Genetically encoded discovery of perfluoroaryl macrocycles that bind to albumin and exhibit extended circulation in vivo.

Peptide-based therapeutics have gained attention as promising therapeutic modalities, however, their prevalent drawback is poor circulation half-life in vivo. In this paper, we report the selection of albumin-binding macrocyclic peptides from genetically encoded libraries of peptides modified by perfluoroaryl-cysteine SNAr chemistry, with decafluoro-diphenylsulfone (DFS). Testing of the binding of the selected peptides to albumin identified SICRFFC as the lead sequence. We replaced DFS with isosteric pentafluorophenyl sulfide (PFS) and the PFS-SICRFFCGG exhibited KD = 4-6 µM towards human serum albumin. When injected in mice, the concentration of the PFS-SICRFFCGG in plasma was indistinguishable from the reference peptide, SA-21. More importantly, a conjugate of PFS-SICRFFCGG and peptide apelin-17 analogue (N3-PEG6-NMe17A2) showed retention in circulation similar to SA-21; in contrast, apelin-17 analogue was cleared from the circulation after 2 min. The PFS-SICRFFC is the smallest known peptide macrocycle with a significant affinity for human albumin and substantial in vivo circulation half-life. It is a productive starting point for future development of compact macrocycles with extended half-life in vivo.

A human protein hydroxylase that accepts D-residues.

Factor inhibiting hypoxia-inducible factor (FIH) is a 2-oxoglutarate-dependent protein hydroxylase that catalyses C3 hydroxylations of protein residues. We report FIH can accept (D)- and (L)-residues for hydroxylation. The substrate selectivity of FIH differs for (D) and (L) epimers, e.g., (D)- but not (L)-allylglycine, and conversely (L)- but not (D)-aspartate, undergo monohydroxylation, in the tested sequence context. The (L)-Leu-containing substrate undergoes FIH-catalysed monohydroxylation, whereas (D)-Leu unexpectedly undergoes dihydroxylation. Crystallographic, mass spectrometric, and DFT studies provide insights into the selectivity of FIH towards (L)- and (D)-residues. The results of this work expand the potential range of known substrates hydroxylated by isolated FIH and imply that it will be possible to generate FIH variants with altered selectivities.

Development and application of a comprehensive machine learning program for predicting molecular biochemical and pharmacological properties.

We establish a comprehensive quantitative structure-activity relationship (QSAR) model termed AlphaQ through the machine learning algorithm to associate the fully quantum mechanical molecular descriptors with various biochemical and pharmacological properties. Preliminarily, a novel method for molecular structural alignments was developed in such a way to maximize the quantum mechanical cross correlations among the molecules. Besides the improvement of structural alignments, three-dimensional (3D) distribution of the molecular electrostatic potential was introduced as the unique numerical descriptor for individual molecules. These dual modifications lead to a substantial accuracy enhancement in multifarious 3D-QSAR prediction models of AlphaQ. Most remarkably, AlphaQ has been proven to be applicable to structurally diverse molecules to the extent that it outperforms the conventional QSAR methods in estimating the inhibitory activity against thrombin, the water-cyclohexane distribution coefficient, the permeability across the membrane of the Caco-2 cell, and the metabolic stability in human liver microsomes. Due to the simplicity in model building and the high predictive capability for varying biochemical and pharmacological properties, AlphaQ is anticipated to serve as a valuable screening tool at both early and late stages of drug discovery.

Structural and stereoelectronic insights into oxygenase-catalyzed formation of ethylene from 2-oxoglutarate.

Ethylene is important in industry and biological signaling. In plants, ethylene is produced by oxidation of 1-aminocyclopropane-1-carboxylic acid, as catalyzed by 1-aminocyclopropane-1-carboxylic acid oxidase. Bacteria catalyze ethylene production, but via the four-electron oxidation of 2-oxoglutarate to give ethylene in an arginine-dependent reaction. Crystallographic and biochemical studies on the Pseudomonas syringae ethylene-forming enzyme reveal a branched mechanism. In one branch, an apparently typical 2-oxoglutarate oxygenase reaction to give succinate, carbon dioxide, and sometimes pyrroline-5-carboxylate occurs. Alternatively, Grob-type oxidative fragmentation of a 2-oxoglutarate-derived intermediate occurs to give ethylene and carbon dioxide. Crystallographic and quantum chemical studies reveal that fragmentation to give ethylene is promoted by binding of l-arginine in a nonoxidized conformation and of 2-oxoglutarate in an unprecedented high-energy conformation that favors ethylene, relative to succinate formation.

