STEP-CD study: ustekinumab use in paediatric Crohn's disease-a multicentre retrospective study from paediatric IBD Porto group of ESPGHAN.

Ustekinumab is an effective therapy for adult Crohn's disease (CD), but data in paediatric CD patients are scarce. The aim of the study was to describe the real-life effectiveness and safety of ustekinumab in paediatric CD. This is a multicentre review of children with Crohn's disease treated with ustekinumab. The aim of our study was to describe the effectiveness and safety of ustekinumab in paediatric real-life practice. This is a study of the Paediatric IBD (inflammatory bowel disease) Porto group of ESPGHAN. Corticosteroid (CS)- and exclusive enteral nutrition (EEN)-free remission, defined as weighted Paediatric Crohn's Disease Activity Index (wPCDAI) < 12.5, and physician global assessment (PGA) were determined at weeks 12 and 52. A total of 101 children were included at a median age of 15.4 years (IQR 12.7-17.2) with a median follow-up of 7.4 months (IQR 5.6-11.8). Ninety-nine percent had received prior anti-TNF, 63% ≥ 2 anti-TNFα therapies and 22% vedolizumab. Baseline median wPCDAI was 39 (IQR 25-57.5) (71 (70%) patients with moderate-severe activity). Weeks 12 and 52 CS- and EEN-free remission were both 40.5%. Clinical response at week 6, iv induction route and older age at onset of ustekinumab treatment were predictive factors associated with clinical remission at week 12. Seven minor adverse events probably related to ustekinumab were reported. One patient died from an unrelated cause.  Conclusion: Our results suggest that ustekinumab is effective and safe in children with chronically active or refractory CD. What is Known: • Ustekinumab is an effective therapy for adult moderate to severe Crohn's disease (CD). • Off-label use of ustekinumab in children is increasing especially in anti-TNF refractory CD. What is New: • Is the largest cohort of real-world use of ustekinumab in paediatric CD to date. • Clinical response at week 6, iv induction and older age at onset of ustekinumab were predictive factors associated with clinical response at week 12.

Novel 1,4-Dihydropyridine Derivatives as Mineralocorticoid Receptor Antagonists.

The mineralocorticoid receptor (MR) belongs to the steroid receptor subfamily of nuclear receptors. MR is a transcription factor key in regulating blood pressure and mineral homeostasis. In addition, it plays an important role in a broad range of biological and pathological conditions, greatly expanding its interest as a pharmacological target. Non-steroidal MR antagonists (MRAs) are of particular interest to avoid side effects and achieve tissue-specific modulation of the receptor. The 1,4-dihydropyridine (1,4-DHP) ring has been identified as an appropriate scaffold to develop non-steroidal MRAs. We report the identification of a novel series of 1,4-DHP that has been guided by structure-based drug design, focusing on the less explored DHP position 2. Interestingly, substituents at this position might interfere with MR helix H12 disposition, which is essential for the recruitment of co-regulators. Several of the newly synthesized 1,4-DHPs show interesting properties as MRAs and have a good selectivity profile. These 1,4-DHPs promote MR nuclear translocation with less efficiency than the natural agonist aldosterone, which explains, at least in part, its antagonist character. Molecular dynamic studies are suggestive of several derivatives interfering with the disposition of H12 in the agonist-associated conformation, and thus, they might stabilize an MR conformation unable to recruit co-activators.

Natural Polyhydroxy Flavonoids, Curcuminoids, and Synthetic Curcumin Analogs as α7 nAChRs Positive Allosteric Modulators.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain, and inflammation. Allosteric modulation of this receptor might be advantageous to reduce the toxicity in comparison with full agonists. Our previous results obtained with some hydroxy-chalcones, which were identified as positive allosteric modulators (PAMs) of α7 nAChR, prompted us to evaluate the potential of some structurally related naturally occurring flavonoids and curcuminoids and some synthetic curcumin analogues, with the aim of identifying new allosteric modulators of the α7 nAChR. Biological evaluation showed that phloretin, demethoxycurcumin, and bis-demethoxicurcuming behave as PAMs of α7 nAChR. In addition, some new curcumin derivatives were able to enhance the signal evoked by ACh; the activity values found for the tetrahydrocurcuminoid analog 23 were especially promising.

Modulating Protein-Protein Interactions by Cyclic and Macrocyclic Peptides. Prominent Strategies and Examples.

