NK cells are an important arm of the innate immune system, and they constitutively express the NKp30 receptor. NKp30-mediated responses are triggered by the binding of specific ligands, such as tumour cell-derived B7-H6, and involve the secretion of cytotoxic mediators TNF-α, IFN-γ, perforins and granzymes. The latter two constitute a target cell-directed response that is critical in the process of immunosurveillance. The structure of NKp30 is presented, focusing on the ligand-binding site, on the ligand-induced structural changes, and on the experimental data available correlating structure and binding affinity. The translation of NKp30 structural changes to disease progression is also reviewed. NKp30 role in immunotherapy has been explored in chimeric antigen receptor T-cell (CAR-T) therapy. However, antibodies or small ligands targeting NKp30 have not yet been developed. The data reviewed unveils the key structural aspects that must be considered for drug design in order to develop novel immunotherapy approaches.
Background and Purpose: Local anesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of Kv potassium channel inhibition by local anesthetics remained unknown. Kv crystal structures predict that some of these residues point away from the central cavity and face into a drug binding site called ‘side pockets´. Thus, the question arises whether the binding site of local anesthetics is exclusively located in the central cavity or also involves the ‘side pockets´. Experimental Approach: A systematic functional alanine mutagenesis approach, scanning 58 mutants, in concert with in silico docking experiments and molecular dynamics simulations were utilized to elucidate the binding site of bupivacaine and ropivacaine. Key Results: Kv1.5 inhibition by local anesthetics requires binding to the central cavity and the ‘side pockets´, where the latter requires interactions with residues of the S5 and the backside of the S6 segment. Mutations in the ‘side pockets´ remove stereoselectivity of Kv1.5 inhibition by bupivacaine. Strikingly, while we found that binding to the ‘side pockets´ is conserved for the different local anesthetics, the binding mode in the central cavity and the ‘side pockets´ shows considerable variations. Conclusion and Implications: Local anesthetics bind to the central cavity and the ‘side pockets´ which provides a crucial key for the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects.
Background and purpose: The pathophysiology of coronary artery spasm ( CAS), with its associated ischaemic crises, is currently poorly understood, and treatment is frequently ineffective. In view of increasing evidence that platelet- platelet based defects may occur in CAS patients,. we investigated platelet reactivity in CAS patients and whether symptomatic crises reflect activation of platelet-endothelial interactions. Experimental approach: CAS patients were evaluated during acute and/or chronic symptomatic phases, and compared with healthy control subjects. Inhibition of platelet aggregation with ADP by the nitric oxide (NO) donor sodium nitroprusside (SNP), . and plasma levels of syndecan-1 (glycocalyx shedding marker), tryptase (mast cell activation marker), and platelet microparticles were measured. Key Results: Inhibition of aggregation by SNP was impaired in chronic CAS, and tended to deteriorate further during symptomatic crises, while plasma levels of syndecan-1, tryptase and platelet microparticles increased. Infusion of high dose N-acetylcysteine (NAC) plus glyceryl trinitrate rapidly restored platelet responsiveness to SNP and decreased plasma syndecan-1 levels. The effect of NAC on platelet responsiveness to SNP was mimicked in vitro by the H2S donor NaHS. Conversely, inhibition of enzymatic release of H2S attenuated NAC effect. Conclusion and Implications: CAS is associated with substantial impairment of platelet NO signaling. During acute symptomatic exacerbations, platelet resistance to NO is aggravated, together with mast cell activation and damage to both vasculature and platelets. NAC reverses platelet resistance to NO via release of H2S, and reverses glycocalyx shedding during symptomatic crises: this suggests that H2S donors may correct the pathophysiological anomalies underlying CAS.
