High-Throughput Optimization of Photochemical Reactions using Segmented-Flow Nanoelectrospray-Ionization Mass Spectrometry.

Within the realm of drug discovery, high-throughput experimentation techniques enable the rapid optimization of reactions and expedited generation of drug compound libraries for biological and pharmacokinetic evaluation. Herein we report the development of a segmented flow mass spectrometry-based platform to enable the rapid exploration of photoredox reactions for early-stage drug discovery. Specifically, microwell plate-based photochemical reaction screens were reformatted to segmented flow format to enable delivery to nanoelectrospray ionization-mass spectrometry analysis. This approach was demonstrated for the late-stage modification of complex drug scaffolds, as well as the subsequent structure-activity relationship evaluation of synthesized analogs. This technology is anticipated to expand the robust capabilities of photoredox catalysis in drug discovery by enabling high-throughput library diversification.

Phenotypic screen identifies calcineurin-sparing FK506 analogs as BMP potentiators for treatment of acute kidney injury.

Acute kidney injury (AKI) is a life-threatening disease with no known curative or preventive therapies. Data from multiple animal models and human studies have linked dysregulation of bone morphogenetic protein (BMP) signaling to AKI. Small molecules that potentiate endogenous BMP signaling should have a beneficial effect in AKI. We performed a high-throughput phenotypic screen and identified a series of FK506 analogs that act as potent BMP potentiators by sequestering FKBP12 from BMP type I receptors. We further showed that calcineurin inhibition was not required for this activity. We identified a calcineurin-sparing FK506 analog oxtFK through late-stage functionalization and structure-guided design. OxtFK demonstrated an improved safety profile in vivo relative to FK506. OxtFK stimulated BMP signaling in vitro and in vivo and protected the kidneys in an AKI mouse model, making it a promising candidate for future development as a first-in-class therapeutic for diseases with dysregulated BMP signaling.

Design of a Compliant Mechanism Based Four-Stage Amplification Piezoelectric-Driven Asymmetric Microgripper.

The existing symmetrical microgrippers have larger output displacements compared with the asymmetrical counterparts. However, the two jaws of a symmetrical microgripper are less unlikely to offer the same forces on the two sides of a grasped micro-object due to the manufacture and assembly errors. Therefore, this paper proposes a new asymmetric microgripper to obtain stable output force of the gripper. Compared with symmetrical microgrippers, asymmetrical microgrippers usually have smaller output displacements. In order to increase the output displacement, a compliant mechanism with four stage amplification is employed to design the asymmetric microgripper. Consequently, the proposed asymmetrical microgripper possesses the advantages of both the stable output force of the gripper and large displacement amplification. To begin with, the mechanical model of the microgripper is established in this paper. The relationship between the driving force and the output displacement of the microgripper is then derived, followed by the static characteristics' analysis of the microgripper. Furthermore, finite element analysis (FEA) of the microgripper is also performed, and the mechanical structure of the microgripper is optimized based on the FEA simulations. Lastly, experimental tests are carried out, with a 5.28% difference from the FEA results and an 8.8% difference from the theoretical results. The results from theoretical calculation, FEA simulations, and experimental tests verify that the displacement amplification ratio and the maximum gripping displacement of the microgripper are up to 31.6 and 632 µm, respectively.

High prevalence of CTX-M belonging to ST410 and ST889 among ESBL producing E. coli isolates from waterfowl birds in China's tropical island, Hainan.

The epidemiology and genetic characteristics of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) have been widely studied in human and veterinary settings. ESBL-producing E. coli are generally reported in pigs, poultry, and dairy farm animals. Here, we report on the prevalence and genetic characteristics of beta-lactamase producing E. coli isolated from waterfowl birds in Hainan, China. After phenotypic confirmation, genes encoding blaCTX-M, blaSHV and blaTEM were detected by polymerase chain reaction (PCR). The isolates were assigned to different phylogenetic groups, and multi-locus sequence typing (MLST). Taken as a whole, 289 (92.9%) out of 311 E. coli isolates from waterfowl birds were confirmed as ESBL phenotypes by double-disk synergy testing. Subsequent PCR analysis revealed that blaCTX-M was the predominant ESBL gene identified in 146 (46.9%) isolates, followed by the combination of blaCTX-M and blaTEM in 70 (22.5%) isolates. The majority of these positive isolates were assigned to phylogroup B2 (46.2%) followed by phylogroup A (43.6%). In addition, MLST assigned representative ESBL positive isolates (n = 40) to 18 STs, and ST410 (ST23cplx) was the most prevalent population (22.5%). The high prevalence of CTX-M and STs frequently associating with E. coli infections should be of concern as it poses threats to animal and public health. To the best of our knowledge, this is the first comprehensive study to report on the occurrence of ESBL producing E. coli from waterfowl birds in China.

Optimal flip angle for robust practical quantitative NMR measurement using a fixed pulse length.

NMR quantification has been traditionally performed by using internal standards. Although methods using external reference in NMR quantification have been developed, the major obstacles in using external referencing method are the measurement deviations associated with changing sample conditions and the requirement of pulse width calibration for every sample in order to compensate these errors. The calibration process is time consuming and in some cases impossible. We developed a quantitative NMR method fixed pulse length (FIXPUL) for all measurements without sample-by-sample calibration. The method is based on the use of an optimal flip angle calibrated for an external standard so that the quantitative errors associated with the pulse width variations are minimized. FIXPUL can be implemented on most basic NMR spectrometers and is robust and easily automated. The method is applicable to a wide range of solution NMR samples in chemistry, biology, and drug research and discovery.