Cyclic and macrocyclic peptides constitute advanced molecules for modulating protein-protein interactions (PPIs). Although still peptide derivatives, they are metabolically more stable than linear counterparts, and should have a lower degree of flexibility, with more defined secondary structure conformations that can be adapted to imitate protein interfaces. In this review, we analyze recent progress on the main methods to access cyclic/macrocyclic peptide derivatives, with emphasis in a few selected examples designed to interfere within PPIs. These types of peptides can be from natural origin, or prepared by biochemical or synthetic methodologies, and their design could be aided by computational approaches. Some advances to facilitate the permeability of these quite big molecules by conjugation with cell penetrating peptides, and the incorporation of ß-amino acid and peptoid structures to improve metabolic stability, are also commented. It is predicted that this field of research could have an important future mission, running in parallel to the discovery of new, relevant PPIs involved in pathological processes.

Pharmacogenetics of trough serum anti-TNF levels in paediatric inflammatory bowel disease.

AIMS: Identifying DNA variants associated with trough serum anti-tumour necrosis factor (TNF) levels could predict response to treatment in inflammatory bowel disease (IBD). To date, no specific studies have been performed in children. The aim of this study was to identify genetic variants associated with trough serum anti-TNF levels and whether these variants are differential markers for infliximab and adalimumab. METHODS: We included 154 children (age < 18 years) from 17 hospitals who had been diagnosed with IBD and actively treated with infliximab or adalimumab. Twenty-one polymorphisms were genotyped using real-time PCR. Trough serum anti-TNF levels were measured using enzyme-linked immunosorbent assay (ELISA). The association between DNA polymorphisms and the therapeutic range or the absolute values of anti-TNF drugs was analysed by Fisher exact test, student's t-test and logistic regression. RESULTS: The variants rs5030728 (TLR4) and rs11465996 (LY96) were associated with subtherapeutic infliximab levels. rs1816702 (TLR2) was associated with supratherapeutic levels and rs3397 (TNFRSF1B) with subtherapeutic levels of adalimumab (P < .05). In addition, rs1816702 (TLR2) and rs2569190 (CD14) were associated with absolute values of trough serum adalimumab, and rs2569190 (CD14) was associated with absolute values of trough serum adalimumab and infliximab (P < .05). CONCLUSION: Genotyping of these DNA variants before starting treatment may help to select the best anti-TNF drug in paediatric patients. The SNP rs1816702 is the most promising marker for tailoring the anti-TNF regimen in children with IBD. For the first time, DNA variants are associated with trough serum anti-TNF levels.

Characterization of Novel Synthetic Polyphenols: Validation of Antioxidant and Vasculoprotective Activities.

Antioxidant compounds, including polyphenols, have therapeutic effects because of their anti-inflammatory, antihypertensive, antithrombotic and antiproliferative properties. They play important roles in protecting the cardiovascular and neurological systems, by having preventive or protective effects against free radicals produced by either normal or pathological metabolism in such systems. For instance, resveratrol, a well-known potent antioxidant, has a counteracting effect on the excess of reactive oxygen species (ROS) and has a number of therapeutic benefits, like anti-inflammatory, anti-cancer and cardioprotective activities. Based on previous work from our group, and on the most frequent OH substitutions of natural polyphenols, we designed two series of synthetically accessible bis-polyhydroxyphenyl derivatives, separated by amide or urea linkers. These compounds exhibit high antioxidant ability (oxygen radical absorbance capacity (ORAC) assay) and interesting radical scavenging activity (RSA) values (2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and α,α-diphenyl-ß-picrylhydrazyl (DPPH) tests). Some of the best polyphenols were evaluated in two biological systems, endothelial cells (in vitro) and whole aorta (ex vivo), highly susceptible for the deleterious effects of prooxidants under different inflammatory conditions, showing protection against oxidative stress induced by inflammatory stimuli relevant in cardiovascular diseases, i.e., Angiotensin II and IL-1ß. Selected compounds also showed strong in vivo antioxidant properties when evaluated in the model organism Saccharomyces cerevisiae.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aß1-42, with cell death almost completely prevented at 10 and 30 µM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Amino acid and peptide prodrugs of diphenylpropanones positive allosteric modulators of α7 nicotinic receptors with analgesic activity.

α7 Nicotinic acetylcholine receptors (nAChRs) are ion channels implicated in a number of CNS pathological processes, including pain and psychiatric, cognitive and inflammatory diseases. Comparing with orthosteric agonism, positive allosteric modulation of these channels constitutes an interesting approach to achieve selectivity versus other nicotinic receptors. We have recently described new chalcones and 1,3-diphenylpropanones as positive allosteric modulators (PAMs) of α7 nAChRs, which proved to have good analgesic activities but poor pharmacokinetic properties. Here we report the preparation of amino acid and peptide derivatives as prodrugs of these modulators with the aim of improving their in vivo biological activity. While the valine derivative showed very short half life in aqueous solutions to be considered a prodrug, Val-Val and Val-Pro-Val are suitable precursors of the parent 1,3-diphenylpropanones, via chemical and enzymatic transformation, respectively. Compounds 19 (Val-Val) and 21 (Val-Pro-Val), prodrugs of the 2',5',4-trihydroxy-1,3-diphenylpropan-1-one 3, showed significant antinociceptive activity in in vivo assays. The best compound, 21, displayed a better profile in the analgesia test than its parent compound 3, exhibiting about the same potency but long-lasting effects.

Disrupting VEGF-VEGFR1 Interaction: De Novo Designed Linear Helical Peptides to Mimic the VEGF13-25 Fragment.

The interaction between vascular endothelial growth factor (VEGF) and its receptors (VEGFR) has important implications in angiogenesis and cancer, which moved us to search for peptide derivatives able to block this protein-protein interaction. In a previous work we had described a collection of linear 13-mer peptides specially designed to adopt helical conformations (Ac-SSEEX5ARNX8AAX12N-NH2), as well as the evaluation of seven library components for the inhibition of the interaction of VEGF with its Receptor 1 (VEGFR1). This study led to the discovery of some new, quite potent inhibitors of this protein-protein system. The results we found prompted us to extend the study to other peptides of the library. We describe here the evaluation of a new selection of peptides from the initial library that allow us to identify new VEGF-VEGFR1 inhibitors. Among them, the peptide sequence containing F, W, and I residues at the 5, 9, and 12 positions, show a very significant nanomolar IC50 value, competing with VEGF for its receptor 1, VEGFR1 (Flt-1), which could represent a new tool within the therapeutic arsenal for cancer detection and therapy.

1,3-diphenylpropan-1-ones as allosteric modulators of α7 nACh receptors with analgesic and antioxidant properties.

UNLABELLED: Nicotine acethylcholine receptors (nAChRs) play critical roles in cognitive processes, neuroprotection and inflammation. RESULTS: According to their substituents, 1,3-diphenylpropan-1-one derivatives act as α7 nAChRs negative allosteric modulators (NAM, OMe) or Type I positive allosteric modulators (PAMs, OH). Compounds 7 and 31 were the most effective (989 and 666% enhancement of ACh-induced currents) and potent (EC50: 12.9 and 6.85 µM) PAMs. They exhibited strong radical scavenging values. Compound 31, selective over other neuronal nAChR subtypes and with acceptable pharmacokinetic profile, showed antinociceptive effects in a model of inflammatory pain. CONCLUSION: Compound 31 is a novel, potent and selective α7 nAChR PAM, displaying antioxidant and analgesic activities. The 1,3-diphenylpropan-1-one scaffold could be the base toward more advanced type I PAMs for the treatment of nAChR-mediated diseases.

Chalcones as positive allosteric modulators of α7 nicotinic acetylcholine receptors: a new target for a privileged structure.

The α7 acetylcholine nicotine receptor is a ligand-gated ion channel that is involved in cognition disorders, schizophrenia, pain and inflammation among other diseases. Therefore, the development of new agents that target this receptor has great significance. Positive allosteric modulators might be advantageous, since they facilitate receptor responses without directly interacting with the agonist binding site. Here we report the search for and further design of new positive allosteric modulators having the relatively simple chalcone structure. From the natural product isoliquiritigenin as starting point, chalcones substituted with hydroxyl groups at defined locations were identified as optimal and specific promoters of α7 nicotinic function. The most potent compound (2,4,2',5'-tetrahydroxychalcone, 111) was further characterized showing its potential as neuroprotective, analgesic and cognitive enhancer, opening the way for future developments around the chalcone structure.

Helical peptides from VEGF and Vammin hotspots for modulating the VEGF-VEGFR interaction.

The design, synthesis, conformational studies and binding affinity for VEGF receptors of a collection of linear and cyclic peptide analogues of the N-terminal α-helix fragments 13-25 of VEGF and 1-13 of Vammin are described. Linear 13(14)-mer peptides were designed with the help of an AGADIR algorithm and prepared following peptide solid-phase synthetic protocols. Cyclic peptide derivatives were prepared on-resin from linear precursors with conveniently located Glu and Lys residues, by the formation of amide linkages. Conformational analysis, CD and NMR, showed that most synthesized peptides have a clear tendency to be structured as α-helices in solution. Some of the peptides were able to bind a VEGFR-1 receptor with moderate affinity. In addition to the described key residues (Phe17, Tyr21 and Tyr25), Val14 and Val20 seem to be relevant for affinity.