NPCARE: database of natural products and fractional extracts for cancer regulation.

BACKGROUND: Natural products have increasingly attracted much attention as a valuable resource for the development of anticancer medicines due to the structural novelty and good bioavailability. This necessitates a comprehensive database for the natural products and the fractional extracts whose anticancer activities have been verified. DESCRIPTION: NPCARE (http://silver.sejong.ac.kr/npcare) is a publicly accessible online database of natural products and fractional extracts for cancer regulation. At NPCARE, one can explore 6578 natural compounds and 2566 fractional extracts isolated from 1952 distinct biological species including plants, marine organisms, fungi, and bacteria whose anticancer activities were validated with 1107 cell lines for 34 cancer types. Each entry in NPCARE is annotated with the cancer type, genus and species names of the biological resource, the cell line used for demonstrating the anticancer activity, PubChem ID, and a wealth of information about the target gene or protein. Besides the augmentation of plant entries up to 743 genus and 197 families, NPCARE is further enriched with the natural products and the fractional extracts of diverse non-traditional biological resources. CONCLUSIONS: NPCARE is anticipated to serve as a dominant gateway for the discovery of new anticancer medicines due to the inclusion of a large number of the fractional extracts as well as the natural compounds isolated from a variety of biological resources.

Investigations on recyclisation and hydrolysis in avibactam mediated serine ß-lactamase inhibition.

ß-Lactams inhibit penicillin-binding proteins (PBPs) and serine ß-lactamases by acylation of a nucleophilic active site serine. Avibactam is approved for clinical use in combination with ceftazidime, and is a breakthrough non ß-lactam ß-lactamase inhibitor also reacting via serine acylation. Molecular dynamics (MD) and quantum chemical calculations on avibactam-mediated inhibition of a clinically relevant cephalosporinase reveal that recyclisation of the avibactam derived carbamoyl complex is favoured over hydrolysis. In contrast, we show that analogous recyclisation in ß-lactam mediated inhibition is disfavoured. Avibactam recyclisation is promoted by a proton shuttle, a 'structural' water protonating the nucleophilic serine, and stabilization of the negative charge developed on aminocarbonyl oxygen. The results imply the potential of calculations for distinguishing between bifurcating pathways during inhibition and in generating hypotheses for predicting resistance. The inability of ß-lactams to undergo recyclisation may be an Achilles heel, but one that can be addressed by suitably functionalized reversibly binding inhibitors.

Cation-π Interactions Contribute to Substrate Recognition in γ-Butyrobetaine Hydroxylase Catalysis.

γ-Butyrobetaine hydroxylase (BBOX) is a non-heme Fe(II) - and 2-oxoglutarate-dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C-H bond of γ-butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N(+) >P(+) >As(+). The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation-π interactions in productive substrate recognition.

Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay.

BACKGROUND: A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. METHODS: We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. RESULTS: We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity. CONCLUSION: Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems.

Unraveling innate substrate control in site-selective palladium-catalyzed C-H heterocycle functionalization.

Understanding the regioselectivity of C-H activation in the absence of directing groups is an important step towards the design of site-selective C-H functionalizations. The Pd(ii)-catalyzed direct arylation of chromones and enaminones provides an intriguing example where a simple substitution leads to a divergence in substrate-controlled site-selectivity. We describe computational and experimental studies which reveal this results from a switch in mechanism and therefore the selectivity-determining step. We present computational results and experimentally measured kinetic isotope effects and labelling studies consistent with this proposal. The C-H activation of these substrates proceeds via a CMD mechanism, which favors more electron rich positions and therefore displays a pronounced kinetic selectivity for the C3-position. However, C2-selective carbopalladation is also a competitive pathway for chromones so that the overall regiochemical outcome depends on which substrate undergoes activation first. Our studies provide insight into the site-selectivity based on the favorability of two competing CMD and carbopalladation processes of the substrates undergoing coupling. This model can be utilized to predict the regioselectivity of coumarins which are proficient substrates for carbopalladation. Furthermore, our model is able to account for the opposite selectivities observed for enaminone and chromone, and explains how a less reactive coupling partner leads to a switch in selectivity.