In-tube derivatization for determination of absolute configuration and enantiomeric purity of chiral compounds by NMR spectroscopy.

We have developed an in-tube derivatization method using commercially available polymer-supported coupling agents to prepare derivatives of chiral compounds directly in NMR tube with high yield and purity. Because the method does not require any workup or purification, the configuration and enatiopurity can be quickly determined by NMR analysis for a small amount of chiral compounds, which is critical for today's fast-paced medicinal chemistry efforts in drug discovery. The application of the method was demonstrated for the derivatization of chiral amines, alcohols, diols, amino alcohols, thiols, and carboxylic acids using various chiral derivatizing agents and coupling agents. This article also serves as a practical guide for in-tube derivatization and selection of suitable chiral derivatizing agents and coupling agents for various types of chiral compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Kriging modeling and SPSA adjusting PID with KPWF compensator control of IPMC gripper for mm-sized objects.

Ionic polymer metal composite (IPMC) as a new smart material has been widely concerned in the micromanipulation field. In this paper, a novel two-finger gripper which contains an IPMC actuator and an ultrasensitive force sensor is proposed and fabricated. The IPMC as one finger of the gripper for mm-sized objects can achieve gripping and releasing motion, and the other finger works not only as a support finger but also as a force sensor. Because of the feedback signal of the force sensor, this integrated actuating and sensing gripper can complete gripping miniature objects in millimeter scale. The Kriging model is used to describe nonlinear characteristics of the IPMC for the first time, and then the control scheme called simultaneous perturbation stochastic approximation adjusting a proportion integration differentiation parameter controller with a Kriging predictor wavelet filter compensator is applied to track the gripping force of the gripper. The high precision force tracking in the foam ball manipulation process is obtained on a semi-physical experimental platform, which demonstrates that this gripper for mm-sized objects can work well in manipulation applications.

Evaluation of solvent effects on protonation using NMR spectroscopy: implication in salt formation.

Investigation of the use of solution NMR spectroscopy to determine the effect of organic solvents on chemical shift changes in bases on addition of acids is reported. This information can be useful in the evaluation of solvents and counterion selection for salt formation. (1)H and (15)N chemical shift changes in three bases (pyrazine, phthalazine, and pyridine) on the addition of acids (1:1 ratio) were determined in various solvents. The effect of acid strength on chemical shift changes was examined. (1)H and (15)N chemical shift changes indicated protonation (salt formation). The media used affected the observed chemical shift changes. In D(2)O the data followed the DeltapK(a) (base-acid) general rule, that the pK(a) value of the acids should be 2 units lower than the pK(a) of the base to ensure proton transfer. Protonation, as measured by chemical shift changes using solution NMR spectroscopy, provided novel insight on potential salt formation in different media. Solution NMR spectroscopy appears to be a useful tool to evaluate counter ion and solvent selection for salt formation reaction.

Suppression of HDAC nuclear export and cardiomyocyte hypertrophy by novel irreversible inhibitors of CRM1.

Histone deacetylase 5 (HDAC5) represses expression of nuclear genes that promote cardiac hypertrophy. Agonism of a variety of G protein coupled receptors (GPCRs) triggers phosphorylation-dependent nuclear export of HDAC5 via the CRM1 nuclear export receptor, resulting in derepression of pro-hypertrophic genes. A cell-based high-throughput screen of a commercial compound collection was employed to identify compounds with the ability to preserve the nuclear fraction of GFP-HDAC5 in primary cardiomyocytes exposed to GPCR agonists. A hit compound potently inhibited agonist-induced GFP-HDAC5 nuclear export in cultured neonatal rat ventricular myocytes (NRVMs). A small set of related compounds was designed and synthesized to evaluate structure-activity relationship (SAR). The results demonstrated that inhibition of HDAC5 nuclear export was a result of compounds irreversibly reacting with a key cysteine residue in CRM1 that is required for its function. CRM1 inhibition by the compounds also resulted in potent suppression of cardiomyocyte hypertrophy. These studies define a novel class of anti-hypertrophic compounds that function through irreversible inhibition of CRM1-dependent nuclear export.

The use of MPA amide for the assignment of absolute configuration of a sterically hindered cyclic secondary amine by 'mix and shake' NMR method.

We present here a new method using methoxyphenylacetic acid (MPA) as the chiral derivatizing agent (CDA) for the assignment of absolute configuration of cyclic secondary amines. The MPA amides were prepared using the purification-free 'mix and shake' method. A detailed conformational analysis for the two diastereomeric amides was conducted by 2D NMR experiments and molecular mechanics calculations. We have established that, in the most stable conformation of each syn rotamer of MPA amides, the H-alpha in the MPA moiety is oriented toward the bulky substituent group at the asymmetric carbon in the chiral amine, presumably to avoid steric and/or electrostatic interactions. The observed NMR data were correlated with the conformational model to allow unambiguous assignment of absolute configuration of secondary amines. The results demonstrate that the MPA can be used as a useful CDA in the case of sterically crowded cyclic secondary amines from which the MTPA amides are usually difficult to make.

Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.

The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism. Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations. The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/extrusion. Here we show that GPR91 (ref. 3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to alpha-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and alpha-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure.