Substituted 1-methyl-4-phenylpyrrolidin-2-ones - Fragment-based design of N-methylpyrrolidone-derived bromodomain inhibitors.

N-Methylpyrrolidone is one of several chemotypes that have been described as a mimetic of acetyl-lysine in the development of bromodomain inhibitors. In this paper, we describe the synthesis of a 4-phenyl substituted analogue - 1-methyl-4-phenylpyrrolidin-2-one - and the use of aryl substitution reactions as a divergent route for derivatives. Ultimately, this has led to structurally complex, chiral compounds with progressively improved affinity as inhibitors of bromodomain-containing protein 4.

Synthesis and elaboration of N-methylpyrrolidone as an acetamide fragment substitute in bromodomain inhibition.

N-Methylpyrrolidone is a solvent molecule which has been shown to compete with acetyl-lysine-containing peptides for binding to bromodomains. From crystallographic studies, it has also been shown to closely mimic the acetamide binding motif in several bromodomains, but has not yet been directly pursued as a fragment in bromodomain inhibition. In this paper, we report the elaboration of N-methylpyrrolidone as a potential lead in fragment-based drug design. Firstly, N-methylpyrrolidone was functionalised to provide points for chemical elaboration. Then, the moiety was incorporated into analogues of the reported bromodomain inhibitor, Olinone. X-ray crystallography revealed that the modified analogues showed comparable binding affinity and structural mimicry to Olinone in the bromodomain binding site.

Beta amino acid-modified and fluorescently labelled kisspeptin analogues with potent KISS1R activity.

Kisspeptin analogues with improved metabolic stability may represent important ligands in the study of the kisspeptin/KISS1R system and have therapeutic potential. In this paper we assess the activity of known and novel kisspeptin analogues utilising a dual luciferase reporter assay in KISS1R-transfected HEK293T cells. In general terms the results reflect the outcomes of other assay formats and a number of potent agonists were identified among the analogues, including ß(2) -hTyr-modified and fluorescently labelled forms. We also showed, by assaying kisspeptin in the presence of protease inhibitors, that proteolysis of kisspeptin activity within the reporter assay itself may diminish the agonist outputs. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

A gel-capture assay for characterizing the sialyl-glycan selectivity of influenza viruses.

Sialic acids (SA) usually linked to galactose (Gal) in an α2,6- or α2,3-configuration are considered the main cell receptors for influenza viruses, in particular for their hemagglutinins (HA). The typing of influenza virus HA receptor selectivity is relevant for understanding the transmissibility of avian and swine viruses to the human population. In this study we developed a simple and inexpensive gel-capture assay (GCA) of the influenza virus HA receptor-binding selectivity. Its principle is the binding of soluble influenza virus to pentasaccharide analogs, representatives of receptors of human and avian influenza viruses, immobilized on a gel resin. The human and avian analogs consisted of a sialyllactose-N-tetraose c (LSTc) [Neu5Ac(α2,6)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc] and a sialyllactose-N-tetraose a (LSTa) [Neu5Ac(α2,3)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc], respectively. Following equilibration, the unbound virus is washed away and the bound one is assayed via HA by densitometry as a function of the analog concentration. Using GCA, the receptor selectivity of three influenza viruses of different HA subtype was investigated. The results showed that the egg-adapted A/California/07/2009 (H1N1) virus exhibited an avian α2,3-linked LSTa selectivity, however, it retained the ability to bind to the α2,6-linked LSTc human receptor analog. Influenza B virus B/Florida/4/2006 showed α2,6-linked LSTc selectivity and a poor α2,3-linked LSTa avidity. The H3N2 virus A/Wisconsin/15/2009 displayed almost comparable avidity for both receptor analogs with a marginally greater α2,3-linked LSTa avidity. The described assay protocol provides a simple and rapid method for the characterization of influenza virus HA receptor binding selectivity.

Use of Kv1.3 blockers for inflammatory skin conditions.