Background and purpose: Amphetamine use disorder is a serious health concern, but surprisingly little is known about the vulnerability to the moderate and compulsive use of this psychostimulant and its underlying mechanisms. Previous research showed that inherited serotonin transporter (SERT) down-regulation increases the motor response to cocaine, as well as moderate and compulsive intake of this psychostimulant. Here we sought to investigate whether these findings generalize to amphetamine and the underlying mechanisms in the nucleus accumbens. Experimental Approach: In serotonin transporter knockout (SERT−/−) and wild-type control (SERT+/+) rats we assessed the locomotor response to acute amphetamine (AMPH) and intravenous AMPH self-administration under short access (ShA: 1 hr daily sessions) and long access (LgA: 6 hr daily sessions) conditions. 24 hrs after AMPH self-administration we analysed the expression of glutamate system components in the nucleus accumbens shell and core. Key results: We found that SERT−/− animals displayed an increased AMPH-induced locomotor response and increased AMPH self-administration under LgA, but not ShA conditions. Further, we observed changes in the vesicular and glial glutamate transporters, NMDA and AMPA receptor subunits and their respective postsynaptic scaffolding proteins as function of serotonin transporter genotype, AMPH exposure (baseline, ShA and LgA) and nucleus accumbens sub region. Conclusion and implications: We demonstrate that SERT gene deletion increases the psychomotor and reinforcing effects of AMPH, and that the latter is potentially mediated, at least in part, by homeostatic changes in the glutamatergic synapse of the nucleus accumbens shell and/or core.
Background and Purpose. Despite widespread abuse of cocaine, there are no approved treatments for cocaine use disorder. Chronic cocaine use is associated with upregulated dopamine D3 receptor (D3R) expression in the brain, and therefore, most D3R-based medication development has focused on D3R antagonists. However, D3R antagonists do not attenuate cocaine intake under “easy” self-administration conditions when response requirements are low. Here we evaluated a novel, highly selective and metabolically stable D3R partial agonist, VK4-40, for its efficacy in reducing cocaine intake and relapse to drug seeking. Experimental Approach. The impact of VK4-40 on cocaine intake and relapse were evaluated using intravenous self-administration procedures under a fixed-ratio 2 reinforcement schedule and cocaine-primed reinstatement conditions in rats. Optogenetic brain-stimulation reward procedures were used to evaluate the interaction of VK4-40 and cocaine in the mesolimbic dopamine system. Sucrose self-administration and a conditioned place preference paradigm was used to evaluate the abuse potential of VK4-40 alone and other unwanted effects. Key Results. VK4-40 dose-dependently reduced cocaine self-administration and cocaine-primed reinstatement of drug-seeking behavior. In addition, VK4-40 inhibited cocaine-enhanced brain-stimulation reward caused by optogenetic stimulation of dopamine neurons in the ventral tegmental area. VK4-40 alone decreased brain-stimulation reward, and produced neither conditioned place preference nor place aversion. This new D3R partial agonist also failed to alter oral sucrose self-administration. Conclusions and Implications. The novel D3R partial agonist, VK4-40, attenuates cocaine reward and relapse in rodents, without significant unwanted effects. These findings support further investigation of D3R partial agonists as putative treatments for cocaine use disorder.
Skin drug delivery is an emerging route in the drug development, due to its great advantages, thus leading to an urgent need to understand the behaviour of active pharmaceutical ingredients into/through the skin. This knowledge is crucial in the early stages of product design and development. Yet, given the skin barrier properties as one of the first body’s natural defence systems, it can act as an obstacle to the successful outcome of a skin drug therapy. To unravel the mechanisms underlying this barrier, reductionist strategies have designed several models with different levels of complexity and integrity, using non-biological and biological components. Besides the detail of information and resemblance to the in vivo Human skin that each in vitro model offers, the technical and economic efforts required should be considered when selecting the most adequate model for the intended research. This review provides an outline of the most commonly applied skin models, including healthy and diseased conditions, lab developed systems and commercialized models, their advantages and limitations and, also an overview of the new trends in skin engineered models.
Cough is an adverse effect that may hinder the delivery of drugs into the lungs. Chemical or mechanical stimulants activate the transient receptor potential in some airway afferent nerves (C or A fibres) to trigger cough. Types of inhaler device and drug, dose, excipients, formulation characteristics including pH, tonicity, aerosol output and particle size may trigger cough by stimulating the cough receptors. Release of inflammatory mediators may increase the sensitivity of the cough receptors to stimulants. The cough-provoking effect of aerosols is enhanced by bronchoconstriction in diseased airways and reduces drug deposition in the target pulmonary regions. In this article, we review the factors by which inhalation products may cause cough.
