Synthesis and structure–activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.     

Structure–activity relationships of substituted oxyoxalamides as inhibitors of the human soluble epoxide hydrolase

We explored both structure–activity relationships among substituted oxyoxalamides used as the primary pharmacophore of inhibitors of the human sEH and as a secondary pharmacophore to improve water solubility of inhibitors. When the oxyoxalamide function was modified with a variety of alkyls or substituted alkyls, compound 6 with a 2-adamantyl group and a benzyl group was found to be a potent sEH inhibitor, suggesting that the substituted oxyoxalamide function is a promising primary pharmacophore for the human sEH, and compound 6 can be a novel lead structure for the development of further improved oxyoxalamide or other related derivatives. In addition, introduction of substituted oxyoxalamide to inhibitors with an amide or urea primary pharmacophore produced significant improvements in inhibition potency and water solubility. In particular, the N,N,O-trimethyloxyoxalamide group in amide or urea inhibitors (26 and 31) was most effective among those tested for both inhibition and solubility. The results indicate that substituted oxyoxalamide function incorporated into amide or urea inhibitors is a useful secondary pharmacophore, and the resulting structures will be an important basis for the development of bioavailable sEH inhibitors.     

Imidazolidine-2,4,5- and pirimidine-2,4,6-triones – New primary pharmacophore for soluble epoxide hydrolase inhibitors with enhanced water solubility

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing imidazolidine-2,4,5-trione or pirimidine-2,4,6-trione has been synthesized. Inhibition potency of the described compounds ranges from 8.4 μM to 0.4 nM. The tested compounds possess higher water solubility than their preceding ureas. Molecular docking indicates new bond between the triones and the active site of sEH that in part explain the observed potency of the new pharmacophores. While less potent than the corresponding ureas, the modifications of urea group reported herein yield compounds with higher water solubility, thus permitting easier formulation.     

Exploration of secondary and tertiary pharmacophores in unsymmetrical N,N′-diaryl urea inhibitors of soluble epoxide hydrolase

The impact of various secondary and tertiary pharmacophores on in vitro potency of soluble epoxide hydrolase (sEH) inhibitors based on the unsymmetrical urea scaffold 1 is discussed. N,N′-Diaryl urea inhibitors of soluble epoxide hydrolase exhibit subtle variations in inhibitory potency depending on the secondary pharmacophore but tolerate considerable structural variation in the second linker/tertiary pharmacophore fragment.     

Chalcone oxides—potent selective inhibitors of cytosolic epoxide hydrolase

The systematic screening of over 150 compounds for inhibitory activity on mammalian cytosolic epoxide hydrolase led to identification of chalcone oxide (trans-1-benzoyl-2-phenyloxirane) as an optimal inhibitory structure. Important structural features for inhibition include two hydrophobic moieties preferably orientating in a trans manner from an electrophilic center such as an activated olefin or epoxide, with the epoxide giving maximal activity. Synthesis of chalcone oxide derivatives bearing a single p-substituent on either phenyl ring has led to very potent inhibitors of the enzyme, the best being 4-phenylchalcone oxide (50% inhibition at 6.4 × 10^?8m). Multiple factorial analysis on the inhibition data for the two series of chalcone oxides prepared (phenyl or benzoyl substituted) revealed both the essentialness of hydrophobic interactions and the apparent nonequivalence of the two hydrophobic sites involved in the inhibitory process. Steric factors were considerably less crucial while electronic effects were unimportant in the compounds examined. The chalcone oxides were either inactive or only weak inhibitors of the other major epoxide-metabolizing enzymes in mouse liver, cytosolic glutathione S-transferase, and microsomal epoxide hydrolase. The nature of the inhibition of cytosolic epoxide hydrolase by chalcone oxides was further investigated through steady-state kinetic analysis and the use of amino acid modifiers. Chalcone oxides give a slowly reversible mixed-noncompetitive inhibition. They may interact covalently with a cysteine residue possibly essential to the catalytic action of cytosolic epoxide hydrolase, and may indeed be alternative substrates with very low turnover. The cytosolic and microsomal epoxide hydrolases can be clearly distinguished by these inhibitors, further indicating different catalytic mechanisms.     

Bioisosterism of urea-based GCPII inhibitors: Synthesis and structure–activity relationship studies

We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure–activity relationship studies of the P1′ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with K_i values below 20 nM. Among them, compound 32d (K_i = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1′ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.     

Unsymmetrical non-adamantyl N,N′-diaryl urea and amide inhibitors of soluble expoxide hydrolase

Incorporation of an adamantyl group in prototypical soluble expoxide hydrolase (sEH) inhibitors afforded improved enzyme potency. We explored replacement of the adamantyl group in unsymmetrical ureas and amides with substituted aryl rings to identify equipotent and metabolically stable sEH inhibitors. We found that aryl rings, especially those substituted in the para position with a strongly electron withdrawing substituent, afforded enzyme IC₅₀ values comparable to the adamantyl compounds in an ether substituted, unsymmetrical N,N′-diaryl urea or amide scaffold.     

Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure–activity relationships

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity relationships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.     

Do the high energy lifestyles of shorebirds result in high maximal metabolic rates? Basal and maximal metabolic rates in least and pectoral sandpipers during migration

Shorebirds have high resting and field metabolic rates relative to many other bird groups, and this is posited to be related to their high‐energy lifestyle. Maximum metabolic outputs for cold or exercise are also often high for bird groups with energetically demanding lifestyles. Moreover, shorebirds demonstrate flexible basal and maximal metabolic rates, which vary with changing energy demands throughout the annual cycle. Consequently, shorebirds might be expected to have high maximum metabolic rates, especially during migration periods. We captured least Calidris minutilla and pectoral C. melanotos sandpipers during spring and fall migration in southeastern South Dakota and measured maximal exercise metabolic rate (MMR; least sandpipers only), summit metabolic rate (M_sum, maximal cold‐induced metabolic rate) and basal metabolic rate (BMR, minimum maintenance metabolic rate) with open‐circuit respirometry. BMR for both least and pectoral sandpipers exceeded allometric predictions by 3–14%, similar to other shorebirds, but M_sum and MMR for both species were either similar to or lower than allometric predictions, suggesting that the elevated BMR in shorebirds does not extend to maximal metabolic capacities. Old World shorebirds show the highest BMR during the annual cycle on the Arctic breeding grounds. Similarly, least sandpiper BMR during migration was lower than on the Arctic breeding grounds, but this was not the case for pectoral sandpipers, so our data only partially support the idea of similar seasonal patterns of BMR variation in New World and Old World shorebirds. We found no correlations of BMR with either M_sum or MMR for either raw or mass‐independent data, suggesting that basal and maximum aerobic metabolic rates are modulated independently in these species.     

Sensing cellular biochemistry with fluorescent chemical–genetic hybrids

Fluorescent biosensors are powerful tools for the detection of biochemical events inside cells with high spatiotemporal resolution. Biosensors based on fluorescent proteins often suffer from issues with photostability and brightness. On the other hand, hybrid, chemical–genetic systems present unique opportunities to combine the strengths of synthetic, organic chemistry with biological macromolecules to generate exquisitely tailored semisynthetic sensors.     

Do plant species with high relative growth rates have poorer chemical defences?

1. Most theories of plant strategies assume the presence of certain 'trade-offs'. One such evolutionary trade-off assumes a decrease in growth rate with increasing investment in chemical defences in species adapted to different levels of habitat fertility.
2. To test this hypothesis, we grew 31 herbaceous species of Asteraceae under controlled conditions of temperature (25 °C), humidity (80%), light (500 μmol m^–2 s^–1) and photoperiod (16 h day^–1) in a modified Hoagland hydroponic solution. The plants grew from seed for 35 days post-germination and were harvested at 14, 21, 28 and 35 days. Relative growth rate (RGR) was calculated as well as a general measure of potential phytochemical toxicity (LC₅₀) using an alcohol extraction of secondary compounds followed by Brine Shrimp bioassay and an assay of total phenolics.
3. The interspecific correlation between RGR and the potential phytochemical toxicity was weak and non-significant ( r _S = 0·12, P = 0·53). The correlation between RGR and total phenolics was weak, positive but significant ( r _S = 0·40, P = 0·03).
4. These results suggest that such an evolutionary trade-off does not exist in this group of Asteraceae.     

State-of-the-art computational methods to predict protein–protein interactions with high accuracy and coverage

Prediction of protein–protein interactions (PPIs) commonly involves a significant computational component. Rapid recent advances in the power of computational methods for protein interaction prediction motivate a review of the state-of-the-art. We review the major approaches, organized according to the primary source of data utilized: protein sequence, protein structure, and protein co-abundance. The advent of deep learning (DL) has brought with it significant advances in interaction prediction, and we show how DL is used for each source data type. We review the literature taxonomically, present example case studies in each category, and conclude with observations about the strengths and weaknesses of machine learning methods in the context of the principal sources of data for protein interaction prediction.     

