CCR4+ CD8+ T cells clonally expand to differentiated effectors in murine psoriasis and in human psoriatic arthritis.

Psoriasis is a chronic inflammatory skin disease with an autoimmune component and associated with joint inflammation in up to 30% of cases. To investigate autoreactive T cells, we developed an imiquimod-induced psoriasis-like inflammation model in K5-mOVA.tg C57BL/6 mice expressing ovalbumin (OVA) on the keratinocyte membrane, adoptively transferred with OT-I OVA-specific CD8+ T cells. We evaluated the expansion of OT-I CD8+ T cells and their localization in skin, blood, and spleen. scRNA-seq and TCR sequencing data from patients with psoriatic arthritis were also analyzed. In the imiquimod-treated K5-mOVA.tg mouse model, OT-I T cells were markedly expanded in the skin and blood at early time points. OT-I T cells in the skin showed mainly CXCR3+ effector memory phenotype, whereas in peripheral blood there was an expansion of CCR4+ CXCR3+ OT-I cells. At a later time point, expanded OVA-specific T-cell population was found in the spleen. In patients with psoriatic arthritis, scRNA-seq and TCR sequencing data showed clonal expansion of CCR4+ TCM cells in the circulation and further expansion in the synovial fluid. Importantly, there was a clonotype overlap between CCR4+ TCM in the peripheral blood and CD8+ T-cell effectors in the synovial fluid. This mechanism could play a role in the generation and spreading of autoreactive T cells to the synovioentheseal tissues in psoriasis patients at risk of developing psoriatic arthritis.

Serum beta-secretase 1 (BACE1) activity increases in patients with mild cognitive impairment.

Beta-secretase 1 (BACE1) is considered as the key enzyme in amyloid-ß formation. Previous works suggest that high BACE1 activity may be present in brain, cerebrospinal fluid and serum of patients with late-onset Alzheimer's disease (LOAD) as well as mild cognitive impairment (MCI). Therefore, we evaluated whether serum BACE1 activity increases in MCI patients and is associated with the progression from MCI to dementia. BACE1 activity was measured in the serum of 259 MCI patients (162 amnestic-aMCI, 97 non-amnestic-naMCI) and 204 healthy Controls. After a median follow-up of 32 months (range: 10-153), 116 MCI progressed to dementia (87 aMCI and 29 naMCI). Serum BACE1 activity was higher in MCI compared with Controls (p < 0.001), and in aMCI with brain atrophy compared with naMCI without brain atrophy (p = 0.04). No difference in BACE1 activity emerged between converter and non-converter MCI, and this was true for both aMCI and naMCI. However, among aMCI with better cognitive performance (n. 163, MMSE score ≥24/30) those converting to dementia had higher BACE1 activity compared to stable ones (p = 0.05). This was not associated with an increased risk to develop dementia (hazard ratio: 1.65; 95% confidence interval: 0.67-4.01). In conclusion, serum BACE1 activity significantly increased in MCI patients (both amnestic and non-amnestic) compared with Controls. Moreover, higher serum BACE1 activity was observed only among aMCI with a better cognitive performance who progressed to dementia, suggesting that a dysregulation of this enzyme might be an early event primarily associated with neurodegeneration.

Thermodynamic Stability and Speciation of Ga(III) and Zr(IV) Complexes with High-Denticity Hydroxamate Chelators.

Increasing attention has been recently devoted to 89Zr(IV) and 68Ga(III) radionuclides, due to their favorable decay characteristics for positron emission tomography (PET). In the present paper, a deep investigation is presented on Ga(III) and Zr(IV) complexes with a series of tri-(H3L1, H3L3, H3L4 and desferrioxamine E, DFOE) and tetrahydroxamate (H4L2) ligands. Herein, we describe the rational design and synthesis of two cyclic complexing agents (H3L1 and H4L2) bearing three and four hydroxamate chelating groups, respectively. The ligand structures allow us to take advantage of the macrocyclic effect; the H4L2 chelator contains an additional side amino group available for a possible further conjugation with a biomolecule. The thermodynamic stability of Ga(III) and Zr(IV) complexes in solution has been measured using a combination of potentiometric and pH-dependent UV-vis titrations, on the basis of metal-metal competition. The Zr(IV)-H4L2 complex is characterized by one of the highest formation constants reported to date for a tetrahydroxamate zirconium chelate (log ß = 45.9, pZr = 37.0), although the complex-stability increase derived from the introduction of the fourth hydroxamate binding unit is lower than that predicted by theoretical calculations. Solution studies on Ga(III) complexes revealed that H3L1 and H4L2 are stronger chelators in comparison to DFOB. The complex stability obtained with the new ligands is also compared with that previously reported for other hydroxamate ligands. In addition to increasing the library of the thermodynamic stability data of Ga(III) and Zr(IV) complexes, the present work allows new insights into Ga(III) and Zr(IV) coordination chemistry and thermodynamics and broadens the selection of available chelators for 68Ga(III) and 89Zr(IV).