Recent results using animal models of inflammatory skin conditions have shown that blockers of the voltage-gated potassium channel, Kv1.3 hold great promise for clinical utility. Kv1.3 blockers act as immunosuppressants by modulating the various subsets of inflammatory T and B cells involved in autoimmune disorders. While peptidic inhibitors based on naturally occurring venoms demonstrate potent and selective Kv1.3 blockade, these require parenteral administration and may face potential immunogenicity problems. Small molecule blockers show considerable diversity, however selectivity over other Kv1-family channels has been difficult to achieve. More recent advances have added to the evidence that Kv1.3 channels are a suitable therapeutic target and that the development of novel and selective agents will herald new drugs for inflammatory skin disorders.

The use of tripeptides for lead discovery of 5-HT4 receptor ligands.

A series of 30 tripeptides were synthesized and tested as novel 5-HT4 receptor ligands. Receptor binding assays showed that a subset of compounds had reasonable potency relative to the agonists serotonin and 5-methoxytryptamine. Structure-activity analyses and molecular docking have highlighted avenues for further synthetic work.

Solid-phase synthesis of cyclic analogues related to the hypoglycaemic peptide hGH(6-13): comparison of two i-->i + 4 lactam cyclization procedures.

The use of 1,3-diisopropylcarbodiimide (DIC) for the synthesis of cyclic analogues of the hypoglycaemic peptide fragment derived from the N-terminus of human growth hormone (hGH), namely hGH[6-13], is described. Different strategies were examined to achieve improved yields for the on resin side-chain to side-chain cyclization of the corresponding linear peptides containing reverse beta-turn motifs. When compared with the more reactive Castro's reagent, the results confirm that DIC in the presence of HOBt can be successfully employed to minimize the formation of intermolecular oligomeric byproducts associated with the preparation of cyclic hGH[6-14] peptide analogues based on an i-->(i + 4)Lys-->Glu or Glu-->Lys cyclization strategy.

Importance of the P4' residue in human granzyme B inhibitors and substrates revealed by scanning mutagenesis of the proteinase inhibitor 9 reactive center loop.

The cytotoxic lymphocyte serine proteinase granzyme B induces apoptosis of abnormal cells by cleaving intracellular proteins at sites similar to those cleaved by caspases. Understanding the substrate specificity of granzyme B will help to identify natural targets and develop better inhibitors or substrates. Here we have used the interaction of human granzyme B with a cognate serpin, proteinase inhibitor 9 (PI-9), to examine its substrate sequence requirements. Cleavage and sequencing experiments demonstrated that Glu(340) is the P1 residue in the PI-9 RCL, consistent with the preference of granzyme B for acidic P1 residues. Ala-scanning mutagenesis demonstrated that the P4-P4' region of the PI-9 RCL is important for interaction with granzyme B, and that the P4' residue (Glu(344)) is required for efficient serpin-proteinase binding. Peptide substrates based on the P4-P4' PI-9 RCL sequence and containing either P1 Glu or P1 Asp were cleaved by granzyme B (k(cat)/K(m) 9.5 x 10(3) and 1.2 x 10(5) s(-1) M(-1), respectively) but were not recognized by caspases. A substrate containing P1 Asp but lacking P4' Glu was cleaved less efficiently (k(cat)/K(m) 5.3 x 10(4) s(-1) M(-1)). An idealized substrate comprising the previously described optimal P4-P1 sequence (Ile-Glu-Pro-Asp) fused to the PI-9 P1'-P4' sequence was efficiently cleaved by granzyme B (k(cat)/K(m) 7.5 x 10(5) s(-1) M(-1)) and was also recognized by caspases. This contrasts with the literature value for a tetrapeptide comprising the same P4-P1 sequence (k(cat)/K(m) 6.7 x 10(4) s(-1) M(-1)) and confirms that P' residues promote efficient interaction of granzyme B with substrates. Finally, molecular modeling predicted that PI-9 Glu(344) forms a salt bridge with Lys(27) of granzyme B, and we showed that a K27A mutant of granzyme B binds less efficiently to PI-9 and to substrates containing a P4' Glu. We conclude that granzyme B requires an extended substrate sequence for specific and efficient binding and propose that an acidic P4' substrate residue allows discrimination between early (high affinity) and late (lower affinity) targets during the induction of apoptosis.