Computational prediction of molecular hydration entropy with hybrid scaled particle theory and free-energy perturbation method.

Despite the importance of the knowledge of molecular hydration entropy (ΔShyd) in chemical and biological processes, the exact calculation of ΔShyd is very difficult, because of the complexity in solute-water interactions. Although free-energy perturbation (FEP) methods have been employed quite widely in the literature, the poor convergent behavior of the van der Waals interaction term in the potential function limited the accuracy and robustness. In this study, we propose a new method for estimating ΔShyd by means of combining the FEP approach and the scaled particle theory (or information theory) to separately calculate the electrostatic solute-water interaction term (ΔSelec) and the hydrophobic contribution approximated by the cavity formation entropy (ΔScav), respectively. Decomposition of ΔShyd into ΔScav and ΔSelec terms is found to be very effective with a substantial accuracy enhancement in ΔShyd estimation, when compared to the conventional full FEP calculations. ΔScav appears to dominate over ΔSelec in magnitude, even in the case of polar solutes, implying that the major contribution to the entropic cost for hydration comes from the formation of a solvent-excluded volume. Our hybrid scaled particle theory and FEP method is thus found to enhance the accuracy of ΔShyd prediction by effectively complementing the conventional full FEP method.

Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

Structure of human RNA N6-methyladenine demethylase ALKBH5 provides insights into its mechanisms of nucleic acid recognition and demethylation.

ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N(6)-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66-292) to 2.0 Å resolution. ALKBH566₋292 has a double-stranded ß-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (ßIV-V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX-nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability. Here, we describe structure-activity relationship studies leading to the discovery of an n-octyl ester form of IOX1 with improved cellular potency (EC50 value of 100 to 4 µM). These findings are supported by in vitro inhibition and selectivity studies, docking studies, activity versus toxicity analysis in cell cultures, and intracellular uptake measurements. The n-octyl ester was found to have improved cell permeability; it was found to inhibit some JmjC demethylases in its intact ester form and to be more selective than IOX1. The n-octyl ester of IOX1 should find utility as a starting point for the development of JmjC inhibitors and as a use as a cell-permeable tool compound for studies investigating the roles of 2OG oxygenases in epigenetic regulation.

Binding of (5S)-penicilloic acid to penicillin binding protein 3.

ß-Lactam antibiotics react with penicillin binding proteins (PBPs) to form relatively stable acyl-enzyme complexes. We describe structures derived from the reaction of piperacillin with PBP3 (Pseudomonas aeruginosa) including not only the anticipated acyl-enzyme complex but also an unprecedented complex with (5S)-penicilloic acid, which was formed by C-5 epimerization of the nascent (5R)-penicilloic acid product. Formation of the complex was confirmed by solution studies, including NMR. Together, these results will be useful in the design of new PBP inhibitors and raise the possibility that noncovalent PBP inhibition by penicilloic acids may be of clinical relevance.

Extended solvent-contact model for protein solvation: test cases for dipeptides.

Solvation effects are critically important in the structural stabilization and functional optimization of proteins. Here, we propose a new solvation free energy function for proteins, and test its applicability in predicting the solvation free energies of dipeptides. The present solvation model involves the improvement of the previous solvent-contact model assuming that the molecular solvation free energy could be given by the sum over the individual atomic contributions. In addition to the existing solvent-contact term, the modified solvation free energy function includes the self-solvation term that reflects the effects of intramolecular interactions in the solute molecule on solute-solvent interactions. Four kinds of atomic parameters should be determined in this solvation model: atomic fragmental volume, maximum atomic occupancy, atomic solvation, and atomic self-solvation parameters. All of these parameters for 16 atom types are optimized with a standard genetic algorithm in such a way to minimize the difference between the solvation free energies of dipeptides obtained from high-level quantum chemical calculations and those predicted by the solvation free energy function. The solvation free energies of dipeptides estimated from the new solvation model are in good agreement with the quantum chemical results. Therefore, the optimized solvation free energy function is expected to be useful for examining the structural and energetic features of proteins in aqueous solution.

New solvation free energy function comprising intermolecular solvation and intramolecular self-solvation terms.