Disulfide and amide-bridged cyclic peptide analogues of the VEGF81₋91 fragment: synthesis, conformational analysis and biological evaluation.

The design, synthesis, conformational studies and binding affinity for VEGFR-1 receptors of a collection of linear and cyclic peptide analogues of the ß-hairpin fragment VEGF(81-91) are described. Cyclic 11-mer peptide derivatives were prepared from linear precursors with conveniently located Cys, Asp or Dap residues, by the formation of disulfide and amide bridges, using solid-phase synthesis. Molecular modelling studies indicated a tendency to be structured around the central ß-turn of the VEGF(81-91) ß-hairpin in most synthesized cyclic compounds. This structural behavior was confirmed by NMR conformational analysis. The NHCO cyclic derivative 7 showed significant affinity for VEGFR-1, slightly higher than the native linear fragment, thus supporting the design of mimics of this fragment as a valid approach to disrupt the VEGF/VEGFR-1 interaction.

Trp-Trp pairs as ß-hairpin stabilisers: hydrogen-bonded versus non-hydrogen-bonded sites.

Trp-Trp pairs have emerged as a successful strategy for ß-hairpin stabilization. Using loop 3 of Vammin as a template, we experimentally demonstrate that the contribution of Trp-Trp pairs to ß-hairpin stability depends on ß-sheet periodicity, that is, they are stabilising at non-hydrogen-bonded sites, but not at hydrogen-bonded positions.

Parallel solid-phase synthesis of a small library of linear and hydrocarbon-bridged analogues of VEGF(81-91): potential biological tools for studying the VEGF/VEGFR-1 interaction.

The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system.

A role for ring-closing metathesis in medicinal chemistry: mimicking secondary architectures in bioactive peptides.

Synthetically versatile and easy to carry out, Ring-Closing Metathesis (RCM) constitutes an attractive chemical tool, easily amenable for multiple substrates in mild conditions. In medicinal chemistry, the use of RCM has been especially prolific during the last few years. An important application that has benefited from this reaction is the stabilization of spatial conformations in bioactive peptides, since their 3D arrangements play relevant roles in biomolecular recognition processes. RCM reaction is being widely used to introduce conformational constraints into small peptides, through the generation of cyclic structures from appropriate linear precursors. As an alternative to strategies like disulfide or lactam-bridged cyclizations, RCM shows the additional advantage of generating hydrocarbon bridges, less prone to metabolic degradation, and metabolically more stable, which could benefit their pharmacokinetic properties. Particularly remarkable is the application of RCM to the preparation of small peptide modulators able to mimic epitopes identified as hotspots within the surface contact areas in protein-protein interactions (PPIs). This review deals with the replacement of S-S and thioether linkages of cyclic peptides by C-C-bridges and with the stabilization of peptide secondary architectures (α-helix, ß-hairpins, ß-turns) through RCM, as a useful strategy for the modulation of therapeutically relevant signaling pathways.

Modulation of protein-protein interactions by stabilizing/mimicking protein secondary structure elements.

In view of the crucial role of protein-protein intercommunication both in biological and pathological processes, the search of modulators of protein-protein interactions (PPIs) is currently a challenging issue. The development of rational strategies to imitate key secondary structure elements of protein interfaces is complementary to other approaches based on the screening of synthetic or virtual libraries. In this sense, the present review provides representative examples of compounds that are able to disturb PPIs of therapeutic relevance, through the stabilization or the imitation of peptide hot-spots detected in contact areas of the interacting proteins. The review is divided into three sections, covering mimetics of the three main secondary structural elements found in proteins, in general, and in protein-protein interfaces, in particular (alpha-helices, beta-sheets, and reverse turns). Once the secondary element has been identified, the first approach typically involves the translation of the primary peptide structure into different cyclic analogues. This is normally followed by gradual decrease of the peptide nature through combination of peptide and non-peptide fragments in the same molecule. The final step usually consists in the development of pertinent organic scaffolds for appending key functional groups in the right spatial disposition, as a means towards totally non-peptide small molecule PPI modulators.

Dynamics of the rise around a fiber: experimental evidence of the existence of several time scales.

We study experimentally the dynamics of the spontaneous rise of a meniscus made of polydimethylsiloxane of various viscosities on the outside of a micrometric fiber made of monofilament in polyethyleneterephthalate. Optical methods are used to measure simultaneously the height of the liquid interface and the associated contact angle versus time. Versus the liquid viscosity, we observe asymptotically, for the dynamic contact angle, either a t(-1/2) behavior in agreement with the hydrodynamic description or a t(-1) behavior predicted by the molecular-kinetic theory, in its linear form, thus confirming the existence of at least two major time scales in the rise phenomenon.