Characterization of epitope regions of thyrotropin beta-subunit recognized by the monoclonal antibodies mAb279 and mAb299: a chimeric peptide approach.

This investigation describes the design, synthesis and evaluation of chimeric peptides related to the bovine thyrotropin beta-subunit, bTSHbeta. The structures of these chimeric peptides were derived from investigations with linear peptides and sequence alignment studies, in association with a homology model of TSHbeta developed from the hCG X-ray crystallographic structure. The structures of these chimeric peptides comprised beta-turn regions of loop L1 [bTSHbeta(14-20)] and loop L3 [bTSHbeta(65-72)] held in close proximity by a bis-beta-alanine linker and the disulfide bond bTSHbeta[Cys16-Cys67]. Linear and cyclic chimeric peptides were evaluated in immunochemical assays for their ability to inhibit the binding of radio-iodinated bTSHbeta [125I-bTSHbeta] to the monoclonal antibodies, mAb279 and mAb299. Previously, mAb279 and mAb299 have been shown to recognize epitopes accessible on the surface of TSHbeta that lie in close proximity to the TSH receptor-binding site. The results indicate that these chimeric peptides can specifically inhibit in a dose-dependent manner the binding of 125I-bTSHbeta to mAb299, while having a lesser effect on the binding with mAb279. Based on these results, it can be concluded that the bTSHbeta-epitope recognized by mAb299 involves contributions from amino residues from the beta-turn regions of the L1 and L3 loops of TSHbeta, and that these loop regions flank part of the receptor binding site of the bTSH beta-subunit.

Comparison of the binding of alpha-helical and beta-sheet peptides to a hydrophobic surface.

The induction and stabilisation of secondary structure for a series of amphipathic alpha-helical and beta-sheet peptides upon their binding to lipid-like surfaces has been characterised by reversed phase high-performance liquid chromatography (RP-HPLC). In addition, a series of peptides which have been shown to switch from beta-sheet to alpha-helical conformation upon transfer from a polar to a non-polar solution environment also have been studied. Binding parameters related to the hydrophobic contact area and affinity for immobilised C18 chains were determined at temperatures that ranged from 5 to 85 degrees C, allowing conformational transitions for the peptides during surface adsorption to be monitored. The results demonstrated that all peptides which adopt secondary structure in solution also exhibited large changes in their interactive properties. Overall, this study demonstrates that the hydrophobic face of each amphipathic peptide dominates the binding process and that hydrophobic interactions are a major factor controlling the surface induction of secondary structure.

Tropane-based amino acids for peptide structure-function studies: inhibitors of platelet aggregation.

Novel tropane (azabicycloheptane) and azabicyclohexane containing amino acids have been prepared and incorporated into analogues of reported inhibitors of platelet aggregation. The influence of these central constraints upon biological activity suggest their utility in peptide structure function studies.

Nurse managed prenatal programs affect outcomes for corporations.

Faced with higher medical costs and increased insurance premiums, corporations are focusing on health promotion and wellness. With increasing numbers of women in the workforce, corporations have identified the need for prenatal programs. By developing, initiating, and evaluating outcome-based prenatal programs nurses can target the health care needs of this select population. One such program documented several outcomes including improved employee health and an 86% reduction in maternal/newborn costs.

Conformational stability of a type II' beta-turn motif in human growth hormone [6-13] peptide analogues at hydrophobic surfaces.