Revisiting glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidases: Crystal structures,physiological substrates and specific inhibitors

Glycoside hydrolase family 20 β-N-acetyl-d-hexosaminidases (GH20s) catalyze the hydrolysis of glycosidic linkages in glycans, glycoproteins and glycolipids. The diverse substrates of GH20s account for their various roles in many important bioprocesses, such as glycoprotein modification, glycoconjugate metabolism, gamete recognition and chitin degradation in fungal cell walls and arthropod exoskeletons. Defects in human GH20s cause lysosomal storage diseases, Alzheimer's disease and osteoarthritis. Similarly, lower levels of GH20s arrest arthropod molting. Although GH20s are promising targets for drug and agrochemical development, designing bioactive molecules to target one specific enzyme is challenging because GH20s share a conserved catalytic mechanism. With the development of structural biology, the last two decades have witnessed a dramatic increase in crystallographic investigations of liganded and unliganded GH20s, providing core information for rational molecular designs. This critical review summarizes recent research advances in GH20s, with a focus on their structural basis of substrate specificity as well as on inhibitor design. As more crystal structures of targeted GH20s are determined and analyzed, dynamics of their catalysis and inhibition will also be elucidated, which will facilitate the development of new drugs, pesticides and agrochemicals.     

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.     

Application of physical–chemical and biological–chemical methods for heavy metals removal from acid mine drainage

Acid mine drainage (AMD) represents a serious environmental problem related to sulfide minerals and coal mining. High content of toxic metals and high acidity in AMD adversely affects surface water, groundwater and soil. The abandoned mine of the Smolník deposit in Slovakia is a typical example in this respect. The quality of AMD needs to be monitored and suitable treatment methods need to be developed.The aim of this paper was to demonstrate the technical feasibility of heavy metals removal from AMD using physical–chemical and biological–chemical methods. The base of the physical–chemical method was electrowinning. The principle of the biological–chemical method was the selective sequential precipitation (SSP) of metals with the application of hydrogen sulfide produced by sulfate-reducing bacteria and sodium hydroxide solution. Both the electrowinning and SSP processes decrease the content of heavy metals in AMD. The pre-treatment of AMD by chemical iron–aluminum precipitation (in the case of electrowinning tests) and chemical iron precipitation (in the case of SSP tests) improved the selectivity of the processes. A further aim of the work was the improvement of the SSP.During the electrochemical experiments, 99% Zn removal – under metallic form – and 94% Mn removal – under MnO₂ form – both with a high degree of purity, were achieved. The SSP process reached the selective precipitation of chosen metals with 99% efficiency – Fe, Al and Mn in the form of metal hydroxides, Cu and Zn as metal sulfides. The results achieved may be used for designing a process appropriate for the selective recovery of metals from the AMD discharged from the Smolník deposit.     

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis

Background

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.

Scope of review

This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.

Major conclusions

The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.

General significance

The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.     

Identification of new candidate drugs for lung cancer using chemical–chemical interactions,chemical–protein interactions and a K-means clustering algorithm

Lung cancer, characterized by uncontrolled cell growth in the lung tissue, is the leading cause of global cancer deaths. Until now, effective treatment of this disease is limited. Many synthetic compounds have emerged with the advancement of combinatorial chemistry. Identification of effective lung cancer candidate drug compounds among them is a great challenge. Thus, it is necessary to build effective computational methods that can assist us in selecting for potential lung cancer drug compounds. In this study, a computational method was proposed to tackle this problem. The chemical–chemical interactions and chemical–protein interactions were utilized to select candidate drug compounds that have close associations with approved lung cancer drugs and lung cancer-related genes. A permutation test and K-means clustering algorithm were employed to exclude candidate drugs with low possibilities to treat lung cancer. The final analysis suggests that the remaining drug compounds have potential anti-lung cancer activities and most of them have structural dissimilarity with approved drugs for lung cancer.     

Bioremediation of synthetic high–chemical oxygen demand wastewater using microalgal species Chlorella pyrenoidosa

The microalgal species Chlorella pyrenoidosa was cultivated in synthetic wastewater of initial chemical oxygen demand (COD), nitrate, and phosphate concentrations of 5000, 100, and 40 mg/L, respectively. The aim of the study was to find out the tolerance of microalgae to different COD concentrations and the extent of COD degradation at those concentrations. Three dilutions of wastewater (initial COD concentrations 5000, 3000, and 1000 mg/L) and three inoculum sizes (0.1, 0.2, and 0.3 g/L) were considered for the study. The experimental parameters such as total organic carbon, total inorganic carbon, COD, optical density, total solids, nitrate, and phosphate were measured on a daily basis. Biodegradation kinetics was determined for all cases using first-order reaction and Monod degradation equations. Optimal results showed that up to 90% reduction in TOC was obtained for 1000 COD wastewater while only 38% reduction in total organic carbon (TOC) was achieved for 5000 COD wastewater. Over 95% reduction in nitrate and nearly 90% removal of phosphate were obtained with the lowest microalgal inoculum concentration (i.e., 0.1 g/L) for all COD dilutions. This study showed that microalgal species C. pyrenoidosa can successfully degrade the organic carbon source (i.e., acetate) with significant removal efficiencies for nitrate and phosphate.