Structure based design of novel 6,5 heterobicyclic mitogen-activated protein kinase kinase (MEK) inhibitors leading to the discovery of imidazo[1,5-a] pyrazine G-479

Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi’s [J. Med. Chem. 2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem. 2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem. 2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450’s 2C9 and 2C19. Lowering the log D by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.     

Structures similar to lipid emulsions and liposomes. Dipalmitoylphosphatidylcholine,cholesterol, Tween 20–Span 20 or Tween 80–Span 80 in aqueous media

In the present work, we show that we obtained nanometric structures made of water, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), cholesterol (Chol), and a mixture of ethoxylated and non-ethoxylated sorbitan fatty acid esters (Tween 20, Span 20, Tween 80, and Span 80) by mixing all of them near the cloud point temperature (cp) of the ethoxylated surfactant. The influence that the constituents had on the size of the particle was determined by a pseudo-ternary phase diagram of water/Tween–Span/DPPC–Chol; the colloidal particles obtained were studied by differential scanning calorimetry, confocal fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. These studies were made for all the systems with at least 23 d of colloidal stability. The most stable system was obtained with the Tween 80–Span 80 pair, behaving as a typical suspension for 48 d; this system was made of water, Tween 80–Span 80 (80:20), DPPC–Chol (95:5) in a corresponding molar ratio of 48:37:100:10. The colloidal particles obtained were a kind of emulsion and liposome structures. The second stable system was obtained with the same mixture, but in a molar ratio of 8:6:9:0, its structure was also a kind of emulsion particles. In both systems and in other less stable ones, the “emulsion particle” was completely new, it structurally corresponds to a nucleus of mixed micelles surrounded by at least one bilayer of DPPC.     

Elucidation of structural and functional properties of albumin bound to gold nanoparticles

Nanoparticle–albumin complexes are being designed for targeted drug delivery and imaging. However, the changes in the functional properties of albumin due to adsorption on nanoparticles remain elusive. Thus, the objective of this work was to elucidate the structural and functional properties of human and bovine serum albumin bound to negatively charged gold nanoparticles (GNPs). Fluorescence data demonstrated static quenching of albumin by GNP with the quenching of buried as well as surface tryptophan in BSA. The binding process was enthalpy and entropy-driven in HSA and BSA, respectively. At lower concentrations of GNP there was a higher affinity for tryptophan, whereas at higher concentrations both tryptophan and tyrosine participated in the interaction. Synchronous fluorescence spectra revealed that the microenvironment of tryptophan in HSA turned more hydrophilic upon exposure to GNP. The α-helical content of albumin was unaltered by GNP. Approximately 37 and 23% reduction in specific activity of HSA and BSA was observed due to GNP binding. In presence of warfarin and ibuprofen the binding constants of albumin–GNP complexes were altered. A very interesting observation not reported so far is the retained antioxidant activity of albumin in presence of GNP i.e. we believe that GNPs did not bind to the free sulfhydryl groups of albumin. However enhanced levels of copper binding were observed. We have also highlighted the differential response in albumin due to gold and silver nanoparticles which could be attributed to differences in the charge of the nanoparticle.     

Phenotypic assays for analyses of pluripotent stem cell–derived cardiomyocytes

Stem cell–derived cardiomyocytes (CMs) hold great hopes for myocardium regeneration because of their ability to produce functional cardiac cells in large quantities. They also hold promise in dissecting the molecular principles involved in heart diseases and also in drug development, owing to their ability to model the diseases using patient‐specific human pluripotent stem cell (hPSC)–derived CMs. The CM properties essential for the desired applications are frequently evaluated through morphologic and genotypic screenings. Even though these characterizations are necessary, they cannot in principle guarantee the CM functionality and their drug response. The CM functional characteristics can be quantified by phenotype assays, including electrophysiological, optical, and/or mechanical approaches implemented in the past decades, especially when used to investigate responses of the CMs to known stimuli (eg, adrenergic stimulation). Such methods can be used to indirectly determine the electrochemomechanics of the cardiac excitation‐contraction coupling, which determines important functional properties of the hPSC‐derived CMs, such as their differentiation efficacy, their maturation level, and their functionality. In this work, we aim to systematically review the techniques and methodologies implemented in the phenotype characterization of hPSC‐derived CMs. Further, we introduce a novel approach combining atomic force microscopy, fluorescent microscopy, and external electrophysiology through microelectrode arrays. We demonstrate that this novel method can be used to gain unique information on the complex excitation‐contraction coupling dynamics of the hPSC‐derived CMs.     

