylating ligands do not all bind at the same position, probably due to their varying sizes. In addition, we found that non-alkylating ligands are capable of binding at the same pocket and directly interacting with Cys124. The comparison of the different ligands demonstrates that stictic acid has a great potential as a p53 activator in terms of less adverse effects although it has poorer pharmacokinetic properties.

P-P σ-bond activation by gold(I) coordination.

Bond activation by gold is of imminent interest with respect to catalytic activity. By coordination of an AuCl fragment onto the P-P σ-bond of an azadiphosphiridine, the P-P bond is cleaved to give a PNP type of ligand in the gold(I) complex. Another equivalent of the gold precursor selectively binds on a P lone pair. The bonding situation was further studied by means of computation, indicating the existence of a 3-center-2-electron bond.

Silver-catalyzed carbon-phosphorus functionalization of N-(p-methoxyaryl)propiolamides coupled with dearomatization: access to phosphorylated aza-decenones.

Silver-catalyzed cascade difunctionalization of N-(p-methoxyaryl)propiolamides coupled with dearomatization was achieved and used to regiospecifically construct a variety of phosphorylated aza-decenones bearing adjacent quaternary stereocenters under mild conditions in moderate to excellent yields.

[15]aneN4S: synthesis, thermodynamic studies and potential applications in chelation therapy.

The purpose of this work was to synthesize and characterize the thiatetraaza macrocycle 1-thia-4,7,10,13-tetraazacyclopentadecane ([15]aneN4S). Its acid-base behaviour was studied by potentiometry at 25 °C and ionic strength 0.10 M in KNO3. The protonation sequence of this ligand was investigated by 1H-NMR titration that also allowed the determination of protonation constants in D2O. Binding studies of [15]aneN4S with Mn2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ and Pb2+ metal ions were further performed under the same experimental conditions. The results demonstrated that this compound has a higher selectivity and thermodynamic stability for Hg2+ and Cu2+, followed by Ni2+. The UV-visible-near IR spectroscopies and magnetic moment data for the Co(II) and Ni(II) complexes indicated a tetragonal distorted coordination geometry for both metal centres. The value of magnetic moment and the X-band EPR spectra of the Cu(II) complex are consistent with a distorted square pyramidal geometry.

Approaches to a multitargeting drug development: first profiled 3- ethoxycarbonyl-1-aza-9-oxafluorenes representing a perspective compound class targeting Alzheimer disease relevant kinases CDK1, CDK5 and GSK-3ß.

Since Alzheimer disease (AD) is a multifactorial disease, recent therapeutical approaches concentrate on the development of a multitargeting drug. Various protein kinases are known to be involved in the progression of AD. A first series of 3-ethoxycarbonyl-1-aza-9-oxafluorenes has been synthesized and biologically evaluated as AD-relevant protein kinase inhibitors. A concentration-dependent inhibition of important AD-relevant kinases has been characterized after the selectivity of kinase inhibition had been demonstrated. Structure-activity relationships of protein kinase inhibition are discussed and first multitargeting inhibitors have been identified.

An expedient synthesis and screening for antiacetylcholinesterase activity of piperidine embedded novel pentacyclic cage compounds.

The aim of this study was to synthesize and evaluate diazapentacyclic analogs for their acetylcholinesterase (AChE) inhibitory activity. The pentacyclic analogs were synthesized by one-pot three-component domino reactions in a microwave synthesizer. Most of the compounds exhibited moderate to good AChE inhibitory activity, compound 5i showed potent inhibitory activity with IC50 1.12 ± 0.01 µM and this may provide a new lead for developing potential inhibitors for Alzheimer's disease.

Laser receptive polyelectrolyte thin films doped with biosynthesized silver nanoparticles for antibacterial coatings and drug delivery applications.

We report a simple method to fabricate multifunctional polyelectrolyte thin films to load and deliver the therapeutic drugs. The multilayer thin films were assembled by the electrostatic adsorption of poly (allylamine hydrochloride) (PAH) and dextran sulfate (DS). The silver nanoparticles (Ag NPs) biosynthesized from novel Hybanthus enneaspermus leaf extract as the reducing agent were successfully incorporated into the film. The biosynthesized Ag NPs showed excellent antimicrobial activity against the range of enteropathogens, which could be significantly enhanced when used with commercial antibiotics. The assembled silver nano composite multilayer films showed rupture and deformation when they are exposed to laser. The Ag NPs act as an energy absorption center, locally heat up the film and rupture it under laser treatment. The antibacterial drug, moxifloxacin hydrochloride (MH) was successfully loaded into the multilayer films. The total amount of MH release observed was about 63% which increased to 85% when subjected to laser light exposure. Thus, the polyelectrolyte thin film reported in our study has significant potential in the field of remote activated drug delivery, antibacterial coatings and wound dressings.

Scorpiand-like azamacrocycles prevent the chronic establishment of Trypanosoma cruzi in a murine model.

Chagas disease is today one of the most important neglected diseases for its upcoming expansion to non-endemic areas and has become a threat to blood recipients in many countries. In this study, the trypanocidal activity of ten derivatives of a family of aza-scorpiand like macrocycles is evaluated against Trypanosoma cruzi in vitro and in vivo murine model in which the acute and chronic phases of Chagas disease were analyzed. The compounds 4, 3 and 1 were found to be more active against the parasite and less toxic against Vero cells than the reference drug benznidazole, 4 being the most active compound, particularly in the chronic phase. While all these compounds showed a remarkable degree of inhibition of the Fe-SOD enzyme of the epimastigote forms of T. cruzi, they produced a negligible inhibition of human CuZn-SOD and Mn-SOD from Escherichia coli. The modifications observed by (1)H NMR and the amounts of excreted catabolites by the parasites after treatment suggested that the mechanism of action could be based on interactions of the side chains of the compounds with enzymes of the parasite metabolism. The ultrastructural alterations observed in treated epimastigote forms confirmed that the compounds having the highest activity are those causing the largest cell damage. A complementary histopathological analysis confirmed that the compounds tested were significantly less toxic to mammals than the reference drug.

π-Excess σ2P ligands: synthesis of biaryl-type 1,3-benzazaphosphole hybrid ligands and formation of P

Acid-catalyzed cyclocondensations of 2-phosphanylanilines 1 with substituted benzaldehydes or heteroaryl aldehydes open a convenient route to new biaryl-type 1H-1,3-benzazaphosphole hybrid ligands 2a-f with o-phosphanylphenyl, pyridyl, imidazolyl, thienyl or o-methoxyphenyl donor groups (in addition to the σ(2)P donor) and to bromophenyl substituted benzazaphospholes 2g,h. Excess aldehyde leads to concomitant reductive N-alkylation, as shown by formation of 3h besides 2h. The reactions proceed via dihydrobenzazaphospholes 4, which can be detected under mild conditions. The aromaticity-driven dehydrogenation does not liberate dihydrogen but is accomplished by transfer hydrogenations, mainly by reduction of some of the aldehyde. N-Secondary 2-phosphanylanilines 5 also react with aldehydes to form the corresponding N-substituted benzazaphospholes 6. The formation of (P^P')M(CO)4-chelate complexes 8a (M = Cr) and 9a,b (M = Mo) was demonstrated by reaction with M(CO)4(norbornadiene). The crystal structure of 9a, determined in addition to the solution structure elucidation by multinuclear NMR spectra, confirms the chelate formation and reveals a trigonal environment for the low coordinated phosphorus, with the P-Mo(0) vector bent out of the benzazaphosphole ring plane by 14.4° (0.57 Å), together with axial chirality of the molecules in the racemic crystals by twisting of the benzazaphosphole and phenyl π-planes around the common C(2)-C(21) bond.

Discovery, synthesis, selectivity modulation and DMPK characterization of 5-azaspiro[2.4]heptanes as potent orexin receptor antagonists.

Starting from a orexin 1 receptor selective antagonist 4,4-disubstituted piperidine series a novel potent 5-azaspiro[2.4]heptane dual orexin 1 and orexin 2 receptor antagonist class has been discovered. SAR and Pharmacokinetic optimization of this series is herein disclosed. Lead compound 15 exhibits potent activity against orexin 1 and orexin 2 receptors along with low cytochrome P450 inhibition potential, good brain penetration and oral bioavailability in rats.

Synthesis and biological evaluation of novel 2,4-disubstituted quinazoline analogues as GPR119 agonists.

GPR119 agonist has emerged as a promising target for the treatment of type 2 diabetes. A series of novel 2,4-disubstituted quinazoline analogues was prepared and evaluated their agonistic activity against human GPR119. The analogues bearing azabicyclic amine substituents (12a, 12c and 12g) exhibited better EC(50) values than that of OEA though they appeared to be partial agonists.

Inhibitors of HIV-1 attachment. Part 11: the discovery and structure-activity relationships associated with 4,6-diazaindole cores.

A series of HIV-1 attachment inhibitors containing a 4,6-diazaindole core were examined in an effort to identify a compound which improved upon the potency and oral exposure of BMS-488043 (2). BMS-488043 (2) is a 6-azaindole-based HIV-1 attachment inhibitor which established proof-of-concept for this mechanism in human clinical studies but required high doses and concomitant administration of a high fat meal to achieve efficacious exposures. Based on previous studies in indole and azaindole scaffolds, SAR investigation was concentrated around the key 7-position in the 4,6-diazaindole series and led to the discovery of molecules with 5- to 20-fold increases in potency and three- to seven-fold increases in exposure over 2 in a rat PK studies.

Determination of moxifloxacin and its oxidation products with kinetic evaluation under potassium permanganate treatment in acidic solution by ultra-performance liquid chromatography/tandem mass spectrometry.

A simple, sensitive, and reproducible ultra-performance LC method for the determination of moxifloxacin (MOXI) oxidation stability under permanganate treatment in acidic conditions (pH 3.0-6.0) was developed. Besides the MOXI peak [retention time (RT) = 2.58], four additional products (RT = 0.86, 0.91, 1.42, and 1.89) were observed in all conditions tested. The oxidation process followed second-order reaction kinetics and depended upon solution acidity. The highest reaction rate constant was observed at pH 3.0, and this value decreased as the pH was increased to 6.0. The oxidation products were characterized, and their fragmentation pathways, derived from MS/MS data, were proposed. Two of these products were identified as hydroxyl derivatives of MOXI and two others as their oxidation product analogs with molecular ions of 418.4 and 416.4 m/z, respectively.