Marino Convertino

Pharmacological Chaperones: Design and Development of New Therapeutic Strategies for the Treatment of Conformational Diseases.

Errors in protein folding may result in premature clearance of structurally aberrant proteins, or in the accumulation of toxic misfolded species or protein aggregates. These pathological events lead to a large range of conditions known as conformational diseases. Several research groups have presented possible therapeutic solutions for their treatment by developing novel compounds, known as pharmacological chaperones. These cell-permeable molecules selectively provide a molecular scaffold around which misfolded proteins can recover their native folding and, thus, their biological activities. Here, we review therapeutic strategies, clinical potentials, and cost-benefit impacts of several classes of pharmacological chaperones for the treatment of a series of conformational diseases.

The Molecular Basis for Dual Fatty Acid Amide Hydrolase (FAAH)/Cyclooxygenase (COX) Inhibition

The design of multitarget‐directed ligands is a promising strategy for discovering innovative drugs. Here, we report a mechanistic study that clarifies key aspects of the dual inhibition of the fatty acid amide hydrolase (FAAH) and the cyclooxygenase (COX) enzymes by a new multitarget‐directed ligand named ARN2508 (2‐[3‐fluoro‐4‐[3‐(hexylcarbamoyloxy)phenyl]phenyl]propanoic acid). This potent dual inhibitor combines, in a single scaffold, the pharmacophoric elements often needed to block FAAH and COX, that is, a carbamate moiety and the 2‐arylpropionic acid functionality, respectively. Molecular modeling and molecular dynamics simulations suggest that ARN2508 uses a noncovalent mechanism of inhibition to block COXs, while inhibiting FAAH via the acetylation of the catalytic Ser241, in line with previous experimental evidence for covalent FAAH inhibition. This study proposes the molecular basis for the dual FAAH/COX inhibition by this novel hybrid scaffold, stimulating further experimental studies and offering new insights for the rational design of novel anti‐inflammatory agents that simultaneously act on FAAH and COX.

μ-Opioid receptor 6-transmembrane isoform: A potential therapeutic target for new effective opioids

The μ-opioid receptor (MOR) is the primary target for opioid analgesics. MOR induces analgesia through the inhibition of second messenger pathways and the modulation of ion channels activity. Nevertheless, cellular excitation has also been demonstrated, and proposed to mediate reduction of therapeutic efficacy and opioid-induced hyperalgesia upon prolonged exposure to opioids. In this mini-perspective, we review the recently identified, functional MOR isoform subclass, which consists of six transmembrane helices (6 TM) and may play an important role in MOR signaling. There is evidence that 6 TM MOR signals through very different cellular pathways and may mediate excitatory cellular effects rather than the classic inhibitory effects produced by the stimulation of the major (7 TM) isoform. Therefore, the development of 6 TM and 7 TM MOR selective compounds represents a new and exciting opportunity to better understand the mechanisms of action and the pharmacodynamic properties of a new class of opioids.

COMT gene locus: new functional variants

Abstract Catechol-O-methyltransferase (COMT) metabolizes catecholaminergic neurotransmitters. Numerous studies have linked COMT to pivotal brain functions such as mood, cognition, response to stress, and pain. Both nociception and risk of clinical pain have been associated with COMT genetic variants, and this association was shown to be mediated through adrenergic pathways. Here, we show that association studies between COMT polymorphic markers and pain phenotypes in 2 independent cohorts identified a functional marker, rs165774, situated in the 3′ untranslated region of a newfound splice variant, (a)-COMT. Sequence comparisons showed that the (a)-COMT transcript is highly conserved in primates, and deep sequencing data demonstrated that (a)-COMT is expressed across several human tissues, including the brain. In silico analyses showed that the (a)-COMT enzyme features a distinct C-terminus structure, capable of stabilizing substrates in its active site. In vitro experiments demonstrated not only that (a)-COMT is catalytically active but also that it displays unique substrate specificity, exhibiting enzymatic activity with dopamine but not epinephrine. They also established that the pain-protective A allele of rs165774 coincides with lower COMT activity, suggesting contribution to decreased pain sensitivity through increased dopaminergic rather than decreased adrenergic tone, characteristic of reference isoforms. Our results provide evidence for an essential role of the (a)-COMT isoform in nociceptive signaling and suggest that genetic variations in (a)-COMT isoforms may contribute to individual variability in pain phenotypes.

ApoE4-specific Misfolded Intermediate Identified by Molecular Dynamics Simulations

The increased risk of developing Alzheimer’s disease (AD) is associated with the APOE gene, which encodes for three variants of Apolipoprotein E, namely E2, E3, E4, differing only by two amino acids at positions 112 and 158. ApoE4 is known to be the strongest risk factor for AD onset, while ApoE3 and ApoE2 are considered to be the AD-neutral and AD-protective isoforms, respectively. It has been hypothesized that the ApoE isoforms may contribute to the development of AD by modifying the homeostasis of ApoE physiological partners and AD-related proteins in an isoform-specific fashion. Here we find that, despite the high sequence similarity among the three ApoE variants, only ApoE4 exhibits a misfolded intermediate state characterized by isoform-specific domain-domain interactions in molecular dynamics simulations. The existence of an ApoE4-specific intermediate state can contribute to the onset of AD by altering multiple cellular pathways involved in ApoE-dependent lipid transport efficiency or in AD-related protein aggregation and clearance. We present what we believe to be the first structural model of an ApoE4 misfolded intermediate state, which may serve to elucidate the molecular mechanism underlying the role of ApoE4 in AD pathogenesis. The knowledge of the structure for the ApoE4 folding intermediate provides a new platform for the rational design of alternative therapeutic strategies to fight AD.