Stress induces reinstatement of extinguished cocaine conditioned place preference by a sequential signaling via neuropeptide S, orexin, and endocannabinoid.

Neurons containing neuropeptide S (NPS) and orexins are activated during stress. Previously, we reported that orexins released during stress, via orexin OX1 receptors (OX1 Rs), contribute to the reinstatement of cocaine seeking through endocannabinoid/CB1 receptor (CB1 R)-mediated dopaminergic disinhibition in the ventral tegmental area (VTA). Here, we further demonstrated that NPS released during stress is an up-stream activator of this orexin-endocannabinoid cascade in the VTA, leading to the reinstatement of cocaine seeking. Mice were trained to acquire cocaine conditioned place preference (CPP) by context-pairing cocaine injections followed by the extinction training with context-pairing saline injections. Interestingly, the extinguished cocaine CPP in mice was significantly reinstated by intracerebroventricular injection (i.c.v.) of NPS (1 nmol) in a manner prevented by intraperitoneal injection (i.p.) of SHA68 (50 mg/kg), an NPS receptor antagonist. This NPS-induced cocaine reinstatement was prevented by either i.p. or intra-VTA microinjection (i.vta.) of SB-334867 (15 mg/kg, i.p. or 15 nmol, i.vta.) and AM 251 (1.1 mg/kg, i.p. or 30 nmol, i.vta.), antagonists of OX1 Rs and CB1 Rs, respectively. Besides, NPS (1 nmol, i.c.v.) increased the number of c-Fos-containing orexin neurons in the lateral hypothalamus (LH) and increased orexin-A level in the VTA. The latter effect was blocked by SHA68. Furthermore, a 30-min restraint stress in mice reinstated extinguished cocaine CPP and was prevented by SHA68. These results suggest that NPS is released upon stress and subsequently activates LH orexin neurons to release orexins in the VTA. The released orexins then reinstate extinguished cocaine CPP via an OX1 R- and endocannabinoid-CB1 R-mediated signaling in the VTA.

Neuropeptide S-initiated sequential cascade mediated by OX1, NK1, mGlu5 and CB1 receptors: a pivotal role in stress-induced analgesia.

BACKGROUND: Stress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model. METHODS: Male C57BL/6 mice of 8-12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice. RESULTS: NPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice. CONCLUSIONS: These results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia.

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Probing the Effect of Sildenafil on Progesterone and Testosterone Production by an Intracellular FRET/BRET Combined Approach.

Forster resonance energy transfer (FRET)-based biosensors have been recently applied to the study of biological pathways. In this study, a new biosensor was validated for the first time in live HEK293 and steroidogenic MLTC-1 cell lines for studying the effect of the PDE5 inhibitor on the hCG/LH-induced steroidogenic pathway. The sensor improves FRET between a donor (D), the fluorescein-like diarsenical probe that can covalently bind a tetracysteine motif fused to the PDE5 catalytic domain, and an acceptor (A), the rhodamine probe conjugated to the pseudosubstrate cGMPS. Affinity constant ( Kd) values of 5.6 ± 3.2 and 13.7 ± 0.8 µM were obtained with HEK293 and MLTC-1 cells, respectively. The detection was based on the competitive displacement of the cGMPS-rhodamine conjugate by sildenafil; the Ki values were 3.6 ± 0.3 nM (IC50 = 2.3 nM) in HEK293 cells and 10 ± 1.0 nM (IC50 = 3.9 nM) in MLTC-1 cells. The monitoring of both cAMP and cGMP by bioluminescence resonance energy transfer allowed the exploitation of the effects of PDE5i on steroidogenesis, indicating that sildenafil enhanced the gonadotropin-induced progesterone-to-testosterone conversion in a cAMP-independent manner.

Glycation affects fibril formation of Aß peptides.