Preparation and Evaluation of Differently Sulfonated Styrene–Divinylbenzene Cross-linked Copolymer Cationic Exchange Resins as Novel Carriers for Drug Delivery

The differently sulfonated styrene–divinylbenzene cross-linked copolymer cationic exchange resins were prepared by oil-in-water polymerization and varied degrees of sulfonation. Several characteristics of the obtained resins were evaluated, i.e., Fourier transform infrared spectra, the ion-exchange capacity, microscopic morphology, size, and swelling. The resin characteristics were altered in relation to the degree of sulfonation, proving that differently sulfonated resins could be prepared. The behavior of chlorpheniramine (CPM) loading and in vitro release in the USP simulated gastric (SGF) and intestinal fluids (SIF) of the obtained resins were also evaluated. The CPM loaded in the resinates (drug-loaded resins) increased with the increasing degree of sulfonic group and hence the drug binding site in the employed resins. The CPM release was lower from the resins with the higher degree of sulfonic group due to the increase in the diffusive path depth. The CPM release was obviously lower in SGF than SIF because CPM, a weak base drug, ionized to a greater extent in SGF and then preferred binding with rather than releasing from the resins. In conclusion, the differently sulfonated resins could be utilized as novel carriers for drug delivery.     

Biological behavior of the curcumin incorporated chitosan/poly(vinyl alcohol) nanofibers for biomedical applications

Electrospun composite scaffolds show high ability to be used in regenerative medicine and drug delivery, due to the nanofibrous structure and high surface area to volume ratio. In this study, we used nanofibrous scaffolds fabricated by chitosan (CS), poly(vinyl alcohol) (PVA), carbopol, and polycaprolactone using a dual electrospinning technique while curcumin (Cur) incorporated inside of the CS/PVA fibers. Scaffolds were fully characterized via scanning electron microscopy, water contact angle, tensile measurement, hydration, protein adsorption, and wrinkled tests. Furthermore, viability of the buccal fat pad-derived mesenchymal stem cells (BFP-MSCs) was also investigated using MTT assay for up to 14 days while cultured on these scaffolds. Cell cycle assay was also performed to more detailed evaluation of the stem cells growth when grown on scaffolds (with and without Cur) compared with the culture plate. Results demonstrated that Cur loaded nanofibrous scaffold had more suitable capability for water absorption and mechanical properties compared with the scaffold without Cur and it could also support the stem cells viability and proliferation. Cur release profile showed a decreasing effect on BFP-MSCs viability in the initial stage, but it showed a positive effect on stem cell viability in a long-term manner. In general, the results indicated that this nanofibrous scaffold has great potential as a delivery of the Cur and BFP-MSCs simultaneously, and so holds the promising potential for use in various regenerative medicine applications.     

Hybrid nanoreservoirs based on dextran-capped dendritic mesoporous silica nanoparticles for CD133-targeted drug delivery

In this study, the chemical features of dendritic mesoporous silica nanoparticles (DMSNs) provided the opportunity to design a nanostructure with the capability to intelligently transport the payload to the tumor cells. In this regard, doxorubicin (DOX)-encapsulated DMSNs was electrostatically surface-coated with polycarboxylic acid dextran (PCAD) to provide biocompatible dextran-capped DMSNs (PCAD-DMSN@DOX) with controlled pH-dependent drug release. Moreover, a RNA aptamer against a cancer stem cell (CSC) marker, CD133 was covalently attached to the carboxyl groups of DEX to produce a CD133-PCAD-DMSN@DOX. Then, the fabricated nanosystem was utilized to efficiently deliver DOX to CD133+ colorectal cancer cells (HT29). The in vitro evaluation in terms of cellular uptake and cytotoxicity demonstrated that the CD133-PCAD-DMSN@DOX specifically targets HT29 as a CD133 overexpressed cancer cells confirmed by flow cytometry and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide assay. The potentially promising intelligent-targeted platform suggests that targeted dextran-capped DMSNs may find impressive application in cancer therapy.     