Background and purpose: Savolitinib (AZD6094, HMPL-504, volitinib) is an oral, potent, and highly selective MET receptor tyrosine kinase inhibitor. This series of studies aimed to develop a pharmacokinetic-pharmacodynamic (PK/PD) model to link inhibition of MET phosphorylation (pMET) by savolitinib with anti-tumour activity. Experimental approach: Cell line-derived xenograft (CDX) experiments using human lung cancer (EBC-1) and gastric cancer (MKN-45) cells were conducted in athymic nude mice using a variety of doses and schedules of savolitinib. Tumour pMET changes and growth inhibition were calculated after 28 days. Population PK/PD techniques were used to construct a PK/PD model for savolitinib. Key results: Savolitinib showed dose- and schedule-dependent anti-tumour activity in the CDX models, with more frequent, lower dosing schedules (e.g. twice daily) being more effective than intermittent, higher dosing schedules (e.g. 4 days on/3 days off or 2 days on/5 days off). There was a clear exposure–response relationship, with maximal suppression of pMET of >90%. Data from additional CDX and patient-derived xenograft (PDX) models overlapped, allowing the calculation of a single EC50 of 0.38 ng/mL. Tumour growth modelling demonstrated that prolonged, high levels of pMET inhibition (>90%) were required for tumour stasis and regression in the models. Conclusion and implications: High and durable levels of MET inhibition by savolitinib are needed for optimal monotherapy anti-tumour activity in preclinical models. The modelling framework developed here can be used to translate tumour growth inhibition from the mouse to human, and thus guide choice of clinical dose and schedule.
Background and Purpose Intra-islet heparan sulfate (HS) plays an important role in the maintenance of the pancreatic islet function. The aim of this study was to investigate the effect mechanism of HS loss on the functioning of islets in diabetic mice. Experimental Approach The hypoglycemic effect of a heparanase inhibitor, OGT2115, was tested in streptozotocin-induced diabetic mice. The islets of pancreas sections were also stained to reveal their morphology. An insulinoma MIN6 cell line and primary isolated murine islets were used to investigate the effect of OGT2115 in vitro. Key Results Intra-islet HS was clearly lost in streptozotocin-induced diabetic mice due to the increased heparanase expression in damaged islets. OGT2115 prevented intra-islet HS loss to improve the glucose profile and insulin secretion in streptozotocin-treated mice. The apoptosis of pancreatic beta cells, the infiltration of mononuclear macrophages, CD4 and CD8 positive T-cells in islets was reduced by OGT2115 in streptozotocin-treated mice, but OGT2115 did not alter the direct streptozotocin-induced damage in vitro. The expression of heparanase was increased in high glucose-treated isolated islets but not in response to direct streptozotocin stimulation. Further experiments showed that high glucose stimuli could decrease the expression of peroxisome proliferator-activated receptor gamma (PPARγ) in cultured islets, thereby relieving the PPARγ-induced inhibition of heparanase gene expression. Conclusion and Implications Hyperglycemia could cause intra-islet HS loss by elevating the expression of heparanase, thereby aggravating inflammatory cell infiltration and islet damage. Inhibition of heparanase might provide benefit for pancreatic beta cell protection in type 1 diabetes.