Increasing evidence shows that ß-amyloid (Aß) peptides, which are associated with Alzheimer disease (AD), are heavily glycated in patients, suggesting a role of this irreversible nonenzymatic post-translational modification in pathology. Previous reports have shown that glycation increases the toxicity of the Aß peptides, although little is known about the mechanism. Here, we used the natural metabolic by-product methylglyoxal as a glycating agent and exploited various spectroscopic methods and atomic force microscopy to study how glycation affects the structures of the Aß40 and Aß42 peptides, the aggregation pathway, and the morphologies of the resulting aggregates. We found that glycation significantly slows down but does not prevent ß-conversion to mature fibers. We propose that the previously reported higher toxicity of the glycated Aß peptides could be explained by a longer persistence in an oligomeric form, usually believed to be the toxic species.

Tetrabranched Hetero-Conjugated Peptides as Bifunctional Agonists of the NOP and Mu Opioid Receptors.

The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt1]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.

NOP-Targeted Peptide Ligands.

The nociceptin/orphanin FQ (N/OFQ)-N/OFQ peptide (NOP) receptor system is widely distributed at both the peripheral and central level where it modulates important biological functions with increasing therapeutic implications. This chapter wants to provide a comprehensive and updated overview focused on the available structure-activity relationship studies on NOP receptor peptide ligands developed through different rational approaches. Punctual modifications and cyclizations of the N/OFQ sequence have been properly combined furnishing potent NOP selective ligands with different pharmacological activities (full and partial agonists, pure antagonists) and enhanced metabolic stability in vivo. The screening of peptide libraries provided a second family of NOP ligands that have been successfully optimized. Moreover, recent findings suggest the possibility to apply different multimerization strategies for the realization of multi-target NOP/opioid receptor ligands or tetrabranched N/OFQ derivatives with extraordinarily prolonged duration of action in vivo. The diverse approaches led to the identification of important pharmacological tools along with drug candidates currently in clinical development such as Rec 0438 (aka UFP-112) for the treatment of overactive bladder and SER 100 (aka ZP120) for the clinical management of systolic hypertension.

Nociceptin/Orphanin FQ and Urinary Bladder.

Following identification as the endogenous ligand for the NOP receptor, nociceptin/orphanin FQ (N/OFQ) has been shown to control several biological functions including the micturition reflex. N/OFQ elicits a robust inhibitory effect on rat micturition by reducing the excitability of the afferent fibers. After intravesical administration N/OFQ increases urodynamic bladder capacity and volume threshold in overactive bladder patients but not in normal subjects. Moreover daily treatment with intravesical N/OFQ for 10 days significantly reduced urine leakage episodes. Different chemical modifications were combined into the N/OFQ sequence to generate Rec 0438 (aka UFP-112), a peptide NOP full agonist with high potency and selectivity and long-lasting duration of action. Rec 0438 mimicked the robust inhibitory effects of N/OFQ on rat micturition reflex; its action is solely due to NOP receptor stimulation, does not show tolerance liability after 2 weeks of treatment, and can be elicited by intravesical administration. Collectively the evidence summarized and discussed in this chapter strongly suggests that NOP agonists are promising innovative drugs to treat overactive bladder.

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.

Structure of the nociceptin/orphanin FQ receptor in complex with a peptide mimetic.

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, δ, κ and µ (δ-OR, κ-OR and µ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and µ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands.

Central noradrenergic activity affects analgesic effect of Neuropeptide S.

BACKGROUND: Neuropeptide S (NPS) is an endogenous neuropeptide controlling anxiolysis, wakefulness, and analgesia. NPS containing neurons exist near to the locus coeruleus (LC) involved in the descending anti-nociceptive system. NPS interacts with central noradrenergic neurons; thus brain noradrenergic signaling may be involved in NPS-induced analgesia. We tested NPS analgesia in noradrenergic neuron-lesioned rats using a selective LC noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). METHODS: A total 66 male Sprague-Dawley rats weighing 350-450 g were used. Analgesic effects of NPS were evaluated using hot-plate and tail-flick test with or without DSP-4. The animal allocated into 3 groups; hot-plate with NPS alone intracerebroventricular (icv) (0.0, 1.0, 3.3, and 10.0 nmol), tail-flick NPS alone icv (0.0 and 10.0 nmol), and hot-plate with NPS and DSP-4 (0 or 50 mg/kg ip). In hot-plate with NPS and DSP-4 group, noradrenaline content in the cerebral cortex, pons, hypothalamus, were measured. RESULTS: NPS 10 nmol icv prolonged hot plate (%MPE) but not tail flick latency at 30 and 40 min after administration. DSP-4 50 mg/kg decreased noradrenaline content in the all 3 regions. The NA depletion inhibited NPS analgesic effect in the hot plate test but not tail flick test. There was a significant correlation between hot plate latency (percentage of maximum possible effect: %MPE) with NPS 10 nmol and NA content in the cerebral cortex (p = 0.017, r 2 = 0.346) which noradrenergic innervation arisen mainly from the LC. No other regions had the correlation. CONCLUSIONS: NPS analgesia interacts with LC noradrenergic neuronal activity.

