IL GIOCO MOLECOLARE TRA DRUG DISCOVERY E CHEMICAL BIOLOGY

La chemical biology \ue8 una disciplina vicina alla chimica farmaceutica strettamente connessa al processo di drug discovery mediante uno scambio di ruolo di inibitori/ligandi e probe molecolari. In tre esempi di applicazione della chemical biology-drug discovery vengono illustrati i linker per la bioconiugazione, le sonde fluorescenti in studi di interazione farmacorecettore cellulare (target engagement) e l\u2019indagine proteomica dei meccanismi biologici d\u2019azione di inibitori specifici

unexpected impact of the number of glutamine residues on metal complex stability

The emerging question which this study aims to answer is: what impact do glutamines have on the stability of metal–peptide complexes? We focused our attention on the N-terminal domain of Hpn and Hpn-like proteins from Helicobacter pylori. Cu2+ and Ni2+ complexes of the model peptides MAHHE-NH2, MAHHEEQ-NH2, MAHHEQQ-NH2 and MAHHEQQHQA-NH2 were studied by means of different thermodynamic and spectroscopic techniques, as well as through molecular modelling computation. Experimental results, in very good agreement with theoretical findings, lead to the not obvious conclusion that the stability of metal complexes distinctly increases with the number of glutamine residues present in the peptide, although glutamine side-chains do not directly take part in coordination. This peculiar finding allows one to look at polyglutamine sequences, not only the ones present in some bacterial chaperones but also those involved in several neurodegenerative diseases, from a new perspective

the unusual metal ion binding ability of histidyl tags and their mutated derivatives

Peptides that consist of repeated sequences of alternating histidines and alanines strongly bind Cu(ii) and form α-helical structures

Acute and subchronic antinociceptive effects of nociceptin/orphanin FQ receptor agonists infused by intrathecal route in rats

Severe pain occurs in the context of many diseases and conditions and is a leading cause of disability. Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the N/OFQ peptide (NOP) receptor. This peptidergic system controls pain transmission and in particular spinally administered N/OFQ has robust antinociceptive properties. The aim of this study was to investigate the spinal antinociceptive properties of NOP peptide agonists after acute and subchronic treatment in rats. Doses unable to alter motor coordination were selected. UFP-112 (full NOP agonist) and UFP-113 (partial NOP agonist) were administered intrathecally (i.t.) by spinal catheterization. Acute injection of UFP-112 induced antinociceptive response at lower dosages (0.03-1nmol i.t.) compared to morphine and similar to N/OFQ. UFP-113 was effective in a 0.001-1nmol i.t. dose range. The antinociceptive effects of NOP ligands were no longer evident in rats knockout for the NOP gene, while those of morphine were maintained. The continuous spinal infusion (by osmotic pumps) of 0.1nmol/h UFP-112 and UFP-113 showed antinociceptive action comparable to 1-3nmol/h morphine or N/OFQ. The antinociceptive effect of morphine progressively decreased and was no longer significant after 6 days of treatment. Similar results were obtained with N/OFQ, UFP-112, and UFP-113. The acute i.t. injection of morphine in animals tolerant to N/OFQ and UFP-112 evoked analgesic effects. Neither morphine nor N/OFQ induced antinociceptive effects in morphine- and UFP-113-tolerant rats. In conclusion this study highlights the analgesic efficacy and potency of UFP-112 and UFP-113 underlining the relevance of NOP system in analgesia

Solution Structure of ZASP PDZ Domain Implications for Sarcomere Ultrastructure and Enigma Family Redundancy

AbstractZ band alternately spliced PDZ-containing protein (ZASP) is a sarcomere Z disk protein expressed in human cardiac and skeletal muscle that is thought to be involved in a dominant familial dilated cardiomyopathy. The N-terminal PDZ domain of ZASP interacts with the C terminus of α-actinin-2, the major component of the Z disk, probably by forming a ternary complex with titin Z repeats. We have determined the structure of ZASP PDZ by NMR and showed that it is a classical class 1 PDZ domain that recognizes the carboxy-terminal sequence of an α-actinin-2 calmodulin-like domain with micromolar affinity. We also characterized the role of each component in the ternary complex ZASP/α-actinin-2/titin, showing that the α-actinin-2/ZASP PDZ interaction involves a binding surface distinct from that recognized by the titin Z repeats. ZASP PDZ structure was used to model other members of the enigma family by homology and to predict their abilities to bind α-actinin-2

Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5

Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively

Role of the ecto-nucleotidases in the cooperative effect of adenosine and neuropeptide-S on locomotor activity in mice.