Background and purpose: Chronic alcohol intake provoked unfavorable geometric and functional changes in the heart along with altered autophagy. Parkin, a cytosolic E3 ubiquitin ligase encoded by PARK2 gene, governs mitochondrial homeostasis and mitophagy although its role in alcoholic cardiomyopathy remains unclear. Experimental approach: This study was designed to examine the role of Parkin in alcohol-induced cardiomyopathy. Adult male wild-type (WT) and PARKIN2 knockout (Parkin-/-) mice were placed on alcohol (4%) or control diet for 8 weeks. Echocardiographic and cardiomyocyte mechanical properties, myocardial and mitochondrial morphology, autophagy and mitophagy were evaluated. GFP-LC3 puncta was employed to assess autophagosome formation. Key results: Our results revealed that alcohol intake led to unfavorable geometric and contractile changes (enlarged left ventricular chamber; decreased fractional shortening, ejection fraction, peak shortening and velocity of shortening/relengthening, prolonged relengthening duration), enlarged cardiomyocyte size and interstitial fibrosis, as well as mitochondrial swelling with cristae disarrangement and mitochondrial depolarization, the effects of which were exacerbated by Parkin deficiency. Alcohol consumption promoted autophagy and PINK1-Parkin-mediated mitophagy, the effects of which were cancelled off by Parkin knockout. Co-immunoprecipitation noted a tight interaction between Parkin and Ambra1 (autophagy and beclin1 regulator 1). In vitro study using neonatal rat cardiomyocytes revealed that Parkin transfection ameliorated ethanol-induced changes in autophagy. However, Ambra1 silencing negated Parkin-induced protection against ethanol-induced autophagy. Conclusions and implications: Taken together, these data suggest an integral role for Parkin in the face of alcoholic challenge possibly through its interaction with Ambra1 to promote autophagy and maintain mitochondrial homeostasis.
Peptide-based cancer therapy has been of great interest due to the unique advantages of peptides, such as the low molecular weight, the ability to specifically target tumor cells, easy availability and low toxicity in normal tissues. Therefore, identify and synthesize novel peptides could provide a promising choice to patients with cancer. The antitumor second generation peptide CIGB-552 has been developed as a candidate to cancer treatment. Proteomic and genomic studies have identified the intracellular protein COMMD1 as the specific target of CIGB-552. This peptide penetrates inside tumor cells to induce the proteasomal degradation of RelA, causing the termination of NF-kB signaling. The antitumor activity of CIGB-552 has been validated in vitro in different human cancer cell lines, as well as in vivo in syngeneic and xenograft tumor mouse models and in cancer-bearing dogs. The aim of this review is to present and discuss the experimental data about CIGB-552, its mechanism of action and its therapeutic potential in human chronic diseases. This peptide is already in phase I of clinical trials as antineoplastic drug, but also has possible application to other inflammatory and metabolic conditions.
An increase in pulmonary artery pressure is a common observation in adult mammals exposed to alveolar hypoxia. It is considered a maladaptive response that places an increased workload on the right ventricle. The mechanisms initiating and maintaining the elevated pressure are of considerable interest to understanding pulmonary vascular homeostasis and developing new treatments for pulmonary hypertension. In particular, it would be helpful to discover the key molecules in the integrated vascular response to hypoxia to inform potential drug targets. One strategy is to take advantage of experiments of nature; specifically, to understand the molecular basis for the inter-individual variation in the pulmonary vascular response to acute and chronic hypoxia. This is the motivation for genetic studies in populations and animals adapted to life at high altitudes. To date, these studies highlight the importance of hypoxia-inducible factor 2α (HIF-2α), encoded by EPAS1, and prolyl hydroxylase domain-containing protein 2 (PHD), encoded by EGLN1, and support efforts to pharmacologically manipulate HIF-2 activity as a treatment for pulmonary hypertension.
Many current animal models of heart failure are hampered by intrinsic methodological complexities, while other models yield only a subtle cardiac phenotype even after prolonged in vivo treatments. A new “chemogenetic” animal model of heart failure recapitulates a critical characteristic shared by many disease states that lead to heart failure in humans: an increase in redox stress in the heart. This “chemogenetic” approach exploits a recombinant yeast enzyme that can be dynamically and specifically activated in vivo to generate the reactive oxygen species (ROS) hydrogen peroxide (H2O2) in cardiac myocytes. Redox stress can be rapidly, selectively, and reversibly manipulated by chemogenetic generation of ROS in cardiac myocytes, yielding a new model of dilated cardiomyopathy. Treatment of animals with the angiotensin receptor blocker valsartan promotes recovery of ventricular function and resolution of adverse cardiac remodeling. This Mini-Review discusses in vivo chemogenetic approaches to manipulate and analyze oxidative stress in the heart.