The interactive properties of several peptides related to human growth hormone (hGH) [6-13] containing a type II' beta-turn motif have been investigated using reversed-phase high-performance liquid chromatography (RP-HPLC). Various chromatographic parameters related to the hydrophobic interactive surface area and binding affinity were measured over the range of temperatures between 5 and 85 degrees C. Variations in these parameters were consistent with significant differences in the relative stability of the type II' beta-turn structures of these peptidomimetics. The effect of changes in peptide conformation were also investigated through the analysis of band-broadening behaviour during the chromatographic process. Significant variations in bandwidth observed at discrete temperatures were related to the rate of interconversion between the type II' beta-turn and more extended conformers. These investigations further document the potential of RP-HPLC for monitoring subtle changes in peptide secondary structure at hydrophobic interfaces.

Breast-feeding in the workplace: how to succeed.

As the numbers of women of childbearing age in the workforce continue to increase, some employers and employees have viewed maternal employment to be incompatible with breast-feeding. This qualitative study investigated factors that hindered as well as facilitated breast-feeding in the workplace. Findings suggest breast-feeding and employment can and should be compatible.

Conformational analysis of human growth hormone [6-13] peptide analogues.

The conformational analysis of a series of ten hGH[6-13] peptide analogues is reported. As part of our earlier studies, the alpha-aminosuccinimide modified fragment Asu11-hGH[6-13] has previously been identified as a potentiator of insulin activity in intravenous insulin tolerance tests, and various analogues have been subsequently designed, synthesised and employed to acquire structure-activity data. These studies have lead to the conclusion that the conformational characteristics at the C-terminus of each of the active peptide analogues is important to the biological activity. In the present investigation, molecular dynamics and simulated annealing techniques have been used to examine the accessible conformational states of the C-terminal region of ten different hGH[6-13] peptide analogues. Of these six are active peptide analogues while the other four show no biological activity. Examination of the conformer groups identified using this molecular dynamics approach showed a common conformational motif for each of the active peptides.

Synthesis of peptide amides using Fmoc-based solid-phase procedures on 4-methylbenzhydrylamine resins.

A two-step low-high protocol for the efficient synthesis of peptide amides is described. The protocol exploits the efficiency of Reagent K for side-chain deprotection with the capability of the hard acid trifluoromethane-sulfonic acid (TFSMA) for cleavage of the peptide from the benzhydrylamine resin. This procedure has proven to be an effective method for the synthesis of peptide amides. The formation of alpha-aminosuccinimide (Asu) derivatives were observed with aspartyl-containing peptides as a minor side reaction product of this procedure, but this Asp-->Asu rearrangement could be successfully suppressed by employing low temperature conditions. The N- to O-acyl rearrangement of threonine and/or serine residues also only occurred to a minor extent under these synthetic conditions.

A simple and inexpensive sample-handling method for the semi-preparative RP-HPLC of polypeptides and non-polar peptide derivatives: pre-adsorption of samples.

A simple method is described for the application onto HPLC columns of very crude or alternatively poorly soluble polypeptide samples prior to their chromatographic purification. The procedure involves the batch pre-adsorption of the crude polypeptide mixture from a dilute solution onto an appropriate preparative-grade chromatographic adsorbent, removal of the solvent by rotary evaporation or lyophilisation and then dry-packing the pre-adsorbed chromatographic material into guard column cartridges of suitable dimensions. The polypeptide products can then be eluted either by isocratic or gradient elution methods through the cartridge coupled in tandem with prepacked semi-preparative HPLC columns. This method has been successfully utilised for the routine RP-HPLC purification of polar and hydrophobic polypeptides prepared by solid phase peptide synthesis (SPPS) methods as well as peptide derivatives and intermediates used as part of SPPS procedures.

Managing asthma triggers in school.

Asthma, a costly, common chronic condition, needs to be managed proactively rather than reactively. Nurses need to teach children and parents how to recognize and avoid asthma triggers. Asthma management is improved when nurses communicate with the child, parents, and school personnel.