Abstract Activation of adenosine receptors modifies the action of classic neurotransmitters (i.e. dopamine, glutamate and acetylcholine) and other neuromodulators, like vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neuropeptide S (NPS). Similarly to adenosine, NPS is involved in the regulation of stimulus and response to fear and arousal. Thus, the present study investigates the effects of NPS on locomotor activity in mice treated with or without α,β-methylene adenosine 5′-diphosphate (AOPCP), the inhibitor of ecto-5′-nucleotidase. Additionally, we evaluate the activity of ecto-5′-nucleotidase in brain slices of mice treated with or without NPS. Male adult CF-1 mice received i.c.v. NPS as 0.1 nmol injection with or without pre-treatment with 1 nmol α,β-methylene adenosine 5′-diphosphate (AOPCP), the selective inhibitor of ecto-5′-nucleotidase, to evaluate locomotor activity. In another set of experiments, mice received i.c.v. infusion of 0.1 nmol NPS to assay enzymatic activity in brain slices. The results demonstrated that the pre-treatment with AOPCP, which was inactive per se, prevented NPS-induced hyperlocomotion in mice. The dose of 0.1 nmol NPS was efficient to induce hyperlocomotion in animals during the observation period in the activity cage. Regarding enzymatic activity, i.c.v. NPS injection did not induce any significant alterations in ATP and AMP hydrolysis in striatum and hippocampus brain slices of mice. The present study shows that the hyperlocomotor effect of NPS depends on the ecto-5′-nucleotidase activity

Design and synthesis of99mTcN-labeled dextran-mannose derivatives for sentinel lymph node detection

Background: New approaches based on the receptor-targeted molecular interaction have been recently developed with the aim to investigate specific probes for sentinel lymph nodes. In particular, the mannose receptors expressed by lymph node macrophages became an attractive target and different multifunctional mannose derivate ligands for the labeling with99mTc have been developed. In this study, we report the synthesis of a specific class of dextran-based, macromolecular, multifunctional ligands specially designed for labeling with the highly stable [99mTc≡N]2+core. Methods: The ligands have been obtained by appending to a macromolecular dextran scaffold pendant arms bearing a chelating moiety for the metallic group and a mannosyl residue for allowing the interaction of the resulting macromolecular99mTc conjugate with specific receptors on the external membrane of macrophages. Two different chelating systems have been selected, S-methyl dithiocarbazate [H2N-NH-C(=S)SCH3=HDTCZ] and a sequence of two cysteine residues, that in combination with a monophosphine coligand, are able to bind the [99mTc≡N]2+core. Conclusions: High-specific-activity labeling has been obtained by simple mixing and heating of the [99mTc≡N]2+group with the new mannose-dextran derivatives

Cyclic Peptides Acting as Allosteric Inhibitors of Human Thymidylate Synthase and Cancer Cell Growth

Thymidylate synthase (TS) is a prominent drug target for different cancer types. However, the prolonged use of its classical inhibitors, substrate analogs that bind at the active site, leads to TS overexpression and drug resistance in the clinic. In the effort to identify anti-TS drugs with new modes of action and able to overcome platinum drug resistance in ovarian cancer, octapeptides with a new allosteric inhibition mechanism were identified as cancer cell growth inhibitors that do not cause TS overexpression. To improve the biological properties, 10 cyclic peptides (cPs) were designed from the lead peptides and synthesized. The cPs were screened for the ability to inhibit recombinant human thymidylate synthase (hTS), and peptide 7 was found to act as an allosteric inhibitor more potent than its parent open-chain peptide [Pro3]LR. In cytotoxicity studies on three human ovarian cancer cell lines, IGROV-1, A2780, and A2780/CP, peptide 5 and two other cPs, including 7, showed IC50 values comparable with those of the reference drug 5-fluorouracil, of the open-chain peptide [d-Gln4]LR, and of another seven prolyl derivatives of the lead peptide LR. These promising results indicate cP 7 as a possible lead compound to be chemically modified with the aim of improving both allosteric TS inhibitory activity and anticancer effectiveness

Lithium attenuates behavioral and biochemical effects of neuropeptide S in mice

Neuropeptide S (NPS) and its receptor NPSR comprise a recently deorphaned G-protein-coupled receptor system. There is a body of evidence suggesting the involvement of NPS in wakefulness, anxiety, locomotor activity and oxidative stress damage. Considering that mood stabilizers block the stimulatory effect of psychostimulants in rodents, the present study aimed to investigate the effects of the pretreatment with lithium and valproate on the hyperlocomotion evoked by NPS. Another relevant action induced by lithium and valproate is the neuroprotection against oxidative stress. Thus, aiming to get further information about the mechanisms of action of NPS, herein we evaluated the effects of NPS, lithium and valproate, and the combination of them on oxidative stress damage. Behavioral studies revealed that the pretreatment with lithium (100 mg/kg, i.p.) and valproate (200 mg/kg, i.p.) prevented hyperlocomotion evoked by NPS 0.1 nmol. Importantly, the dose of valproate used in this study reduced mouse locomotion, although it did not reach the statistical significance. Biochemical analyses showed that lithium attenuated thiobarbituric reactive species (TBARS) formation in the striatum, cerebellum and hippocampus. NPS per se reduced TBARS levels only in the hippocampus. Valproate did not significantly affect TBARS levels in the brain. However, the combination of mood stabilizers and NPS blocked, instead of potentiate, the neuroprotective effects of each one. No relevant alterations were observed in carbonylated proteins after all treatments. Altogether, the present findings suggested that mainly the mood stabilizer lithium evoked antagonistic effects on the mediation of hyperlocomotion and protection against lipid peroxidation induced by NPS