A substrate-free activity-based protein profiling screen for the discovery of selective PREPL inhibitors

Peptidases play vital roles in physiology through the biosynthesis, degradation, and regulation of peptides. Prolyl endopeptidase-like (PREPL) is a newly described member of the prolyl peptidase family, with significant homology to mammalian prolyl endopeptidase and the bacterial peptidase oligopeptidase B. The biochemistry and biology of PREPL are of fundamental interest due to this enzyme's homology to the biomedically important prolyl peptidases and its localization in the central nervous system. Furthermore, genetic studies of patients suffering from hypotonia-cystinuria syndrome (HCS) have revealed a deletion of a portion of the genome that includes the PREPL gene. HCS symptoms thought to be caused by lack of PREPL include neuromuscular and mild cognitive deficits. A number of complementary approaches, ranging from biochemistry to genetics, will be required to understand the biochemical, cellular, physiological, and pathological mechanisms regulated by PREPL. We are particularly interested in investigating physiological substrates and pathways controlled by PREPL. Here, we use a fluorescence polarization activity-based protein profiling (fluopol-ABPP) assay to discover selective small-molecule inhibitors of PREPL. Fluopol-ABPP is a substrate-free approach that is ideally suited for studying serine hydrolases for which no substrates are known, such as PREPL. After screening over 300,000 compounds using fluopol-ABPP, we employed a number of secondary assays to confirm assay hits and characterize a group of 3-oxo-1-phenyl-2,3,5,6,7,8-hexahydroisoquinoline-4-carbonitrile and 1-alkyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile PREPL inhibitors that are able to block PREPL activity in cells. Moreover, when administered to mice, 1-isobutyl-3-oxo-3,5,6,7-tetrahydro-2H-cyclopenta[c]pyridine-4-carbonitrile distributes to the brain, indicating that it may be useful for in vivo studies. The application of fluopol-ABPP has led to the first reported PREPL inhibitors, and these inhibitors will be of great value in studying the biochemistry of PREPL and in eventually understanding the link between PREPL and HCS.     

Silane Treatment of 3D-Printed Sandstone Models for Improved Spontaneous Imbibition of Water

Transport in Porous Media - Due to the natural heterogeneity of hydrocarbon reservoirs, accurate modeling and simulation of geomaterials can lead to sophisticated problems when multiple variables...     

The ArNe-N2O van der Waals trimer: a high resolution spectroscopic study of its rotational spectrum,structure and dynamics

A new ternary van der Waals complex of the type rare gas-rare gas'-linear molecule, ArNe-N₂O, was investigated using a pulsed molecular beam cavity Fourier transform microwave spectrometer. The rotational spectra of six isotopomers of the trimer were studied in detail. These include Ar²⁰Ne-¹⁴N¹⁴NO, Ar²²Ne-¹⁴N¹⁴NO, Ar²⁰Ne-15N¹⁴NO, Ar²²Ne-¹⁵N¹⁴N0, Ar²⁰Ne-¹⁴N¹⁵NO and Ar²²Ne-¹⁴N¹⁵NO. Nuclear quadrupole hyperfine structures of the rotational transitions that are due to the one or two ¹⁴N nuclei were resolved and analysed. The resulting spectroscopic constants were used to provide structural and dynamical information about the trimer. Based on the quartic centrifugal distortion constants, a harmonic force field analysis was performed to estimate the frequencies of the van der Waals vibrational modes. A perturbation of the electronic charge distribution at the site of the central ¹⁴N nucleus of N²0 upon complex formation was detected and discussed. Differences of structural parameters of the trimer as compared to those of the respective dimer units are indicative of the presence of significant three-body non-additive contributions to the interaction energy.     

High-performance liquid chromatography of amino acid peptides and proteins

Summary The retention behaviour of seven globular proteins ranging in molecular weight from 12,000 to 69,000 was investigated using Mono-Q anion-exchange resin as the stationary phase and sodium chloride as the displacer salt. In particular the influence of changes in ionic strength and mobile phase pH on the isocratic retention properties was assessed. Several proteins were found to have significant retention when the pH of the mobile phase was below the reported pl values of the proteins. This behaviour results from the non-uniform charge distribution on the protein surface, which allows interaction with the charged stationary phase even though the protein net charge is equal to or greater than zero. The influence of pH and ionic strength on experimentally observed bandwidths was also investigated. The dependence of the effective reduced plate height on solute capacity factor was found to vary significantly with the mobile phase pH, a behaviour consistent with the interplay of complex multisite binding kinetics. These results provide a basis for further detailed investigations into the mechanism of interaction of proteins not only with charged surfaces associated with adsorptive chromatographic media but also with other macromolecules. For Part LXXXII, see ref. [27].     

Robust chaos synchronization based on adaptive fuzzy delayed feedback ${\bf{\mathcal{H}}}_{\bf{\infty}}$ control

In this paper, we propose a new adaptive H_￥\mathcal H_\infty synchronization strategy, called an adaptive fuzzy delayed feedback H_￥\mathcal H_\infty synchronization (AFDFHS) strategy, for chaotic systems with uncertain parameters and external disturbances. Based on Lyapunov–Krasovskii theory, Takagi–Sugeno (T–S) fuzzy model and adaptive delayed feedback H_￥\mathcal H_\infty control scheme, the AFDFHS controller is presented such that the synchronization error system is asymptotically stable with a guaranteed H_￥\mathcal{H}_{\infty } performance. It is shown that the design of the AFDFHS controller with adaptive law can be achieved by solving a linear matrix inequality (LMI), which can be easily facilitated by using some standard numerical packages. An illustrative example is given to demonstrate the effectiveness of the proposed AFDFHS approach.     

Potent and selective inhibitors of glutathione S-transferase omega 1 that impair cancer drug resistance

Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC(50) = 21 nM) and in situ (IC(50) = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in chemotherapy resistance.     

Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.     

Effects of reverberation on perceptual segregation of competing voices

Two experiments investigated the effect of reverberation on listeners' ability to perceptually segregate two competing voices. Culling et al. [Speech Commun. 14, 71-96 (1994)] found that for competing synthetic vowels, masked identification thresholds were increased by reverberation only when combined with modulation of fundamental frequency (F0). The present investigation extended this finding to running speech. Speech reception thresholds (SRTs) were measured for a male voice against a single interfering female voice within a virtual room with controlled reverberation. The two voices were either (1) co-located in virtual space at 0 degrees azimuth or (2) separately located at +/-60 degrees azimuth. In experiment 1, target and interfering voices were either normally intonated or resynthesized with a fixed F0. In anechoic conditions, SRTs were lower for normally intonated and for spatially separated sources, while, in reverberant conditions, the SRTs were all the same. In experiment 2, additional conditions employed inverted F0 contours. Inverted F0 contours yielded higher SRTs in all conditions, regardless of reverberation. The results suggest that reverberation can seriously impair listeners' ability to exploit differences in F0 and spatial location between competing voices. The levels of reverberation employed had no effect on speech intelligibility in quiet.     

Valuing flexibility: an impulse control framework

This paper presents a general framework for modeling and evaluating investments which involve flexibility to switch between alternative states of operation (for example, different sets of production inputs and outputs). The model employs a contingent claims pricing approach and encompasses previous real option models as special cases. We discuss practical solution techniques for the valuation problem under different assumptions regarding the cost and frequency of switching.