Solvation free energy is a fundamental thermodynamic quantity that should be determined to estimate various physicochemical properties of a molecule and the desolvation cost for its binding to macromolecular receptors. Here, we propose a new solvation free energy function through the improvement of the solvent-contact model, and test its applicability in estimating the solvation free energies of organic molecules with varying sizes and shapes. This new solvation free energy function is constructed by combining the existing solute-solvent interaction term with the self-solvation term that reflects the effects of intramolecular interactions on solvation. Four kinds of atomic parameters should be determined in this solvation model: atomic fragmental volume, maximum atomic occupancy, atomic solvation, and atomic self-solvation parameters. All of these parameters for total 37 atom types are optimized by the operation of a standard genetic algorithm in such a way to minimize the difference between the experimental solvation free energies and those calculated by the solvation free energy function for 362 organic molecules. The solvation free energies estimated from the new solvation model compare well with the experimental results with the associated squared correlation coefficients of 0.88 and 0.85 for training and test sets, respectively. The present solvation model is thus expected to be useful for estimating the solvation free energies of organic molecules.

5-Carboxy-8-hydroxyquinoline is a Broad Spectrum 2-Oxoglutarate Oxygenase Inhibitor which Causes Iron Translocation.

2-Oxoglutarate and iron dependent oxygenases are therapeutic targets for human diseases. Using a representative 2OG oxygenase panel, we compare the inhibitory activities of 5-carboxy-8-hydroxyquinoline (IOX1) and 4-carboxy-8-hydroxyquinoline (4C8HQ) with that of two other commonly used 2OG oxygenase inhibitors, N-oxalylglycine (NOG) and 2,4-pyridinedicarboxylic acid (2,4-PDCA). The results reveal that IOX1 has a broad spectrum of activity, as demonstrated by the inhibition of transcription factor hydroxylases, representatives of all 2OG dependent histone demethylase subfamilies, nucleic acid demethylases and γ-butyrobetaine hydroxylase. Cellular assays show that, unlike NOG and 2,4-PDCA, IOX1 is active against both cytosolic and nuclear 2OG oxygenases without ester derivatisation. Unexpectedly, crystallographic studies on these oxygenases demonstrate that IOX1, but not 4C8HQ, can cause translocation of the active site metal, revealing a rare example of protein ligand-induced metal movement.

Docosahexaenoic acid sensitizes colon cancer cells to sulindac sulfide-induced apoptosis.

Sulindac analogs represent one of the most efficacious groups of NSAIDs reducing the risk of colon cancer. Recent studies have shown that sulindac sulfide, a sulindac analog effective at lower doses compared to its parent compound, triggers the death receptor (DR)5-dependent extrinsic apoptotic pathway. Induction of apoptosis via activation of the DR-mediated pathway would be an ideal therapeutic strategy to eliminate cancer cells. In this study, we investigated the possibility that colon cancer cells are sensitized to sulindac sulfide-induced apoptosis by docosahexaenoic acid (DHA), via activation of the DR/extrinsic apoptotic pathway. Our data demonstrated that DHA combination sensitized colon cancer cells to sulindac sulfide-induced apoptosis, leading to enhanced growth suppression of human colon cancer xenografts. The combination effect was primarily attributed to increased cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-8 activation. Moreover, pretreatment with z-IETD-FMK (caspase-8 inhibitor) or stable expression of dominant negative caspase-8 genes blocked DHA/sulindac sulfide cotreatment-induced apoptosis. In view of the finding that DR5 silencing abrogated the combination-stimulated apoptosis, we propose that apoptotic synergy induced by sulindac sulfide plus DHA is mediated via DR5. Our findings collectively support the utility of a combination of sulindac sulfide and DHA in the effective prevention and treatment of colon cancer.

Structure-based virtual screening approach to the discovery of p38 MAP kinase inhibitors.

p38 Mitogen-activated protein kinase (MAPK) has been considered to be a promising target for the development of therapeutics for various immunologic diseases. Herein we report an example for a successful application of the virtual screening with protein-ligand docking to identify the novel inhibitors of p38α MAPK. These inhibitors were screened for having desirable physicochemical properties as a drug candidate and compound 1-3 revealed a moderate inhibitory activity with IC(50) values ranging from 0.7 to 20 µM. Therefore, they deserve a consideration for further development by structure-activity relationship (SAR) studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the newly identified inhibitors in the ATP-binding site of p38 MAPK are addressed in detail.