Current and emerging commercial optical biosensors.

The field of commercial optical biosensors is rapidly evolving, with new systems and detection methods being developed each year. This review outlines the currently available biosensor hardware and highlights unique features of each platform. Affinity-based biosensor technology, with its high sensitivity, wide versatility and high throughput, is playing a significant role in basic research, pharmaceutical development, and the food and environmental sciences. Likewise, the increasing popularity of biosensors is prompting manufacturers to develop new instrumentation for dedicated applications. We provide a preview of some of the emerging commercial systems that are dedicated to drug discovery, proteomics, clinical diagnostics and routine biomolecular interaction analysis.     

Effects of CETP inhibition with anacetrapib on metabolism of VLDL-TG and plasma apolipoproteins C-II,C-III,and E

Cholesteryl ester transfer protein (CETP) mediates the transfer of HDL cholesteryl esters for triglyceride (TG) in VLDL/LDL. CETP inhibition, with anacetrapib, increases HDL-cholesterol, reduces LDL-cholesterol, and lowers TG levels. This study describes the mechanisms responsible for TG lowering by examining the kinetics of VLDL-TG, apoC-II, apoC-III, and apoE. Mildly hypercholesterolemic subjects were randomized to either placebo (N = 10) or atorvastatin 20 mg/qd (N = 29) for 4 weeks (period 1) followed by 8 weeks of anacetrapib, 100 mg/qd (period 2). Following each period, subjects underwent stable isotope metabolic studies to determine the fractional catabolic rates (FCRs) and production rates (PRs) of VLDL-TG and plasma apoC-II, apoC-III, and apoE. Anacetrapib reduced the VLDL-TG pool on a statin background due to an increased VLDL-TG FCR (29%; P = 0.002). Despite an increased VLDL-TG FCR following anacetrapib monotherapy (41%; P = 0.11), the VLDL-TG pool was unchanged due to an increase in the VLDL-TG PR (39%; P = 0.014). apoC-II, apoC-III, and apoE pool sizes increased following anacetrapib; however, the mechanisms responsible for these changes differed by treatment group. Anacetrapib increased the VLDL-TG FCR by enhancing the lipolytic potential of VLDL, which lowered the VLDL-TG pool on atorvastatin background. There was no change in the VLDL-TG pool in subjects treated with anacetrapib monotherapy due to an accompanying increase in the VLDL-TG PR.     

Inhibitors for the bacterial ectonucleotidase Lp1NTPDase from Legionella pneumophila

Legionella pneumophila is an aerobic, Gram-negative bacterium of the genus Legionella, which constitutes the major causative agent of Legionnaires’ disease. Recently a nucleoside triphosphate diphosphohydrolase (NTPDase) from L. pneumophila was identified and termed Lp1NTPDase; it was found to be a structural and functional homolog of mammalian NTPDases catalyzing the hydrolysis of ATP to ADP and ADP to AMP. Its activity is believed to contribute to the virulence of Legionella pneumophila. Therefore Lp1NTPDase inhibitors are considered as novel antibacterial drugs. However, only weakly potent compounds are available so far. In the present study, a capillary electrophoresis (CE)-based enzyme assay for monitoring the Lp1NTPDase activity was established. The enzymatic reaction was performed in a test tube followed by separation of substrate and products by CE and subsequent quantification by UV analysis. After kinetic characterization of the enzyme, a series of 1-amino-4-ar(alk)ylamino-2-sulfoanthraquinone derivatives structurally related to the anthraquinone dye Reactive Blue 2, a non-selective ecto-NTPDase inhibitor, was investigated for inhibitory activity on Lp1NTPDase using the CE-based enzyme assay. Derivatives bearing a large lipophilic substituent (e.g., fused aromatic rings) in the 4-position of the 1-amino-2-sulfoanthraquinone showed the highest inhibitory activity. Compounds with IC₅₀ values in the low micromolar range were identified. The most potent inhibitor was 1-amino-4-[phenanthrene-9-yl-amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (28, PSB-16131), with an IC₅₀-value of 4.24 μM. It represents the most potent Lp1NTPDase inhibitor described to date. These findings may serve as a starting point for further optimization. Lp1NTPDase inhibition provides a novel approach for the (immuno)therapy of Legionella infections.