Structural and functional interactions between six-transmembrane μ-opioid receptors and β2-adrenoreceptors modulate opioid signaling.

The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β2-adrenergic receptors (β2-ARs) through an interaction with the fifth and sixth helices of β2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy.

Docking and Scoring with Target-Specific Pose Classifier Succeeds in Native-Like Pose Identification But Not Binding Affinity Prediction in the CSAR 2014 Benchmark Exercise.

The CSAR 2014 exercise provided an important benchmark for testing current approaches for pose identification and ligand ranking using three X-ray characterized proteins: Factor Xa (FXa), Spleen Tyrosine Kinase (SYK), and tRNA Methyltransferase (TRMD). In Phase 1 of the exercise, we employed Glide and MedusaDock docking software, both individually and in combination, with the special target-specific pose classifier trained to discriminate native-like from decoy poses. All approaches succeeded in the accurate detection of native and native-like poses. We then used Glide SP and MedusaScore scoring functions individually and in combination with the pose-scoring approach to predict relative binding affinities of the congeneric series of ligands in Phase 2 of the exercise. Similar to other participants in the CSAR 2014 exercise, we found that our models showed modest prediction accuracy. Quantitative structure-activity relationship (QSAR) models developed for the FXa ligands using available bioactivity data from ChEMBL showed relatively low prediction accuracy for the CSAR 2014 ligands of the same target. Interestingly, QSAR models built with CSAR data only yielded Spearman correlation coefficients as high as ρ = 0.69 for FXa and ρ = 0.79 for SYK based on 5-fold cross-validation. Virtual screening of the DUD library using the FXa structure was successful in discriminating between active compounds and decoys in spite of poor ranking accuracy of the underlying scoring functions. Our results suggest that two of the three common tasks associated with molecular docking, i.e., native-like pose identification and virtual screening, but not binding affinity prediction, could be accomplished successfully for the CSAR 2014 challenge data set.

Differences in the Antinociceptive Effects and Binding Properties of Propranolol and Bupranolol Enantiomers

UNLABELLED Recent efforts have suggested that the β-adrenergic receptor (β-AR) system may be a novel and viable therapeutic target for pain reduction; however, most of the work to date has focused on the β(2)-adrenergic receptor (AR). Here, we compared the antinociceptive effects of enantiomeric configurations of propranolol and bupranolol, two structurally similar nonselective β-blocking drugs, against mouse models of inflammatory and chronic pain. In addition, we calculated in silico docking and measured the binding properties of propranolol and bupranolol for all 3 β-ARs. Of the agents examined, S-bupranolol is superior in terms of its antinociceptive effect and exhibited fewer side effects than propranolol or its associated enantiomers. In contrast to propranolol, S-bupranolol exhibited negligible β-AR intrinsic agonist activity and displayed a full competitive antagonist profile at β(1)/β(2)/β(3)-ARs, producing a unique blockade of β(3)-ARs. We have shown that S-bupranolol is an effective antinociceptive agent in mice without negative side effects. The distinctive profile of S-bupranolol is most likely mediated by its negligible β-AR intrinsic agonist activity and unique blockade of β(3)-AR. These findings suggest that S-bupranolol instead of propranolol may represent a new and effective treatment for a variety of painful conditions. PERSPECTIVE The S enantiomer of bupranolol, a β-receptor antagonist, shows greater antinociceptive efficacy and a superior preclinical safety profile and it should be considered as a unique β-adrenergic receptor compound to advance future clinical pain studies.

Differential Regulation of 6- and 7-Transmembrane Helix Variants of μ-Opioid Receptor in Response to Morphine Stimulation.

The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids.

miRNA-711 Binds and Activates TRPA1 Extracellularly to Evoke Acute and Chronic Pruritus

Increasing evidence suggests that extracellular miRNAs may serve as biomarkers of diseases, but the physiological relevance of extracellular miRNA is unclear. We find that intradermal cheek injection of miR-711 induces TRPA1-depedent itch (scratching) without pain (wiping) in naive mice. Extracellular perfusion of miR-711 induces TRPA1 currents in both Trpa1-expressing heterologous cells and native sensory neurons through the core sequence GGGACCC. Computer simulations reveal that the core sequence binds several residues at the extracellular S5-S6 loop of TRPA1, which are critical for TRPA1 activation by miR-711 but not allyl isothiocyanate. Intradermal inoculation of human Myla cells induces lymphoma and chronic itch in immune-deficient mice, associated with increased serum levels of miR-711, secreted from cancer cells. Lymphoma-induced chronic itch is suppressed by miR-711 inhibitor and a blocking peptide that disrupts the miR-711/TRPA1 interaction. Our findings demonstrated an unconventional physiological role of extracellular naked miRNAs as itch mediators and ion channel modulators.