Urotensin-II peptidomimetic incorporating a non-reducible 1,5-triazole disulfide bond reveals a pseudo-irreversible covalent binding mechanism to the urotensin G-protein coupled receptor.

The urotensin-II receptor (UTR) is a class A GPCR that predominantly binds to the pleiotropic cyclic peptide urotensin-II (U-II). U-II is constrained by a disulfide bridge that induces a ß-turn structure and binds pseudo-irreversibly to UTR and is believed to result in a structural rearrangement of the receptor. However, it is not well understood how U-II binds pseudo-irreversibly and the nature of the reorganization of the receptor that results in G-protein activation. Here we describe a series of U-II peptidomimetics incorporating a non-reducible disulfide bond structural surrogate to investigate the feasibility that native U-II binds to the G protein-coupled receptor through disulfide bond shuffling as a mechanism of covalent interaction. Disubstituted 1,2,3-triazoles were designed with the aid of computational modeling as a non-reducible mimic of the disulfide bridge (Cys5-Cys10) in U-II. Solid phase synthesis using CuAAC or RuAAC as the key macrocyclisation step provided four analogues of U-II(4-11) incorporating either a 1,5-triazole bridge (5, 6) or 1,4-triazole bridge (9, 10). Biological evaluation of compounds 5, 6, 9 and 10 was achieved using in vitro [125I]UII binding and [Ca2+]i assays at recombinant human UTR. Compounds 5 and 6 demonstrated high affinity (KD ∼ 10 nM) for the UTR and were also shown to bind reversibly as predicted and activate the UTR to increase [Ca2+]i. Importantly, our results provide new insight into the mechanism of covalent binding of U-II with the UTR.

Selective breeding for high anxiety introduces a synonymous SNP that increases neuropeptide S receptor activity.

Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behavior (HAB) versus low anxiety-related behavior, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behavior. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity-an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioral consequence of these differences revealed that intracerebroventricular NPS reversed the hyperanxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date.

Structure activity studies of nociceptin/orphanin FQ(1-13)-NH2 derivatives modified in position 5.

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1-13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1-13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X(5)]N/OFQ(1-13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [(35)S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X(5)]N/OFQ(1-13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity.

Mass spectrometric/bioinformatic identification of a protein subset that characterizes the cellular activity of anticancer peptides.

The preclinical study of the mechanism of action of anticancer small molecules is challenging due to the complexity of cancer biology and the fragmentary nature of available data. With the aim of identifying a protein subset characterizing the cellular activity of anticancer peptides, we used differential mass spectrometry to identify proteomic changes induced by two peptides, LR and [d-Gln(4)]LR, that inhibit cell growth and compared them with the changes induced by a known drug, pemetrexed, targeting the same enzyme, thymidylate synthase. The quantification of the proteome of an ovarian cancer cell model treated with LR yielded a differentially expressed protein data set with respect to untreated cells. This core set was expanded by bioinformatic data interpretation, the biologically relevant proteins were selected, and their differential expression was validated on three cis-platinum sensitive and resistant ovarian cancer cell lines. Via clustering of the protein network features, a broader view of the peptides' cellular activity was obtained. Differences from the mechanism of action of pemetrexed were inferred from different modulation of the selected proteins. The protein subset identification represents a method of general applicability to characterize the cellular activity of preclinical compounds and a tool for monitoring the cellular activity of novel drug candidates.

Ligands raise the constraint that limits constitutive activation in G protein-coupled opioid receptors.

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in δ (DOP) and µ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4-5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the "two state" extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type. We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form.

A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ.

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores (PWT1, PWT2 and PWT3) containing a maleimido moiety. [Cys(18)]N/OFQ-NH2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar pKB values (8.02-8.48). In vivo after supraspinal administration PWT2-N/OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N/OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness.